Cell evaluation method and cell evaluation program

Abstract

To provide a cell evaluation method and a cell evaluation program that can evaluate the respiration activity of mitochondria noninvasively and quantitatively.SOLUTION: A cell evaluation method includes an extraction step and an evaluation step. The extraction step is to extract peaks derived from reduced cytochrome from a spectrum of Raman scattering light obtained by irradiating cells with excitation light having a wavelength included in an electronic absorption band of the reduced cytochrome. The evaluation step is to evaluate the respiration activity of mitochondria present in the cells on the basis of the intensity of the extracted peaks.SELECTED DRAWING: Figure 2

Description

[Technical Field]

[0001] The present invention relates to a cell evaluation method and a cell evaluation program. [Background technology]

[0002] Oocyte maturity is an important factor that influences the accuracy of assisted reproductive technology. Conventionally, oocyte maturity has been evaluated by, for example, destructive analysis or morphological characteristics. [Prior art documents] [Patent documents]

[0003] [Patent Document 1] Patent No. 6759497 Summary of the Invention [Problem to be solved by the invention]

[0004] However, conventional evaluation methods involve invasive evaluation of oocytes by destructive analysis or subjective evaluation by an observer who observes morphological characteristics, and therefore have problems in assessing oocyte maturity non-invasively and quantitatively. Note that these problems may also arise when assessing mitochondrial respiratory activity present in various types of cells, not just oocyte maturity.

[0005] The present application has been made in view of the above, and aims to provide a cell evaluation method and a cell evaluation program that can evaluate mitochondrial respiratory activity non-invasively and quantitatively. [Means for solving the problem]

[0006] The cell evaluation method according to the present application includes an extraction step and an evaluation step. The extraction step involves irradiating a cell with excitation light having a wavelength included in the electronic absorption band of the reduced cytochrome, and extracting a peak derived from the reduced cytochrome from the spectrum of Raman scattered light obtained by irradiating the cell with excitation light having a wavelength included in the electronic absorption band of the reduced cytochrome. The evaluation step involves evaluating the respiratory activity of mitochondria present in the cell based on the intensity of the extracted peak. [Effects of the Invention]

[0007] According to one aspect of the embodiment, it is possible to obtain an effect of being able to evaluate mitochondrial respiratory activity non-invasively and quantitatively. [Brief explanation of the drawings]

[0008] [Figure 1] FIG. 1 is a diagram showing an overview of a cell evaluation method according to an embodiment. [Figure 2] FIG. 2 is a functional block diagram showing an example of the configuration of the cell evaluation device according to the embodiment. [Figure 3] FIG. 3 is a first explanatory diagram for explaining the reason for using 532 nm excitation light. [Figure 4] FIG. 4 is a second explanatory diagram for explaining the reason for using 532 nm excitation light. [Figure 5] FIG. 5 is a third explanatory diagram for explaining the reason for using 532 nm excitation light. [Figure 6] FIG. 6 shows a first result of the verification of Raman spectra at each stage of oocyte maturity. [Figure 7] FIG. 7 shows a second result of the verification of Raman spectra at each stage of oocyte maturity. [Figure 8] FIG. 8 is a first explanatory diagram for explaining the basis of the evaluation process in the evaluation unit. [Figure 9] FIG. 9 is a second explanatory diagram for explaining the basis of the evaluation process in the evaluation unit. [Figure 10] FIG. 10 is a third explanatory diagram for explaining the basis of the evaluation process in the evaluation unit. [Figure 11]FIG. 11 is a fourth explanatory diagram for explaining the basis of the evaluation process in the evaluation unit. [Figure 12] FIG. 12 is a fifth explanatory diagram for explaining the basis of the evaluation process in the evaluation unit. [Figure 13] FIG. 13 is a sixth explanatory diagram for explaining the basis of the evaluation process in the evaluation unit. [Figure 14] FIG. 14 is a seventh explanatory diagram for explaining the basis of the evaluation process in the evaluation unit. [Figure 15] FIG. 15 shows non-invasive evaluation of Raman spectroscopy. [Figure 16] FIG. 16 is a flowchart showing the procedure of the ovum evaluation process executed by the cell evaluation device according to the embodiment. DETAILED DESCRIPTION OF THE INVENTION

[0009] Hereinafter, a detailed description will be given of a mode for carrying out the cell evaluation method and cell evaluation program according to the present application (hereinafter referred to as "embodiment") with reference to the drawings. Note that the cell evaluation method and cell evaluation program according to the present application are not limited to these embodiments. Furthermore, the same components in the following embodiments are given the same reference numerals, and duplicated descriptions will be omitted.

[0010] (Embodiment) First, an overview of a cell evaluation method according to an embodiment will be described using Fig. 1. Fig. 1 is a diagram showing an overview of a cell evaluation method according to an embodiment. In the cell evaluation method according to an embodiment, the respiratory activity of mitochondria present in a cell 100 shown in Fig. 1 is evaluated by Raman spectroscopy. In particular, in the present disclosure, the maturity of an oocyte, which is the cell 100, is evaluated based on the respiratory activity of the mitochondria. Note that the cell 100 is not limited to an oocyte, and may be other cells such as iPS (Induced Pluripotent Stem) cells.

[0011] Here, evaluation of oocyte maturity is an extremely important factor that determines the accuracy of assisted reproductive technology. In other words, the ability to accurately determine the M2 stage, when oocytes have high fertilization potential (embryo development potential), will lead to improved accuracy in assisted reproductive technology. However, conventional evaluation methods often involve destructive analysis such as RNA assays, oocyte morphological observation, or culture medium analysis, and none of these methods allows for non-invasive and quantitative evaluation of oocyte maturity.

[0012] Therefore, in the present disclosure, peaks derived from reduced cytochromes are extracted from the spectrum of Raman scattered light obtained by Raman spectroscopy, and the maturity of oocytes (or mitochondrial respiratory activity) is evaluated based on the intensity of the peaks. In the following embodiment, a cell evaluation method using reduced cytochrome c, one of the reduced cytochromes, will be described.

[0013] Specifically, in the cell evaluation method according to the embodiment, first, peaks derived from reduced cytochrome c are extracted from the spectrum of Raman scattered light obtained by irradiating cell 100 with excitation light having a wavelength included in the electronic absorption band of reduced cytochrome c (step S1).

[0014] As shown in Fig. 1, in Raman spectroscopy, when excitation light is irradiated onto a cell 100, Rayleigh scattered light, which has the same wavelength as the excitation light, and Raman scattered light, which has a wavelength different from that of the excitation light, are measured. In the present disclosure, the spectrum of Stokes scattered light (Raman spectrum), which has a wavelength longer than that of the excitation light, is used. Fig. 1 shows the Raman spectrum, with the horizontal axis representing the Raman shift and the vertical axis representing the peak intensity.

[0015] In the cell evaluation method according to the embodiment, the Raman shifts are 747, 1125, 1309 and 1582 cm -1The peak with a smaller Raman shift is extracted as the peak of reduced cytochrome c. Note that the smaller the Raman shift of a peak, the closer the peak's wavelength is to the wavelength of the excitation light (Raman shift of zero). The Raman shift of the peak derived from reduced cytochrome c shown in Figure 1 is an example, and the Raman shift value may vary depending on the measurement conditions of the Raman spectroscopy.

[0016] Then, in the cell evaluation method according to the embodiment, the respiratory activity of mitochondria present in the cell 100 is evaluated based on the intensity of the extracted peak (step S2). For example, in the cell evaluation method according to the embodiment, the higher the peak intensity, the higher the mitochondrial respiratory activity is evaluated. This is because the higher the activity of reduced cytochrome c (the stronger the peak intensity) and the higher the fertilization ability of the egg (the closer it is to the M2 stage), the higher the mitochondrial respiratory activity.

[0017] In this way, in the cell evaluation method according to the embodiment, the respiratory activity of mitochondria present in the cell 100 can be quantitatively evaluated by measuring the intensity of the peak derived from the reduced cytochrome obtained by Raman spectroscopy.

[0018] Furthermore, as will be described in detail later, even if excitation light is irradiated onto the cell 100 in Raman spectroscopy, it does not affect the embryonic developmental ability (fertilization ability of the egg) of the cell 100. In other words, by using Raman spectroscopy, it is possible to evaluate mitochondrial respiratory activity without destroying the cell 100. In other words, according to the cell evaluation method of the embodiment, it is possible to evaluate mitochondrial respiratory activity non-invasively and quantitatively.

[0019] Furthermore, in the cell evaluation method according to the embodiment, peaks derived from lipids are extracted from the Raman spectrum shown in Fig. 1, and the maturity of the oocyte is evaluated based on the relationship between the intensities of the peaks derived from reduced cytochrome c and lipids (step S3). In the example shown in Fig. 1, the peaks derived from lipids are at 1447 and 1657 cm -1This is because in overripe oocytes that have passed the M2 stage, lipid metabolism decreases, resulting in an increased amount of lipids (increasing the intensity of the peak). -1 The peaks in the graph are overlapping peaks of proteins in addition to lipids.

[0020] In the cell evaluation method according to the embodiment, the maturity of an oocyte can be evaluated using the intensity ratio of the peaks derived from reduced cytochrome c and lipids, or the product of the intensity ratios, and details of this point will be described later.

[0021] In this way, the cell evaluation method according to the embodiment can non-invasively and quantitatively evaluate the maturity of eggs by measuring the intensities of peaks derived from reduced cytochrome c and lipids obtained by Raman spectroscopy.

[0022] Next, a configuration example of a cell evaluation device that executes a cell evaluation method according to an embodiment will be described with reference to Fig. 2. Fig. 2 is a functional block diagram showing a configuration example of a cell evaluation device 1 according to an embodiment.

[0023] As shown in Fig. 2, the cell evaluation device 1 according to the embodiment is connected to a Raman spectroscopic device 10. Note that Fig. 2 shows an example in which the cell evaluation device 1 according to the embodiment and the Raman spectroscopic device 10 are configured as separate devices, but the cell evaluation device 1 and the Raman spectroscopic device 10 may be configured integrally as a single device.

[0024] The Raman spectrometer 10 is a device that irradiates the cell 100 with excitation light, which is laser light, and measures the generated Raman scattered light. The Raman spectrometer 10 outputs the Raman spectrum of the measured Raman scattered light to the cell evaluation device 1.

[0025] The Raman spectroscopic device 10 may not only output information about the measured Raman spectrum to the cell evaluation device 1, but may also print the Raman spectrum on paper media or store it in a portable storage medium. In such cases, a person (evaluator) may scan the Raman spectrum printed on paper media into the cell evaluation device 1, or connect the storage medium to the cell evaluation device 1 to transfer the data.

[0026] 2, the cell evaluation device 1 according to the embodiment includes a control unit 2 and a storage unit 3. The control unit 2 includes an acquisition unit 21, an extraction unit 22, an evaluation unit 23, and an output unit 24. The storage unit 3 stores threshold information 31.

[0027] In the block diagram of FIG. 2, only the components necessary for explaining the features of this embodiment are shown as functional blocks, and descriptions of general components are omitted.

[0028] In other words, each component shown in the block diagram of Figure 2 is a functional concept and does not necessarily have to be physically configured as shown. For example, the specific form of distribution and integration of each functional block is not limited to that shown, and all or part of it can be functionally or physically distributed and integrated in any unit depending on various loads, usage conditions, etc.

[0029] Here, the cell evaluation device 1 includes, for example, a computer having a CPU (Central Processing Unit), ROM (Read Only Memory), RAM (Random Access Memory), a hard disk drive, input / output ports, and various other circuits.

[0030] The CPU of the computer functions as the acquisition unit 21, extraction unit 22, evaluation unit 23, and output unit 24 of the control unit 2, for example, by reading and executing a reception program stored in the ROM.

[0031] Furthermore, at least some or all of the acquisition unit 21, extraction unit 22, evaluation unit 23 and output unit 24 of the control unit 2 can be configured with hardware such as an ASIC (Application Specific Integrated Circuit) or an FPGA (Field Programmable Gate Array).

[0032] The storage unit 3 is configured with a storage device such as a semiconductor element memory, a hard disk drive, etc. The storage unit 3 stores threshold value information 31, various programs (cell evaluation programs), and various information required for processing by the control unit 2.

[0033] Furthermore, the threshold information 31 stored in the storage unit 3 is information including a threshold for evaluating the cell 100 by the evaluation unit 23, which will be described later. Specifically, the threshold information 31 is information including a threshold for evaluating the maturity of an ovum. The threshold indicated by the threshold information 31 will be described in detail below.

[0034] Next, each function of the control unit 2 (acquisition unit 21, extraction unit 22, evaluation unit 23, and output unit 24) will be described.

[0035] The acquisition unit 21 acquires a spectrum of Raman scattered light (Raman spectrum) obtained by irradiating the cell 100 with excitation light having a wavelength included in the electronic absorption band of reduced cytochrome c. The wavelength of the excitation light can be, for example, 532 nm.

[0036] Here, the rationale for using 532 nm excitation light will be explained using Figures 3 to 5. Figures 3 to 5 are explanatory diagrams 1 to 3 for explaining the rationale for using 532 nm excitation light. Figure 3 shows the ultraviolet-visible absorption band (electronic absorption band) spectra of various cytochromes, including reduced cytochromes. Figure 4 shows Raman spectra obtained when various cytochromes are irradiated with 532 nm excitation light. Figure 5 shows Raman spectra obtained when a mouse ovum (an example of cell 100) is irradiated with 532 nm excitation light and a comparative 785 nm excitation light.

[0037] FIG. 3 shows the ultraviolet-visible absorption band spectra of reduced cytochrome b, reduced cytochrome c, oxidized cytochrome b, and oxidized cytochrome c. As shown in FIG. 3, reduced cytochrome c has an electronic absorption band in the wavelength range of 460 nm to 600 nm. That is, by using excitation light of 532 nm, a wavelength included in the electronic absorption band of reduced cytochrome c, a resonance effect occurs, thereby enhancing the intensity of the peak derived from reduced cytochrome c. Note that although the present disclosure illustrates the use of excitation light of 532 nm, excitation light of any wavelength included in the electronic absorption band of reduced cytochrome c can be used.

[0038] Next, Figure 4 shows the Raman spectra measured by irradiating various cytochromes with 532 nm excitation light. As shown in Figure 4, when reduced cytochrome c was irradiated with 532 nm excitation light, the Raman shifts were particularly pronounced at 750, 1123, 1312, and 1582 cm. -1 It was confirmed that the intensities of the four peaks were increased due to the resonance effect. Therefore, in the present disclosure, the extraction unit 22 in the subsequent stage extracts at least one of these four peaks as the peak of reduced cytochrome c.

[0039] As shown in Figure 4, when reduced cytochrome b was irradiated with 532 nm excitation light, the Raman shifts were particularly high at 747, 1127, and 1583 cm -1 It was confirmed that the intensities of the three peaks above were increased due to the resonance effect. Therefore, instead of reduced cytochrome c, at least one of these three peaks may be extracted as a peak of reduced cytochrome b to evaluate mitochondrial respiratory activity (oocyte maturity).

[0040] Next, Figure 5 shows the Raman spectra measured by irradiating mouse oocytes with excitation light of 532 nm and 785 nm (for comparison). Note that Jcl:ICR mice were used in this experiment. The oocytes used were 15 hours after hCG administration.

[0041] As shown in Figure 5, when oocytes were irradiated with 532 nm excitation light, the 748, 1126, 1310, and 1584 cm , which are derived from reduced cytochrome c, were observed compared to 785 nm excitation light. -1 It was confirmed that the intensities of the four peaks above were strong. Therefore, by using 532 nm excitation light, it is possible to extract the peaks derived from reduced cytochrome c.

[0042] Next, the results of Raman spectrum verification using 532 nm excitation light at each stage of egg maturity will be described with reference to Figures 6 and 7. Figures 6 and 7 show first and second results of Raman spectrum verification at each stage of egg maturity.

[0043] As shown in Figure 6, in this study, mice were administered PMSG, followed 48 hours later by hCG, and eggs were collected 13, 15, 18, and 24 hours after hCG administration. The eggs collected 13 hours later correspond to the M1 stage, those collected 15 hours later to the M2 stage, those collected 18 hours later to the M3 stage, and those collected 24 hours later to the M4 stage.

[0044] In Figure 7, the Raman spectra measured by irradiating the oocytes with 532 nm excitation light after each time point are arranged vertically (vertically on the page). As shown in Figure 7, in all of the Raman spectra after each time point, peaks at 747, 1125, 1310, and 1582 cm originating from reduced cytochrome c were observed. -1 It was confirmed that the intensities of the four peaks (±α) were strong. In other words, it was confirmed that the peaks derived from reduced cytochromes were strong at each stage of oocyte maturity.

[0045] 2, the processing performed by the extraction unit 22 and subsequent units will be described. The extraction unit 22 is a processing unit that executes an extraction step. The extraction unit 22 extracts peaks derived from reduced cytochrome c from the Raman spectrum acquired by the acquisition unit 21.

[0046] Specifically, the extraction unit 22 extracts Raman shifts of 750, 1123, 1312, and 1582 cm from the Raman spectrum. -1 At least one peak in the range (±α allowed) is extracted as a peak of reduced cytochrome c.

[0047] The extraction unit 22 also extracts at least one peak derived from lipids from the Raman spectrum acquired by the acquisition unit 21. The peak derived from lipids has Raman shifts of 1447 and 1657 cm -1 The inventors have already found that

[0048] In the following description, the peak derived from reduced cytochrome c may be referred to as the "first peak," and the peak derived from lipids may be referred to as the "second peak."

[0049] The evaluation unit 23 is a processing unit that executes the evaluation step. The evaluation unit 23 evaluates the respiratory activity of mitochondria present in the cell 100 based on the intensity of the first peak extracted by the extraction unit 22. The evaluation unit 23 also evaluates the maturity of the oocyte based on the relationship between the intensities of the first peak and the second peak.

[0050] Here, the basis for the evaluation process in the evaluation unit 23 will be described with reference to Fig. 8 to Fig. 14. Fig. 8 to Fig. 14 are explanatory diagrams No. 1 to No. 7 for explaining the basis for the evaluation process in the evaluation unit 23.

[0051] Figures 8 and 9 show the results of principal component analysis using the Raman spectra (see Figure 7) obtained after 13 and 24 hours as data sets. As a result of the principal component analysis shown in Figure 8, PC (Principal Component) 2 showed the first peak (750, 1127, and 1583 cm) derived from reduced cytochrome c. -1 ) was confirmed to decrease in peak intensity from 13 hours to 24 hours after the treatment. In addition, in PC2, the second peak (1438 and 1657 cm) derived from lipids was observed. -1 ) was confirmed to show an increase in peak intensity from 13 hours to 24 hours.

[0052] Furthermore, as shown in Figure 9, when the horizontal axis is PC2 and the vertical axis is PC3, the Raman spectrum after 13 hours tends to be biased toward the lower left region, and the Raman spectrum after 24 hours tends to be biased toward the upper right region. This tendency is due to the increase and decrease in the intensity of the peaks derived from reduced cytochrome c and lipids.

[0053] In other words, it was confirmed that eggs at the M4 stage (24 hours after maturation) had a decreased intensity of the first peak derived from reduced cytochrome c and an increased intensity of the second peak derived from lipids compared to eggs at the M1 stage (13 hours after maturation).

[0054] Furthermore, in Figures 10 and 11, the first peaks at 747 and 1125 cm -1 The results of comparing the peaks at 13 hours, 15 hours, 18 hours, and 24 hours after maturation are shown. Also, in Figure 12, the second peak at 1438 cm -1 The results are shown comparing the peaks at 13 hours, 15 hours, 18 hours, and 24 hours after maturity.

[0055] As shown in Figures 10 and 11, the intensity of the first peak tended to gradually decrease as the maturity of the oocyte increased. On the other hand, as shown in Figure 12, the intensity of the second peak tended to gradually increase as the maturity of the oocyte increased between 13 and 18 hours, although there was some error.

[0056] Therefore, based on the above-mentioned grounds, the evaluation unit 23 evaluates that the stronger the intensity of the first peak, the higher the mitochondrial respiratory activity and the higher the quality of the ovum with high fertility. Furthermore, the evaluation unit 23 evaluates that the lower the intensity of the second peak, the higher the lipid metabolism and the higher the quality of the ovum with high fertility. Note that the evaluation unit 23 may determine the maturity level of the ovum from the intensities of the first and second peaks by setting a threshold value for determining the maturity level of the ovum.

[0057] As shown in Figures 10 and 11, the first peak appears at 1125 cm -1 Compared to 747cm -1 The peaks in the figure show the difference in peak intensity for each maturity level of the oocyte with high accuracy. Therefore, the first peaks extracted by the extraction unit 22 are the peak with the smallest Raman shift (747 cm) among the peaks derived from reduced cytochrome c. -1 It is preferable that the peak includes at least the peaks (peaks (a) and (b)). This allows the evaluation unit 23 to evaluate the maturity of the ovum with high accuracy.

[0058] Furthermore, the evaluation unit 23 can evaluate the maturity of an ovum based on the relationship between the intensities of the first peak and the second peak. Specifically, the evaluation unit 23 evaluates the maturity of an ovum based on the output value of a function that includes the intensities of the first peak and the second peak as variables. The output value of such a function is, for example, the intensity ratio between the first peak and the second peak. Here, the intensity ratio between the first peak and the second peak will be described with reference to FIG. 13 .

[0059] Fig. 13 shows the intensity ratio between the first and second peaks. -1 The first peak at 1445 cm -1 The horizontal axis shows the intensity ratio of the second peak at 1123 cm -1 The first peak at 1445 cm -1 The vertical axis represents the intensity ratio of the second peak of the sample. The plots in the graph represent sample data after 13 hours, 15 hours, 18 hours, and 24 hours, respectively.

[0060] As shown in Figure 13, the region R1 in the upper right corner of the graph is a region where both intensity ratios are high, and contains a large amount of sample data from 13 hours and 15 hours later. In other words, the region R1 in the upper right corner of the graph indicates high-quality oocytes with high respiratory activity and lipid metabolism.

[0061] On the other hand, the region R2 at the bottom left of the graph is an area where both intensity ratios are low, and where there is a lot of sample data from 24 hours later. In other words, region R2 at the bottom left of the graph indicates overripe oocytes with low respiratory activity and lipid metabolism.

[0062] Therefore, the evaluation unit 23 calculates the intensity ratio for each combination of the second peak and each of the plurality of first peaks, and evaluates the maturity of the ovum based on the calculated plurality of intensity ratios. Specifically, the evaluation unit 23 stores information on the regions R1 and R2 as threshold information 31, and evaluates the maturity of the ovum based on which region R1 or R2 a set of the calculated plurality of intensity ratios is plotted in.

[0063] That is, the evaluation unit 23 evaluates the maturity of the ovum based on the threshold information 31 in which the regions R1 and R2 indicating the maturity are linked in a multidimensional space with each axis representing a plurality of output values ​​(intensity ratios). This makes it possible to determine the maturity of the ovum with high accuracy.

[0064] 13 shows an example in which the maturity of an ovum is evaluated using two intensity ratios (two dimensions), but it can also be evaluated using three or more intensity ratios (three or more dimensions). Note that the maturity of an ovum may also be evaluated using one intensity ratio (one dimension).

[0065] Furthermore, the evaluation unit 23 can evaluate the maturity of an ovum based on the product of a plurality of intensity ratios. This point will be explained using FIG. 14. FIG. 14 is a diagram showing the product of two intensity ratios. In FIG. 14, the product of the intensity ratios is calculated for each sample data shown in FIG. 13 and is shown as a histogram. In FIG. 14, the product of the intensity ratios is shown on the horizontal axis, and the number of sample data is shown on the vertical axis.

[0066] As shown in Figure 14, when the product of the intensity ratios is less than 0.6, the number of sample data for eggs after 24 hours is large, while when the product of the intensity ratios is 0.6 or more, the number of sample data for eggs after 24 hours is small.

[0067] Therefore, the evaluation unit 23 evaluates the egg as being of good quality when the product of the two intensity ratios is equal to or greater than the threshold value of the threshold information 31 (for example, 0.6 or greater), and evaluates the egg as being overripe when the product of the two intensity ratios is less than the threshold value. As a result, even if the product of the two intensity ratios is plotted at a position outside the two regions R1 and R2 shown in Fig. 13, the maturity of the egg can be evaluated with high accuracy by calculating the product of the intensity ratios.

[0068] If it is desired to discriminate ova of higher quality (closer to the M2 stage), this can be achieved by increasing the threshold value of the threshold information 31 (for example, 1.0).

[0069] Furthermore, although the above describes the case where the output value of the function is an intensity ratio or a product of intensity ratios, any function can be used as long as the output value of the function includes the intensity of the first peak and the intensity of the second peak as variables.

[0070] Returning to Fig. 2, the output unit 24 will be described. The output unit 24 outputs the evaluation result by the evaluation unit 23. For example, the output unit 24 displays the evaluation result by the evaluation unit 23 on a display unit (not shown). Furthermore, the output unit 24 may transmit the evaluation result by the evaluation unit 23 to a terminal device of an evaluator or the like.

[0071] Next, non-invasive evaluation using Raman spectroscopy will be explained using Figure 15. Figure 15 is a diagram showing non-invasive evaluation using Raman spectroscopy. Figure 15 graphs the embryonic development rates for an irradiation group where oocytes were irradiated with 532 nm excitation light (Raman in Figure 15) and a non-irradiation group where no excitation light was irradiated (Cont. in Figure 15). Figure 15 also shows the embryonic development rates of blastocysts and morulae after in vitro fertilization and five days of culture after irradiation with excitation light (no irradiation in the non-irradiation group).

[0072] As shown in Figure 15, no significant difference was observed in the embryonic development rate between the irradiated and non-irradiated groups at 13, 15, 18, and 24 hours after irradiation. This confirms that the excitation light is not invasive to the oocytes. Therefore, Raman spectroscopy can be used to non-invasively and quantitatively evaluate oocytes.

[0073] Next, the procedure of the ovum evaluation process executed by the cell evaluation device 1 according to the embodiment will be described with reference to Fig. 16. Fig. 16 is a flowchart showing the procedure of the ovum evaluation process executed by the cell evaluation device 1 according to the embodiment.

[0074] As shown in FIG. 16, first, the acquisition unit 21 acquires a Raman spectrum obtained by irradiating an ovum with excitation light having a wavelength included in the electronic absorption band of reduced cytochrome c from the Raman spectrometer 10 (step S101).

[0075] Next, the extraction unit 22 extracts peaks derived from reduced cytochrome c and lipids from the Raman spectrum (step S102).

[0076] Next, the evaluation unit 23 calculates the intensity ratio of the peaks derived from reduced cytochrome c and lipids (step S103). Note that in step S103, the intensity ratio is calculated for each of a plurality of combinations of reduced cytochrome c and lipids.

[0077] Next, the evaluation unit 23 calculates the product of the calculated intensity ratios (step S104). Next, the evaluation unit 23 evaluates the maturity of the ovum from the product of the intensity ratios using the threshold information 31 stored in the storage unit 3 (step S105), and ends the evaluation process.

[0078] (effect) As described above, the cell evaluation method according to the embodiment includes an extraction step and an evaluation step. In the extraction step, peaks derived from reduced cytochromes are extracted from the spectrum of Raman scattered light obtained by irradiating the cell 100 with excitation light having a wavelength included in the electronic absorption band of the reduced cytochrome. In the evaluation step, the respiratory activity of mitochondria present in the cell 100 is evaluated based on the intensity of the extracted peaks. This allows for non-invasive and quantitative evaluation of mitochondrial respiratory activity.

[0079] Although some of the embodiments of the present application have been described in detail above with reference to the drawings, these are merely examples, and the present invention can be implemented in other forms that include the embodiments described in the Disclosure of the Invention section and that have been modified and improved in various ways based on the knowledge of those skilled in the art.

[0080] (others) Furthermore, among the processes described in the above embodiments, all or part of the processes described as being performed automatically can be performed manually, or all or part of the processes described as being performed manually can be performed automatically using known methods. In addition, the information including the processing procedures, specific names, various data, and parameters shown in the above documents and drawings can be changed as desired unless otherwise specified. For example, the various information shown in each drawing is not limited to the information shown in the drawings.

[0081] Furthermore, the components of each device shown in the figure are conceptual functional components and do not necessarily have to be physically configured as shown in the figure. In other words, the specific form of distribution and integration of each device is not limited to that shown in the figure, and all or part of them can be functionally or physically distributed and integrated in any unit depending on various loads, usage conditions, etc.

[0082] Furthermore, the processes described in the above-described embodiments can be combined as appropriate within the scope of not causing any contradiction in the process contents.

[0083] Furthermore, the above-mentioned "section, module, unit" can be read as "means" or "circuit," etc. For example, the extraction unit 22 can be read as extraction means or extraction circuit. [Explanation of symbols]

[0084] 1. Cell evaluation device 2. Control Unit 3 Storage section 10 Raman spectrometer 21 Acquisition Department 22 Extraction part 23 Evaluation Department 24 Output section 31 Threshold Information 100 cells

Claims

1. an extraction step of extracting a peak derived from the reduced cytochrome from a spectrum of Raman scattered light obtained by irradiating a cell with excitation light having a wavelength included in the electronic absorption band of the reduced cytochrome; an evaluation step of evaluating the respiratory activity of mitochondria present in the cells based on the intensity of the extracted peaks; Including, the cell is an ovum, the reduced cytochrome is reduced cytochrome c, The extraction step comprises: extracting a first peak derived from the reduced cytochrome c and a second peak derived from lipids from the spectrum; The evaluation step includes: evaluating the maturity of the ovum based on an output value of a function including the intensity of the first peak and the intensity of the second peak as variables; A cell evaluation method characterized by:

2. The extraction step comprises: extracting a plurality of first peaks having different Raman shifts from the spectrum; The evaluation step includes: calculating the output value for each combination of the second peak and each of the plurality of first peaks, and evaluating the maturity of the oocyte based on the calculated plurality of output values; The cell evaluation method according to claim 1,

3. The evaluation step includes: Evaluating the maturity of the ovum based on threshold information associated with a region indicating the maturity in a multidimensional space having each of the plurality of output values ​​as an axis. The cell evaluation method according to claim 2,

4. The output value is is an intensity ratio between the first peak and the second peak, The evaluation step includes: evaluating the maturity of the ovum based on a product of a plurality of the intensity ratios; The cell evaluation method according to claim 2 or 3,

5. The plurality of first peaks are Among the peaks derived from the reduced cytochrome c, at least the peak with the smallest Raman shift is included. The cell evaluation method according to any one of claims 2 to 4, characterized in that:

6. The wavelength of the excitation light is 532 nm The cell evaluation method according to any one of claims 1 to 5,

7. an extraction step of extracting a peak derived from the reduced cytochrome from a spectrum of Raman scattered light obtained by irradiating a cell with excitation light having a wavelength included in the electronic absorption band of the reduced cytochrome; an evaluation procedure for evaluating the respiratory activity of mitochondria present in the cells based on the intensity of the extracted peaks; on the computer, the cell is an ovum, the reduced cytochrome is reduced cytochrome c, The extraction procedure comprises: extracting a first peak derived from the reduced cytochrome c and a second peak derived from lipids from the spectrum; The evaluation procedure includes: evaluating the maturity of the ovum based on an output value of a function including the intensity of the first peak and the intensity of the second peak as variables; A cell evaluation program featuring:

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