Analysis method, analysis device, and program

Abstract

Provided is an analysis method that uses a computer to calculate the magnitude ratio of the macroscopic speed to the microscopic speed of cells in a cell sheet and evaluate the quality characteristics of the cells in the cell sheet on the basis of the ratio.

Description

Analysis method, analysis device, and program

[0001] This application claims priority to Japanese Patent Application No. 2024-066110, filed on April 16, 2024, the contents of which are incorporated herein by reference.

[0002] In regenerative medicine, methods for evaluating the cell quality, such as proliferation and differentiation potential, of cells for transplantation into patients include evaluating the cell viability after extracting and processing cultured cells in some way, or the percentage of positive cells after staining with specific markers. While these evaluation methods allow for detailed analysis, they involve cell destruction, making the evaluated cells unsuitable for transplantation into patients. Therefore, a non-invasive method for evaluating cells is needed to ensure that the evaluated cells remain suitable for transplantation into patients.

[0003] For example, Patent Documents 1 to 4 and Non-Patent Document 1 disclose methods for non-invasively evaluating cells.

[0004] JP 2020-22412 A International Publication No. 2018 / 012601 JP 2017-23055 A Japanese Patent No. 6218208 A

[0005] Daisuke Nanba, Fujio Toki, Sota Tate, Matome Imai, Natsuki Matsushita, Ken Shiraishi, Koji Sayama, Hiroshi Toki, Shigeki Higashiyama, Yann Barrandon; Cell motion predicts human epidermal stemness. J Cell Biol 27 April 2015; 209 (2): 305-315.

[0006] However, the evaluation methods disclosed in Patent Documents 1-4 and Non-Patent Document 1 are insufficient for evaluating the quality characteristics of cells in cell sheets. An object of the present invention is to provide an analysis method, an analysis device, and a program that solve the above-mentioned problems.

[0007] One aspect of the present invention is an analytical method that calculates, using a computer, the ratio of the magnitude of the macroscopic velocity of cells in a cell sheet to the microscopic velocity of cells, and evaluates the quality characteristics of the cells in the cell sheet based on said ratio.

[0008] One aspect of the present invention is an analytical device that calculates the ratio of the magnitude of the macroscopic velocity of cells in a cell sheet to the microscopic velocity of cells, and evaluates the quality characteristics of the cells in the cell sheet based on the ratio.

[0009] According to the present invention, the quality characteristics of cells in a cell sheet can be evaluated.

[0010] 1 is a diagram showing the configuration of an evaluation system 1 according to the present embodiment. FIG. 2 is a diagram showing the configuration of an analysis device 12 according to the present embodiment. FIG. 3 is a diagram showing the speed calculated by a speed calculation unit 122. FIG. 4 is a diagram showing the relationship between a value s indicating a spatial scale and Ratio(s). FIG. 5 is a flowchart showing the operation of the analysis device 12 according to the present embodiment. FIG. 6 is a diagram showing experimental results. FIG. 7 is a diagram showing experimental results.

[0011] Hereinafter, an embodiment of the present invention will be described in detail with reference to the drawings. FIG. 1 is a diagram showing the configuration of an evaluation system 1 according to this embodiment. The evaluation system 1 is a system for evaluating cell sheets. The cell sheet to be evaluated is a cell sheet used in regenerative medicine. Examples of cell sheets to be evaluated include, but are not limited to, mesenchymal stem cell sheets, epithelial cell sheets, and epidermal cell sheets. The cell sheet to be evaluated is preferably a cell sheet with strong cell-to-cell adhesion, such as an epithelial cell sheet or an epidermal cell sheet. Examples of epithelial cell sheets include corneal epithelial cell sheets and oral mucosa epithelial cell sheets.

[0012] The evaluation system 1 includes a photographing device 11 and an analysis device 12. The photographing device 11 photographs multiple cell sheets. The photographing device 11 photographs each cell sheet at least twice at the same time interval, regardless of the cell sheet. The photographing device 11 includes, for example, a microscope, and photographs the enlarged cell sheet. The magnification of the photographed cell sheet may be such that the image contains several hundred cells in the cell sheet. The magnification of the photographed cell sheet is basically 4x for oral mucosal epithelial cell sheets and basically 1.6x for mesenchymal stem cell sheets. Note that oral mucosal epithelial cell sheets may be photographed at 1.6x, and mesenchymal stem cell sheets may be photographed at 4x.

[0013] When the photographing device 11 photographs N images of one cell sheet, the time at which each image is photographed is defined as t n (n is an integer between 1 and N). n indicates the order in which the images were taken. In this case, the cell sheet is photographed at the same time interval, so t n+1 -t n (n is any integer between 1 and N-1) is a constant.

[0014] The photographing device 11 outputs the photographed image of the cell sheet to the analyzing device 12 .

[0015] The analysis device 12 analyzes the photographed cell sheet based on the images input from the photographing device 11. Figure 2 is a diagram showing the configuration of the analysis device 12 according to this embodiment. The analysis device 12 includes an image acquisition unit 121, a velocity calculation unit 122, a velocity ratio calculation unit 123, a cell sheet evaluation unit 124, and an output unit 125. For simplicity of explanation, the images input from the photographing device 11 will be described below as two images of one cell sheet.

[0016] The image acquisition unit 121 acquires two images input from the photographing device 11. At this time, the two acquired images are referred to as image A1 and image A2 in order of their earliest photographing time.

[0017] The velocity calculation unit 122 calculates the microscopic and macroscopic velocities of the cell based on the image A1 and the image A2. For example, the velocity calculation unit 122 calculates the microscopic velocity of the cell, and then calculates the sum of the microscopic velocities calculated in a predetermined region to calculate a more macroscopic velocity.

[0018] A specific calculation method will be described below. The velocity calculation unit 122 first calculates an optical flow from the pixels of image A1 and image A2. The velocity calculation unit 122 calculates the optical flow from the pixels of image A1 and image A2 using, for example, the Farneback method. This allows the microscopic velocity of the cell to be calculated.

[0019] The optical flow (microscopic velocity) calculated from image A1 and image A2 is defined as V(i, j), where i and j are pixel positions in the images, and i is 1 to N. x is an integer equal to or greater than 1, and j is an integer equal to or greater than N y In other words, the number of pixels in image A1 and image A2 is N x ×N y V(i, j) is a two-dimensional vector, and V(i, j)= (V x (i,j), V y (ij)) is defined as V x (i,j) and V y (ij) is a scalar quantity, and indicates the velocity in the x direction and the velocity in the y direction at the pixel position (i, j).

[0020] The velocity calculation unit 122 calculates the macroscopic velocity of the cell by calculating the average of the optical flow for a predetermined number of adjacent pixels. For example, the velocity calculation unit 122 calculates the macroscopic velocity of the cell by calculating the average of the optical flow calculated from a total of 25 adjacent 5 x 5 pixels. The velocity calculation unit 122 can calculate a more macroscopic velocity of the cell by increasing the number of pixels for which the average optical flow is calculated.

[0021] Macroscopic velocity V m (i, j, s) is a two-dimensional vector like V(i, j), and V m (i, j, s)= (V mx(i, j, s), V my (i, j, s)). V mx (i, j, s) and V my (i, j, s) is a scalar quantity. V m (i, j, s) is defined by equation (1).

[0022] In formula (1), s is V m For example, if s = 5, the average value of the optical flow calculated from the 25 adjacent pixels (5 × 5) is V. m When s=1, the average value of the optical flow calculated from one pixel is V m Therefore, V m (i, j, 1) = V(i, j). In equation (1), 1≦i≦N x -s, where 1 ≤ j ≤ N y -s. By increasing the value of s, V m Since s is the average value of the optical flow calculated from more pixels, V m It can be said that this value indicates the spatial scale that serves as the basis for calculation.

[0023] The velocity ratio calculation unit 123 calculates the macroscopic velocity V(i, j) relative to the microscopic velocity V(i, j) in the image. m The magnitude of (i, j, s) is calculated. Specifically, the velocity ratio calculation unit 123 calculates the average value of the absolute values ​​of the velocities calculated for each macroscopic scale. Specifically, the velocity ratio calculation unit 123 calculates the average value Average(s) of the absolute values ​​of the velocities calculated for each value s indicating the spatial scale using equation (2).

[0024] In equation (2), |V m (i, j, s)| is the absolute value of the velocity calculated for each value s indicating the spatial scale, and is a value calculated by equation (3).

[0025] In this way, the average value Average(s) of the absolute values ​​of the velocities calculated for each value s indicating the spatial scale is calculated.

[0026] FIG. 3 is a diagram showing the speed calculated by the speed calculation unit 122. FIG. 3(a) shows a microscopic speed vector, and FIG. 3(b) shows a macroscopic speed vector. When the directions of the microscopic speeds vary among adjacent pixels, the magnitude of the calculated macroscopic speed becomes small because the magnitudes of the microscopic speeds cancel each other out. On the other hand, when the directions of the microscopic speeds are aligned, the magnitude of the calculated macroscopic speed becomes large because the magnitudes of the microscopic speeds do not cancel each other out.

[0027] The speed ratio calculation unit 123 compares the average values of the absolute values of the speeds calculated for each value s indicating the spatial scale. The speed ratio calculation unit 123 calculates the ratio Ratio(s) of Average(s) to Average(1). Ratio(s) is represented by Equation (4).

[0028] The smaller Ratio(s) is, the smaller the magnitude of the macroscopic speed relative to the magnitude of the microscopic speed, which means that there are many variations in the direction of the cell speed. The larger Ratio(s) is, the larger the magnitude of the macroscopic speed relative to the magnitude of the microscopic speed, which means that there are few variations in the direction of the cell speed.

[0029] In Equation (4), Ratio(s) is the ratio of Average(s) to Average(1), but it may be the ratio of Average(s) to Average(α) (α≠1). That is, the speed ratio calculation unit 123 calculates the magnitude of the macroscopic speed V m (i, j, α) with respect to the microscopic speed V m (i, j, s) (α < s) and may calculate Ratio(s).

[0030] The image acquisition unit 121 acquires images of different cell sheets, the speed calculation unit 122 calculates Average(s) for different cell sheets, and the speed ratio calculation unit 123 calculates Ratio(s) for different cell sheets.

[0031] The cell sheet evaluation unit 124 evaluates the quality characteristics of the cell sheet based on the magnitude of the macroscopic velocity relative to the microscopic velocity of the cells calculated for each cell sheet. The quality characteristics of the cell sheet indicate, for example, the operability or strength of the cell sheet. Specifically, the cell sheet evaluation unit 124 evaluates the quality characteristics of the cell sheet based on the Ratio(s) calculated for each cell sheet. The degree to which a group of cells in a cell sheet move synchronously and in the same direction affects the suitability of the cell sheet for transplantation. When many of the cell groups move synchronously and in the same direction, that is, when there is little variation in the direction of cell velocity and the Ratio(s) calculated for a value s indicating the same spatial scale is small, the quality characteristics of the cell sheet are evaluated as, for example, good operability or high robustness.

[0032] Figure 4 shows the relationship between the value s indicating the spatial scale and Ratio(s). In the graph shown in Figure 4, the horizontal axis represents the value s indicating the spatial scale, and the vertical axis represents Ratio(s). The Ratio(s) indicated by the solid line decreases gradually with changes in the value s indicating the spatial scale. Therefore, in the cell sheet for which the Ratio(s) indicated by the solid line is calculated, there is little variation in the direction of cell velocity. In addition, the Ratio(s) indicated by the dashed line decreases rapidly with changes in the value s indicating the spatial scale. Therefore, in the cell sheet for which the Ratio(s) indicated by the dashed line is calculated, there is much variation in the direction of cell velocity.

[0033] The speed ratio calculation unit 123 may calculate the rate of change of Ratio(s) with respect to the value s indicating the spatial scale. For example, the speed ratio calculation unit 123 calculates the rate of change a by first-order approximation of the relationship between Ratio(s) and s to calculate the equation Ratio(s)=a S + b. In the first-order approximation, the rate of change a is calculated by applying the least squares method to the combination of Ratio(s) and s, for example.

[0034] The cell sheet evaluation unit 124 may evaluate the cell sheet based on the rate of change a calculated for each cell sheet. When the absolute value of the rate of change a is small, Ratio(s) decreases gradually with respect to changes in the value s indicating the spatial scale, and there is little variation in the direction of cell velocity in the cell sheet. When the absolute value of the rate of change a is large, Ratio(s) decreases rapidly with respect to changes in the value s indicating the spatial scale, and there is much variation in the direction of cell velocity in the cell sheet.

[0035] The cell sheet evaluation unit 124 may evaluate the quality characteristics of the cell sheet based on a predetermined reference value. When the value s indicating the spatial scale is a predetermined value and Ratio(s) is equal to or greater than a predetermined reference value, the cell sheet evaluation unit 124 may evaluate the quality characteristics of the cell sheet as having good operability or high robustness. For example, when the absolute value of the rate of change a is equal to or less than a predetermined reference value, the cell sheet evaluation unit 124 may evaluate the quality characteristics of the cell sheet as having good operability or high robustness.

[0036] The output unit 125 outputs the evaluation results of the cell sheets. The output unit 125 may output the calculation results for each cell sheet (for example, Average(s), Ratio(s), or rate of change a).

[0037] 5 is a flowchart showing the operation of the analysis device 12 according to this embodiment. The image acquisition unit 121 acquires images from the imaging device 11 (step S11). Here, the image acquisition unit 121 acquires at least two images of the same position on one cell sheet, taken at different times. The velocity calculation unit 122 calculates the microscopic and macroscopic velocities of the cells based on the at least two images (step S12). The velocity calculation unit 122 calculates the microscopic cell velocity by, for example, calculating the optical flow of pixels between images, and calculates the macroscopic cell velocity by calculating the average of the optical flow for a predetermined number of adjacent pixels.

[0038] The velocity ratio calculation unit 123 calculates the magnitude of the macroscopic velocity relative to the microscopic velocity in the image (step S13). Specifically, the velocity ratio calculation unit 123 calculates the average value Average(s) of the absolute values ​​of the velocities calculated for each value s indicating the spatial scale. The velocity ratio calculation unit 123 compares the average values ​​of the absolute values ​​of the velocities calculated for each value s indicating the spatial scale (step S14). Specifically, the velocity ratio calculation unit 123 calculates the ratio Ratio(s) of Average(s) to Average(1).

[0039] The cell sheet evaluation unit 124 evaluates the quality characteristics of the cell sheet based on the magnitude of the macroscopic velocity relative to the microscopic velocity of the cells calculated for each cell sheet (step S15). The output unit 125 outputs the evaluation results of the quality characteristics of the cell sheet (step S16).

[0040] In cell sheets, particularly epithelial cell sheets such as oral mucosal epithelial cell sheets, the amount of movement of adhered cells in the same direction is strongly correlated with the quality characteristics of the cells. Therefore, the analysis device 12 can evaluate the quality characteristics of the cell sheet by calculating the magnitude of the macroscopic velocity relative to the microscopic velocity of the cells in the cell sheet.

[0041] The velocity calculation unit 122 may perform image blur correction processing on the microscopic velocity V(i, j). Specifically, the velocity calculation unit 122 may calculate the corrected microscopic velocity V'(i, j) by subtracting the average of V(i, j) for all pixels from V(i, j) for each pixel using equation (5). If the field of view is sufficiently wide, the average velocity over the entire field of view can be expected to be 0, and the average of V(i, j) for all pixels is assumed to be the blur over the entire image.

[0042] In the above description, the image acquisition unit 121 acquires two images for one cell sheet, but three or more images may be acquired for one cell sheet. In this case, the velocity calculation unit 122 may calculate the microscopic and macroscopic velocities of the cells based on one image and the image captured next, calculate Average(s) from the two images, and calculate the average value of the Average(s) calculated for each combination of images. For example, the image acquisition unit 121 acquires N images for one cell sheet, and the velocity calculation unit 122 calculates the average value of the Average(s) calculated for each combination of images at time t n The image taken at time t n+1 The velocity calculation unit 122 calculates the microscopic and macroscopic velocities of the cells based on the images captured by the image capturing unit 121 and calculates Average(s). The velocity calculation unit 122 calculates Average(s) for each s from n=1 to n=N-1, thereby calculating N-1 Average(s) for each s. The velocity calculation unit 122 calculates the average of the N-1 Average(s) for each s, thereby calculating the average of the absolute values ​​of the velocities calculated for each value s indicating the spatial scale.

[0043] Alternatively, the velocity calculation unit 122 may calculate the microscopic and macroscopic velocities of the cells based on one image and the image captured next, calculate Average(s) from the two images, calculate Ratio(s) from the calculated Average(s), and calculate the average value of the Ratio(s) calculated for each combination of images. For example, the image acquisition unit 121 acquires N images for one cell sheet, and the velocity calculation unit 122 calculates the microscopic and macroscopic velocities of the cells based on the image captured next at time t n The image taken at time t n+1 Based on the images captured in the image capturing unit 121, the microscopic and macroscopic velocities of the cells are calculated, Average(s) is calculated, and Ratio(s) is calculated. The velocity calculation unit 122 calculates Ratio(s) for each s from n=1 to n=N-1, thereby calculating N-1 Ratio(s) for each s. The velocity calculation unit 122 calculates the average of the N-1 Ratio(s) for each s, thereby calculating the ratio of Average(s) to Average(1).

[0044] Experimental Example The following describes the experiment conducted. Oral mucosal epithelial cell sheets were used. Two oral mucosal epithelial cell sheets were prepared using FBS-supplemented medium and high Ca medium. FBS-supplemented medium was supplemented with fetal bovine serum. High Ca medium was a serum-free medium with CaCl2 added to increase the Ca concentration. Oral mucosal epithelial cell sheets cultured in FBS-supplemented medium generally exhibited better operability and robustness than oral mucosal epithelial cell sheets cultured in high Ca medium. Subsequently, video recording was performed on the cell sheets prepared using each medium, and the macroscopic velocity relative to the microscopic velocity was measured for at least two images to examine the quality characteristics of the cell sheets. The results of the experiment showed that the cell sheets prepared in FBS-supplemented medium exhibited greater unidirectional migration of adhered cells than the cell sheets prepared in high Ca medium. Based on the analysis results obtained by the analysis device 12, the quality characteristics of the cell sheets could be noninvasively estimated.

[0045] In this experiment, image blur correction was performed. Figure 6 shows the experimental results. Figure 6(a) is a graph showing the relationship between s and Ratio(s) for oral mucosal epithelial cell sheets cultured in FBS-supplemented medium and high Ca medium before image blur correction, and Figure 6(b) is a graph showing the relationship between s and Ratio(s) for oral mucosal epithelial cell sheets cultured in FBS-supplemented medium and high Ca medium after image blur correction. Before blur correction, the oral mucosal epithelial cell sheets cultured in FBS-supplemented medium had a smaller Ratio(s) value for the same value of s than the oral mucosal epithelial cell sheets cultured in high Ca medium. However, after blur correction, the oral mucosal epithelial cell sheets cultured in FBS-supplemented medium had a larger Ratio(s) value for the same value of s than the oral mucosal epithelial cell sheets cultured in high Ca medium. The results after blur correction showed that oral mucosal epithelial cell sheets cultured in FBS-supplemented medium were generally easier to handle or more robust than oral mucosal epithelial cell sheets cultured in high Ca medium.

[0046] Furthermore, the quality characteristics of oral mucosal epithelial cell sheets prepared using FBS-supplemented medium at different times after the start of preparation were examined. Specifically, the quality characteristics of oral mucosal epithelial cell sheets prepared 2 days, 4 days, approximately 8 days, and approximately 12 days after the start of preparation were examined.

[0047] After videotaping the oral mucosal epithelial cell sheet at each elapsed time, the magnitude of macroscopic velocity relative to microscopic velocity was measured for at least two images to examine the quality characteristics of the cell sheet. Figure 7 shows the experimental results. In the legend in Figure 7, "48 hrs" indicates oral mucosal epithelial cell sheets two days after the start of production, "96 hrs" indicates oral mucosal epithelial cell sheets four days after the start of production, "196 hrs" indicates oral mucosal epithelial cell sheets approximately eight days after the start of production, and "292 hrs" indicates oral mucosal epithelial cell sheets approximately 12 days after the start of production. The oral mucosal epithelial cell sheet with the shortest elapsed time, 2 days, had a large rate of change a of Ratio(s) relative to the value s indicating spatial scale. However, as the elapsed time increased to 4 or approximately 8 days, the rate of change a decreased, and at approximately 12 days, the rate of change a increased again. This indicates that the quality characteristics of oral mucosal epithelial cell sheets improved in operability or robustness over time, and then deteriorated in operability or robustness.

[0048] For example, a reference value is set between the magnitude of the rate of change a in oral mucosal epithelial cell sheets after 2 days and approximately 12 days and the magnitude of the rate of change a in oral mucosal epithelial cell sheets after 4 days and approximately 8 days, and oral mucosal epithelial cell sheets whose rate of change a is less than the reference value are judged to have good operability or high robustness, and oral mucosal epithelial cell sheets whose rate of change a is equal to or greater than the reference value are judged to have poor operability or low robustness. This allows the quality characteristics of oral mucosal epithelial cell sheets to be judged.

[0049] Furthermore, the quality characteristics of oral mucosal epithelial cell sheets prepared using FBS-supplemented media from cells with different passage numbers were examined. Specifically, the quality characteristics of oral mucosal epithelial cell sheets prepared from cells at passage 2, which were prepared from cells that had been passaged twice, and oral mucosal epithelial cell sheets prepared from cells at passage 5, which were obtained by further passage 3 times from the cells at passage 2, were examined.

[0050] Oral mucosal epithelial cell sheets with different passage numbers were videotaped, and the magnitude of macroscopic velocity relative to microscopic velocity was measured for at least two images to examine the quality characteristics of the cell sheets. Figure 8 shows the experimental results. In the legend in Figure 8, "P5 54 hrs" indicates an oral mucosal epithelial cell sheet prepared using cells at passage 5, and "P2 96 hrs" indicates an oral mucosal epithelial cell sheet prepared using cells at passage 2. The rate of change a of Ratio(s) relative to the value s indicating the spatial scale for the oral mucosal epithelial cell sheet prepared using cells at passage 2 was smaller than the rate of change a of Ratio(s) relative to the value s indicating the spatial scale for the oral mucosal epithelial cell sheet prepared using cells at passage 5. This indicates that the quality characteristics of oral mucosal epithelial cell sheets prepared using cells with fewer passage numbers are easier to handle or more robust.

[0051] In the example shown in FIG. 8, for example, when the magnitude of the rate of change a is 0.125 μm －１ The quality characteristics of oral mucosal epithelial cell sheets with a change rate a of less than 0.125 μm are judged to be easy to handle or highly robust. －１ The quality characteristics of the oral mucosal epithelial cell sheet can be determined by determining that the quality characteristics of the oral mucosal epithelial cell sheet are poor in operability or poor in robustness.

[0052] Other Embodiments One embodiment of the present invention has been described in detail above with reference to the drawings, but the specific configuration is not limited to that described above, and various design changes and the like can be made within the scope that does not deviate from the gist of the present invention.

[0053] Part or all of the configuration of the analysis device 12 in the above-described embodiment may be implemented by a computer. In this case, a program for implementing this function may be recorded on a computer-readable recording medium, and the program may be loaded into a computer system and executed. Note that the term "computer system" as used herein includes hardware such as an OS and peripheral devices. Furthermore, "computer-readable recording medium" refers to portable media such as flexible disks, optical magnetic disks, ROMs, and CD-ROMs, as well as storage devices such as hard disks built into computer systems. Furthermore, "computer-readable recording medium" may also include devices that dynamically store programs for a short period of time, such as communication lines used when transmitting programs via networks such as the Internet or telephone lines, or devices that store programs for a fixed period of time, such as volatile memory within a computer system that serves as a server or client. The program may be designed to implement part of the aforementioned functions, or may be capable of implementing the aforementioned functions in combination with a program already stored in the computer system, or may be implemented using a programmable logic device such as an FPGA (Field Programmable Gate Array).

[0054] According to the present invention, the quality characteristics of cells in a cell sheet can be evaluated.

[0055] 1 Evaluation system, 11 Imaging device, 12 Analysis device, 121 Image acquisition unit, 122 Speed ​​calculation unit, 123 Speed ​​ratio calculation unit, 124 Cell sheet evaluation unit, 125 Output unit

Claims

1. An analytical method comprising: calculating, by a computer, the ratio of the magnitude of the macroscopic velocity of cells in a cell sheet to the microscopic velocity of cells; and evaluating the quality characteristics of cells in the cell sheet based on said ratio.

2. The analysis method of claim 1, wherein the microscopic velocity is calculated by calculating optical flow from the image of the cell sheet, the macroscopic velocity is calculated by averaging the optical flow at adjacent pixels, and the ratio is calculated by averaging the magnitude of the macroscopic velocity relative to the average magnitude of the microscopic velocity.

3. The analysis method of claim 2, wherein different macroscopic velocities are calculated depending on a value indicating a spatial scale, which is the number of adjacent pixels; the ratio is calculated as the average magnitude of the macroscopic velocities relative to the average magnitude of the microscopic velocities for each value indicating the spatial scale; and the quality characteristics of the cells in the cell sheet are evaluated based on the rate of change of the ratio relative to the value indicating the spatial scale.

4. The analysis method according to claim 3, wherein the rate of change is a coefficient of the value indicating the spatial scale when the relationship between the value indicating the spatial scale and the ratio is approximated to a first order.

5. The analysis method according to any one of claims 1 to 4, wherein the cell sheet is an epithelial cell sheet.

6. An analytical device that calculates the ratio of the magnitude of the macroscopic velocity of cells in a cell sheet to the microscopic velocity of cells, and evaluates the quality characteristics of cells in the cell sheet based on said ratio.

7. A program for causing a computer to execute the analysis method according to any one of claims 1 to 4.

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