Passage timing determination method, passage timing determination system, and cell culture system

Abstract

The passage is required to be carried out at a proper time according to the actual cell state. This problem is solved by a passage timing determination method comprising: an imaging step for imaging cells at a plurality of prescribed locations in a culture vessel at a first imaging timing, and acquiring cell images at each of the plurality of prescribed locations at the first imaging timing; a primary information acquisition step for acquiring primary information of cells captured in the cell image at each location at a first imaging time; a statistical index value acquisition step for calculating a statistical index value of the primary information of the cells; and a determination step in which the imaging time corresponding to the time at which the statistical index value reaches the predetermined threshold value is determined as the time at which the passage timing is reached.

Description

Technical Field

[0001] The present invention relates to a method and system for determining the timing of cell subculture in cell culture, and a culture system using the same. In particular, the present invention relates to a method and system for determining the timing of cell subculture that expands or proliferates in a culture medium in a culture container, and a culture system using the same. Background Art

[0002] In the culture of cells represented by stem cells, cells are inoculated in a culture medium put into a culture container such as a culture dish and cultured in an incubator. The inoculated cells adhere to the culture surface of the part of the bottom surface of the culture medium in the culture container, and after a certain period of time, the culture surface is covered by the extension or proliferation of the cells. Moreover, when the contact between adjacent cells increases, the extension or proliferation stops. Therefore, in order to make up for the insufficient space, passage is carried out, that is, the cells are dispersed and re-inoculated in a new container and culture is continued. Therefore, in order to efficiently stretch or proliferate the cells, it is important to pass on the culture at the appropriate time. Usually, this time is set to the moment when the occupancy rate of the cells reaches about 80% relative to the confluent state in the culture surface. And, in the past, a culture method for passage was adopted in which the period expected to reach this moment was set as the period of passage based on experience.

[0003] Prior art literature

[0004] Patent Literature

[0005] Patent Document 1: Japanese Patent Application Publication No. 2005-192485

[0006] Patent Document 2: Japanese Patent Application Publication No. 2001-275659 Summary of the invention

[0007] Problems to be solved by the invention

[0008] However, the rate of cell expansion or proliferation varies from cell to cell, and the timing of passage may vary from one culture vessel to another due to differences in the state of cells when they are seeded in a culture vessel. Fig. 6A and Figure 6B is a diagram showing the distribution of cells in the observation site Op. Fig. 6AAs shown, in the culture of cells, the following state is an ideal culture: the deviation of the distance between cells is small, and the distance between cells gradually shortens on average without the deviation of the distance between cells from the state where the cells are inoculated. In such an ideal culture, the moment when the occupancy rate of cells in the culture container reaches a specified value is the appropriate time for subculture. As an example of this moment, there is a moment when the occupancy rate of cells reaches about 80% relative to the confluent state. However, as mentioned above, usually, the moment when the occupancy rate of cells reaches a specified benchmark based on experience is used as the time for subculture, rather than the moment when the occupancy rate of cells is determined to reach a specified benchmark as the time for subculture. In this context, it is difficult to simply grasp the occupancy rate of cells in a short time. As an example of a technical method for solving this problem, Patent Documents 1 and 2 disclose a method for objectively judging the timing of subculture that was previously judged based on experience based on image data of cells in a culture medium.

[0009] Patent document 1 discloses a culture state detection device that calculates the cell density based on the correlation between the run length of cells on a straight measurement line of image data that crosses the culture medium and the cell density per unit area of ​​the culture medium, and compares it with a predetermined judgment value to determine the timing of subculturing. However, in Patent document 1, although image data as two-dimensional data is used, since a straight measurement line that crosses the image data is used, the data is one-dimensional data, and there is a problem that the accuracy is reduced in the process of accurately capturing the distribution of cells on the culture surface, and there is a problem that the reduction in accuracy has an adverse effect on the grasp of the accurate timing of subculturing.

[0010] On the other hand, patent document 2 discloses the following culture method and culture device: according to the image data of the cells on the culture surface in the culture medium, the number of cells in the culture medium, the cell concentration, the adhered cell occupied area or the adhered cell occupancy rate are calculated, and the timing of passage is determined according to the calculation result. However, in patent document 2, when the timing of passage is determined each time, the image data of the cells photographed are compared with the image data of the cells photographed before, and pattern recognition is performed to investigate the difference point, and the degree of extension or proliferation of the cells is measured. In addition, further, when the timing of passage is predicted afterwards, the timing of passage is predicted not only according to the image data, but also according to the consumption of the culture medium. That is, in patent document 2, it is not to perform fixed-point observation at a plurality of locations specified in the culture surface, but to further predict the timing of passage according to the consumption of the culture medium, so there is a problem that the calculation load of determination becomes larger and the system structure outside the image becomes larger. Therefore, it is required to determine the timing of appropriate passage only according to the image data of the cells on the culture surface under the condition of not using the state of the culture medium by a simple structure.

[0011] In addition, further, if Figure 6BAs shown in the figure, in the case where the deviation of the distance between cells in the culture surface is large, the appropriate timing of subculturing will come before the occupancy rate of cells in the culture medium reaches about 80%. However, Patent Documents 1 and 2 do not grasp the deviation of cells in the culture surface and use it to determine the timing of subculturing. Therefore, it is required to grasp the deviation of cells in the culture surface to determine the appropriate timing of subculturing.

[0012] In addition, even if the time of starting culture is the same, the timing of subculture is not necessarily consistent between culture containers. Therefore, if the timing of subculture is uniformly determined as a certain period, the best time for subculture will be missed. Therefore, it is required to determine the timing of subculture based on the change of cells in the culture container over time for each specified period, and as an element for determining the timing of subculture, multiple specified locations are extracted for fixed-point observation, and subculture is determined at an appropriate timing of subculture based on the actual state of cells in the culture surface.

[0013] Means for solving problems

[0014] One embodiment of the present invention is a method for determining the timing of subculture, which determines the timing of subculture of cells that extend or proliferate in a culture medium in a culture container, wherein the method for determining the timing of subculture has the following steps: a shooting step, at a first shooting time, shooting cells at a plurality of specified locations of the culture container to obtain cell images at each of the plurality of specified locations at the first shooting time; a primary information acquisition step, obtaining primary information of the cells captured in the cell images at each of the plurality of specified locations at the first shooting time; a statistical index value acquisition step, calculating the statistical index value of the primary information of the cells; and a determination step, determining the shooting time corresponding to the time when the statistical index value reaches a specified threshold as the time when the subculture timing has arrived.

[0015] Another aspect of the present invention is a method for determining the timing of passage, which determines the timing of passage of cells that extend or proliferate in a culture medium in a culture container, wherein the method for determining the timing of passage comprises the following steps: a photographing step, photographing cells at a plurality of predetermined locations of the culture container at a first photographing time and a second photographing time after the first photographing time, and acquiring cell images of each of the plurality of predetermined locations at the first photographing time and the second photographing time; a primary information acquiring step, acquiring primary information of the cells photographed in the cell images of the each of the plurality of predetermined locations at the first photographing time and the second photographing time; a statistical index value acquiring step, calculating the statistical index value of the primary information at the first photographing time and the second photographing time; a time-varying calculation step, calculating the time-varying index value based on the statistical index values ​​at the first photographing time and the second photographing time; and a determining step, determining the photographing time corresponding to the time when the time-varying index value reaches a predetermined threshold as the time when the passage timing has arrived.

[0016] Another aspect of the present invention is a method for determining the timing of passage, which determines the timing of passage of cells that extend or proliferate in a culture medium in a culture container, wherein the method for determining the timing of passage comprises the following steps: a photographing step, photographing cells at a plurality of predetermined locations of the culture container at a first photographing time and a second photographing time after the first photographing time, and acquiring cell images of each of the plurality of predetermined locations at the first photographing time and the second photographing time; a primary information acquiring step, acquiring primary information of the cells photographed in the cell images of the each of the plurality of predetermined locations at the first photographing time and the second photographing time; a time-varying calculation step, calculating a time-varying index value based on the primary information at the first photographing time and the second photographing time; a statistical index value acquiring step, calculating a statistical index value of the time-varying index value at the first photographing time and the second photographing time; and a determining step, determining the photographing time corresponding to the time when the statistical index value reaches a predetermined threshold as the time when the passage timing has arrived.

[0017] Another aspect of the present invention is a method for determining the timing of passage, which determines the timing of passage of cells extending or proliferating in a culture medium in a culture container, wherein the method for determining the timing of passage comprises the following steps: a photographing step, photographing cells at a plurality of predetermined locations of the culture container at a first photographing time, a second photographing time after the first photographing time, and a third photographing time after the second photographing time, to obtain cell images at each of the plurality of predetermined locations at each of the first photographing time, the second photographing time, and the third photographing time; a primary information acquisition step, obtaining primary information of the cells photographed in the cell images at each of the plurality of predetermined locations at each of the first photographing time, the second photographing time, and the third photographing time; and a statistical index value acquisition step, Calculating the statistical index value of the first information of each of the first shooting moment, the second shooting moment and the third shooting moment; a first time-varying calculation step, calculating the first time-varying index value of the statistical index value of the first shooting moment and the second shooting moment, and the first time-varying index value of the statistical index value of the second shooting moment and the third shooting moment; a second time-varying calculation step, calculating the second time-varying index value based on the first time-varying index value of the first shooting moment and the second shooting moment, and the first time-varying index value of the second shooting moment and the third shooting moment; and a determination step, determining the shooting moment corresponding to the moment when the second time-varying index value reaches a specified threshold as the moment when the passage opportunity has arrived.

[0018] Another aspect of the present invention is a method for determining the timing of passage, which determines the timing of passage of cells extending or proliferating in a culture medium in a culture container, wherein the method for determining the timing of passage comprises the following steps: a photographing step, photographing cells at a plurality of predetermined locations of the culture container at a first photographing time, a second photographing time after the first photographing time, and a third photographing time after the second photographing time, to obtain cell images at each of the plurality of predetermined locations at each of the first photographing time, the second photographing time, and the third photographing time; a primary information acquisition step, obtaining primary information of the cells photographed in the cell images at each of the plurality of predetermined locations at each of the first photographing time, the second photographing time, and the third photographing time; and a time-varying change calculation step. , calculating the first time-varying index value of the first information at the first shooting moment and the second shooting moment, and the first time-varying index value of the first information at the second shooting moment and the third shooting moment; a second time-varying calculation process, calculating the second time-varying index value according to the first time-varying index value at the first shooting moment and the second shooting moment, and the first time-varying index value at the second shooting moment and the third shooting moment; a statistical index value acquisition process, calculating the statistical index value of the second time-varying index value of each of the first shooting moment, the second shooting moment and the third shooting moment; and a determination process, determining the shooting moment corresponding to the moment when the statistical index value reaches a specified threshold as the moment when the passage opportunity is reached.

[0019] Another aspect of the present invention is a method for determining the timing of passage, which determines the timing of passage of cells extending or proliferating in a culture medium in a culture container, wherein the method for determining the timing of passage comprises the following steps: a photographing step, at a first photographing time, a second photographing time after the first photographing time, and a third photographing time after the second photographing time, photographing cells at a plurality of predetermined locations of the culture container, and acquiring cell images at each of the plurality of predetermined locations at each of the first photographing time, the second photographing time, and the third photographing time; a primary information acquisition step, acquiring primary information of the cells photographed in the cell images at each of the plurality of predetermined locations at each of the first photographing time, the second photographing time, and the third photographing time; and a time-varying change calculation step. A process for calculating a first time variation index value of the first information at the first shooting moment and the second shooting moment, and a first time variation index value of the first information at the second shooting moment and the third shooting moment; a statistical index value acquisition process for calculating the statistical index value of the first time variation index value of each of the first shooting moment, the second shooting moment and the third shooting moment; a second time variation calculation process for calculating second time variation index values ​​based on the statistical index values ​​of the first shooting moment and the second shooting moment, and the statistical index values ​​of the second shooting moment and the third shooting moment; and a determination process for determining the shooting moment corresponding to the moment when the second time variation index value reaches a specified threshold as the moment when the passage opportunity is reached.

[0020] Another embodiment of the present invention is a subculture timing determination system, which determines the subculture timing of cells extending or proliferating in a culture medium in a culture container, wherein the subculture timing determination system comprises: a photographing component, which photographs cells on a culture surface at each of a plurality of specified locations of the culture medium in the culture container to obtain a cell image; and a processing device, which obtains primary information of the cells photographed in the cell image, wherein the photographing component photographs cells at a plurality of specified locations of the culture container at a first photographing time to obtain cell images at each of the plurality of specified locations at the first photographing time, wherein the processing device obtains primary information of the cells photographed in the cell images at each of the plurality of specified locations at the first photographing time, calculates a statistical index value of the primary information of the cells, and wherein the processing device determines the photographing time corresponding to the time when the statistical index value reaches a specified threshold as the time when the subculture timing has arrived.

[0021] Another aspect of the present invention is a subculture timing determination system, which determines the subculture timing of cells extending or proliferating in a culture medium in a culture container, wherein the subculture timing determination system comprises: a photographing component, which photographs cells on a culture surface at each of a plurality of predetermined locations of the culture medium in the culture container to obtain a cell image; and a processing device, which obtains primary information of the cells photographed in the cell image, wherein the photographing component photographs the cells at a plurality of predetermined locations of the culture container at a first photographing time and at a second photographing time after the first photographing time to obtain the first photographing time information. The processing device obtains the cell images of each of the plurality of locations at each of the first shooting time and the second shooting time, obtains the first information of the cells captured in the cell images of each of the locations at each of the first shooting time and the second shooting time, calculates the statistical index values ​​of the first shooting time and the second shooting time, and calculates the time-varying index value based on the statistical index values ​​of the first shooting time and the second shooting time, and the processing device determines the shooting time corresponding to the time when the time-varying index value reaches a prescribed threshold as the time when the passage opportunity has arrived.

[0022] Another aspect of the present invention is a subculture timing determination system, which determines the subculture timing of cells extending or proliferating in a culture medium in a culture container, wherein the subculture timing determination system comprises: a photographing component, which photographs cells on a culture surface at each of a plurality of predetermined locations of the culture medium in the culture container to obtain a cell image; and a processing device, which obtains primary information of the cells photographed in the cell image, wherein the photographing component photographs the cells at a plurality of predetermined locations of the culture container at a first photographing time and at a second photographing time after the first photographing time to obtain the first photographing time information. The processing device obtains the first information of the cells captured in the cell images of the respective locations at the first shooting time and the second shooting time, calculates the time-varying index value based on the first information at the first shooting time and the second shooting time, and calculates the statistical index value of the time-varying index value at the first shooting time and the second shooting time, and the processing device determines the shooting time corresponding to the time when the statistical index value reaches a prescribed threshold as the time when the passage opportunity arrives.

[0023] Another aspect of the present invention is a subculture timing determination system, which determines the subculture timing of cells extending or proliferating in a culture medium in a culture container, wherein the subculture timing determination system comprises: a photographing component, which photographs cells on a culture surface at each of a plurality of predetermined locations of the culture medium in the culture container to obtain a cell image; and a processing device, which obtains primary information of the cells photographed in the cell image, the photographing component photographs cells at a plurality of predetermined locations of the culture container at a first photographing time, a second photographing time after the first photographing time, and a third photographing time after the second photographing time to obtain cell images at each of the plurality of predetermined locations at the first photographing time, the second photographing time, and the third photographing time, and the processing device obtains primary information of the cells photographed in the cell image. The processing device calculates the first information of the cells captured in the cell images at the respective locations at the respective shooting times and the third shooting times, calculates the statistical index values ​​of the first information at the respective first shooting times, the second shooting times, and the third shooting times, calculates the first time-varying index values ​​of the statistical index values ​​at the first shooting times and the second shooting times, and the first time-varying index values ​​of the statistical index values ​​at the second shooting times and the third shooting times, and calculates the second time-varying index values ​​based on the first time-varying index values ​​at the first shooting times and the second shooting times, and the first time-varying index values ​​at the second shooting times and the third shooting times, and determines the shooting time corresponding to the time when the second time-varying index values ​​reach a specified threshold as the time when the passage opportunity has arrived.

[0024] Another embodiment of the present invention is a subculture timing determination system, which determines the subculture timing of cells extending or proliferating in a culture medium in a culture container, wherein the subculture timing determination system comprises: a photographing component, which photographs cells on a culture surface of a plurality of predetermined locations of the culture medium in the culture container to obtain a cell image; and a processing device, which obtains primary information of the cells photographed in the cell image, the photographing component photographs cells at a plurality of predetermined locations of the culture container at a first photographing time, a second photographing time after the first photographing time, and a third photographing time after the second photographing time to obtain cell images at each of the plurality of predetermined locations at the first photographing time, the second photographing time, and the third photographing time, and the processing device obtains primary information of the cells photographed in the cell image. The processing device comprises: a processing device for processing a cell captured in the cell image at each location at each of the first and second shooting times and the third shooting time, calculating a first time-varying index value of the first information at the first shooting time and the second shooting time, and a first time-varying index value of the first information at the second and the third shooting time, and calculating a second time-varying index value based on the first time-varying index value at the first shooting time and the second shooting time, and the first time-varying index value at the second and the third shooting time, and calculating a statistical index value of the second time-varying index value at each of the first shooting time, the second shooting time and the third shooting time, and determining the shooting time corresponding to the time when the statistical index value reaches a specified threshold value as the time when the passage opportunity has arrived.

[0025] Another embodiment of the present invention is a subculture timing determination system, which determines the subculture timing of cells extending or proliferating in a culture medium in a culture container, wherein the subculture timing determination system comprises: a photographing component, which photographs cells on a culture surface at each of a plurality of predetermined locations of the culture medium in the culture container to obtain a cell image; and a processing device, which obtains primary information of the cells photographed in the cell image, the photographing component photographs cells at a plurality of predetermined locations of the culture container at a first photographing time, a second photographing time after the first photographing time, and a third photographing time after the second photographing time to obtain cell images at each of the plurality of predetermined locations at the first photographing time, the second photographing time, and the third photographing time, and the processing device obtains primary information of the cells photographed in the cell image. The processing device calculates the first information of the cells captured in the cell images of the respective locations at the second shooting time and the third shooting time, calculates the first time-varying index value of the first information at the first shooting time and the second shooting time, and the first time-varying index value of the first information at the second shooting time and the third shooting time, calculates the statistical index value of the first time-varying index value at the first shooting time, the second shooting time and the third shooting time, and calculates the second time-varying index value based on the statistical index value at the first shooting time and the second shooting time, and the statistical index value at the second shooting time and the third shooting time, and determines the shooting time corresponding to the time when the second time-varying index value reaches a specified threshold as the time when the passage opportunity has arrived.

[0026] Effects of the Invention

[0027] According to the present invention, it is possible to determine the appropriate timing of subculturing cells during cell culture. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1A The schematic external structure of the cell culture system of the present invention is shown.

[0029] Figure 1B An example of a functional block diagram of the cell culture system of the present invention is shown.

[0030] Figure 1C Another example of a functional block diagram of the cell culture system of the present invention is shown.

[0031] Figure 1D Another example of the functional block diagram of the cell culture system of the present invention is shown.

[0032] Figure 2A The following is an overview of the flow of the culture process in the cell culture system of the present invention.

[0033] Figure 2B The entire outline of the processing flow of the passage determination process of the present invention is shown.

[0034] Figure 2C The schematic structure of the cell passage timing determination unit of the present invention is shown.

[0035] Figure 2D The following shows the concept of an image captured by the imaging means of the cell transfer timing determination unit of the present invention.

[0036] Figure 2E The concept of time lapse regarding the photographing time of the image acquired in the cell transfer timing determination unit of the present invention is shown.

[0037] Figure 2F The flow of the determination data generation process according to the first embodiment of the present invention is shown.

[0038] Figure 3A The flow of the determination data generation process in the first example of the second embodiment of the present invention is shown.

[0039] Figure 3B The following is a flow of a process of generating determination data in the second example of the second embodiment of the present invention.

[0040] Figure 3C An example of the concept of selecting a focus imaging time in the most recent comparison method according to the second embodiment of the present invention is shown.

[0041] Figure 3D Another example of the concept of selecting the focus imaging time in the latest comparison method according to the second embodiment of the present invention is shown.

[0042] Figure 3E An example of the concept of selecting a focus imaging time in the reference comparison method according to the second embodiment of the present invention is shown.

[0043] Figure 3F The concept of generating a time-varying index value according to an example of the second embodiment of the present invention is shown.

[0044] Figure 3G The concept of generating the specific growth rate and doubling time of the temporal change index value is shown as another example of the second embodiment of the present invention.

[0045] Figure 4A The flow of the determination data generation process in the first example of the third embodiment of the present invention is shown.

[0046] Figure 4BThe following is a flow of a process of generating determination data in the second example of the third embodiment of the present invention.

[0047] Figure 4C The following is a flow of a process of generating determination data in the third example of the third embodiment of the present invention.

[0048] Figure 4D An example of the concept of selecting a focus imaging time in the closest comparison method according to the third embodiment of the present invention is shown.

[0049] Figure 4E Another example of the concept of selecting the focus imaging time in the latest comparison method according to the third embodiment of the present invention is shown.

[0050] Figure 4F An example of the concept of selecting a focus imaging time in the reference comparison method according to the third embodiment of the present invention is shown.

[0051] Figure 4G Another example of the concept of selecting the focus imaging time in the reference comparison method according to the third embodiment of the present invention is shown.

[0052] Figure 4H The concept of generating the difference change amount and the change amount rate between the primary temporal variation index value and the secondary temporal variation index value according to the third embodiment of the present invention is shown.

[0053] Fig. 4I The concept of generating the rate of change per unit time as the primary temporal variation index value and the difference in rate of change as the secondary temporal variation index value according to the third embodiment of the present invention is shown.

[0054] Figure 4J The concepts of the specific growth rate and doubling time as a primary temporal change index value according to the third embodiment of the present invention are shown.

[0055] Figure 5A An example of a modified concept of selection of the focus imaging time according to the fourth embodiment of the present invention is shown.

[0056] Figure 5B The concept of the reference value integration method according to the fourth embodiment of the present invention is shown.

[0057] Figure 5C The concept of the matrix temporal variation method according to the fourth embodiment of the present invention is shown.

[0058] Figure 5D The concept of the matrix temporal variation method according to the fifth embodiment of the present invention is shown.

[0059] Figure 5EThe concept of the matrix temporal variation method according to the fifth embodiment of the present invention is shown.

[0060] Fig. 6A 2 is a diagram schematically showing the distribution of cells in the observation site Op, and shows a case where there is no deviation in the distribution of cells.

[0061] Figure 6B 2 is a diagram schematically showing the distribution of cells in the observation site Op, and shows a case where the distribution of cells is largely biased. DETAILED DESCRIPTION

[0062] [First embodiment]

[0063] (Cell Culture System)

[0064] Reference Figures 1A to 2F The method and system for determining the timing of subculture according to the first embodiment of the present invention are described. In the present invention, subculture is particularly suitable for culturing adherent cells in a culture container, but is not necessarily limited to adherent cells. The determination of the timing of subculture is achieved by a subculture timing determination unit 3 as a subculture timing determination system and a cell culture system 1 having the subculture timing determination unit 3. The determination of the timing of subculture according to the first embodiment is described below using the subculture timing determination unit 3 and the cell culture system 1. Figure 1A This is a schematic diagram of the appearance of the cell culture system 1. Figure 1B This is an example of a functional block diagram showing the electrical connection of the cell culture system 1 . Figure 1C and Figure 1D An example of another functional block diagram showing the electrical connections of the cell culture system 1 is shown. Figure 2A It is a diagram schematically showing the flow of the culture and subculture process of the cell culture system 1 of the present invention. Figure 2B It is a diagram showing the flow of the process of determining the passage according to the first embodiment.

[0065] For example, Figure 1B As shown, the cell culture system 1 includes internal units such as a culture medium unit 2, a subculture timing determination unit 3, a culture unit 4 as an incubator, a cell seeding device 5, a transport unit 6, a centrifuge 7, and a processing device 8. The cell culture system 1 does not necessarily need to include all of these units, and may include at least some of these units.

[0066] In the cell culture system 1, the processing device 8 is electrically connected to the unit group (culture medium unit 2, subculture timing determination unit 3, culture unit 4, cell inoculation device 5, conveying unit 6, centrifugal device 7) constituting the cell culture system 1 in such a manner that the processing device 8 can send and receive signals with the unit group (culture medium unit 2, subculture timing determination unit 3, culture unit 4, cell inoculation device 5, conveying unit 6, centrifugal device 7). The culture medium unit 2 is a device for adding culture medium into the culture container and removing and recovering the old culture medium for culture medium replacement and adding new culture medium. The subculture timing determination unit 3 is a device for determining the timing of subculture of the culture container, and has a camera 31 as a photographing component inside. The culture unit 4 is a device for storing the culture container in an internal chamber and maintaining the room at a specified temperature. The cell inoculation device 5 is a device for inoculating cells by, for example, dropping a cell turbid solution into the culture container at the beginning or at the time of subculture. The conveying unit 6 is a device for moving the culture container between the unit groups. The centrifugal device is a device for performing separation for recovering cells peeled off from the culture container when the culture container is subcultured.

[0067] In the processing device 8, for example Figure 1B As shown, the processing device 8 sends control signals for controlling the internal units to the culture medium unit 2, the subculture timing determination unit 3, the culture unit 4, the cell seeding device 5, the conveying unit 6, and the centrifugal device 7, respectively, so as to control the respective actions of the internal units. In addition, the processing device 8 receives signals of the respective states of the internal units from the culture medium unit 2, the subculture timing determination unit 3, the culture unit 4, the cell seeding device 5, the conveying unit 6, and the centrifugal device 7, respectively.

[0068] The processing device 8 controls the unit group (culture medium unit 2, subculture timing determination unit 3, culture unit 4, cell inoculation device 5, transport unit 6, centrifuge device 7) constituting the cell culture system 1 of the present invention, and receives and sends the signals required for the control. In addition, in addition to this, it is also possible to receive signals of detected data in each unit. That is, it is also possible to receive detected data (such as the number of cells, etc.) in the culture medium unit 2 and the subculture timing determination unit 3, etc. In addition, as described later, the processing device 8 is particularly capable of processing the data of each unit in the subculture timing determination unit 3, etc., and making the required judgments.

[0069] Next, refer to Figure 2A, the general functions of each unit and the culture and subculture process of the present invention are described. First, in the cell inoculation process as step 1, a culture medium containing cells is formed in the evaluation culture container Pe. That is, here, a cell suspension containing cells is added to the evaluation culture container Pe by the cell inoculation device 5, or a culture medium is added to the evaluation culture container Pe and cells are inoculated therein. For example, the evaluation culture container Pe is prepared by using the dispensing tube 21a of the dispensing machine 21 to inject a cell suspension into a flat culture container such as a culture dish or a shallow bottom plate, or by injecting a cell suspension into the culture medium after injecting the culture medium.

[0070] In the culture process as step 2, the evaluation culture container Pe inoculated with cells in step 1 is placed on the rack 41 in the culture unit 4 and stored under a constant temperature for a predetermined period. During this period, the cells in the evaluation culture container Pe adhere to the bottom of the evaluation culture container Pe and spread or proliferate in the evaluation culture container Pe containing the culture medium. When the evaluation culture container Pe has passed the predetermined period, the subculture timing determination process as step 3 is performed. The processing device 8 performs a timed count of the predetermined period, sends a control signal to the transport unit 6, removes the evaluation culture container Pe to be determined from the culture unit 4, and transports the evaluation culture container Pe to the subculture timing determination unit 3.

[0071] In step 3, the evaluation culture container Pe is taken out from the culture unit 4 and transported to the subculture timing determination unit 3, and the subculture timing determination unit 3 takes a picture of the culture medium of the evaluation culture container Pe and determines whether the time for subculture has arrived. Here, if it is determined that the evaluation culture container Pe has not arrived at the time for subculture, the culture process of step 2 is returned. Steps 2 and 3 are repeatedly executed until it is determined that the time for subculture has arrived. In the culture process of step 2, if the period of the determination of the time for subculture has not yet arrived but the culture medium needs to be replaced, the culture medium is replaced by the culture unit 2 in step 4 as needed, and the culture process of step 2 is returned. In addition, if the result of the determination of the time for subculture is that the time for subculture has not yet arrived but the culture medium needs to be replaced, the culture medium is also replaced by the culture unit 2 in step 4, and then the culture process of step 2 is returned. On the other hand, in the subculture timing determination process, if it is determined that the evaluation culture container Pe has arrived at the time for subculture, it is transferred to the subculture process as step 5. In the subculture process of step 5, for example, the following process can be performed. First, the cells in the evaluation culture container Pe are taken out and recovered, and the cell suspension containing the taken out cells is adjusted, and the cell suspension is added to a new evaluation culture container Pe by the cell seeding device 5 to seed the cells. When adjusting the cell suspension, the centrifugal device 7 is used to separate the cells as needed. When the cells are seeded in the new evaluation culture container Pe, the subculture is completed. After the subculture, the culture process returns to step 2, and steps 2 to 5 are repeated as needed.

[0072] Between each step from step 1 to step 5, the transport unit 6 transports the evaluation culture container Pe between the culture medium unit 2, the subculture timing determination unit 3, the culture unit 4, and the cell seeding device 5. The transport unit 6 is provided with a retractable arm 62 mounted on a moving table 61 that moves along an elevating track, for example, and can move in front of each unit to take out and put in the evaluation culture container Pe. The front end of the arm 62 is provided with an actuator 63 that can hold the evaluation culture container Pe.

[0073] like Figure 1BAs shown, the processing device 8 is, for example, arranged outside each unit group so as to manage the overall control of the unit group (culture medium unit 2, subculture timing determination unit 3, etc.) constituting the cell culture system 1. However, in addition to or instead of the processing device 8, an internal processor can be arranged in each unit for the control and data processing of each unit. In this manner, the unit equipped with the processor can function independently of other units. For example, in the case of the subculture timing determination unit 3, a device for performing the processing can also be arranged in the subculture timing determination unit 3 in a manner dedicated to the control and data processing of the subculture timing determination unit 3. The subculture timing determination unit 3 can be used independently of other units.

[0074] That is, in this example, Figure 1C As shown, the processing device 8 configures the processing device 33 as an internal processor in the subculture timing determination unit 3, so that the processing device 33 can replace the processing device 8 to control and process data of the subculture timing determination unit 3, thereby enabling the subculture timing determination unit 3 to function independently of other units.

[0075] In addition, further, Figure 1D As shown, the processing device 33 may exist outside the subculture timing determination device 3a which is physically and externally identifiable as the subculture timing determination unit 3, and outside the cell culture apparatus 1a which is physically and externally identifiable as the cell culture system 1. In this case, the processing device 33 is connected to the subculture timing determination device 3a via the communication component and the network N in a manner capable of communicating. For example, the subculture timing determination device 3a is connected to the camera 31 which is a photographing component via the communication component and the network N. In this case, the network N and the processing device 33 are also configured as a cloud. In this example, even if the processing device 33 exists outside the subculture timing determination device 3a and the cell culture apparatus 1a in appearance (physically and externally), the subculture timing determination unit 3 actually (electrically and systematically) includes the processing device 33. In addition, if the cell culture system 1 includes the subculture timing determination unit 3, the cell culture system 1 becomes identical to the subculture timing determination unit 3 in terms of the substantial system structure. Figure 1C Thus, the processing device 33 can be configured separately from the passage timing determination device 3a equipped with the camera 31, and the processing and control in the passage timing determination device 3a can be performed from the outside.

[0076] Next, the determination of the timing of subculturing performed by the subculturing timing determination unit 3 will be described below. Here, an example in which the processing device 33 is arranged in the subculturing timing determination unit 3 ( Figure 1CAlthough the above description is made with reference to the structure of FIG. 3 , the same is true when the processing device 33 is not arranged in the passage timing determination unit 3 and the processing is performed by the processing device 8 arranged outside the passage timing determination unit 3. In this case, it is sufficient to make the processing device 8 have the following functions performed by the processing device 33.

[0077] (Cell passaging timing determination unit)

[0078] Next, refer to Figure 2B The subculture timing determination unit 3 and the subculture timing determination process in the subculture timing determination method as the aforementioned step 3 are described. The subculture determination process is composed of a photographing step (S11) of first acquiring a cell image, a determination data generating step (S12) of generating determination data based on the cell image, and a determination step (S13) of determining whether the timing of subculture has arrived.

[0079] First, in the imaging step ( S11 ), an image of cells in the evaluation culture container Pe is captured. Figure 2C The schematic structure of the subculture timing determination unit 3 is shown. The subculture timing determination unit 3 has a photographing table 32, a processing device 33, and a camera 31 as a photographing member. The evaluation culture container Pe can be arranged on the photographing table 32, and the camera 31 can photograph the surface of the culture medium in the evaluation culture container Pe. A predetermined portion of the surface of the culture medium in the evaluation culture container Pe transported by the arm 62 of the transport unit 6 is photographed by the camera 31 and acquired as an image. The data of the acquired image is stored in a storage device (not shown).

[0080] The processing device 33 executes the determination data generation step (S12) and the determination step (S13). The processing device 33 acquires the image acquired in the imaging step (S11), and processes the data obtained from the image to determine the timing of passage. Figure 2C to Figure 2F , which is described in detail below.

[0081] (Photography process)

[0082] First, in the photographing process (S11), the camera 31 performs a photographing process for all evaluation culture containers Pe in the culture unit 4 of the cell culture system 1. In this photographing process, the image of the cells on the culture surface of the culture medium is photographed and obtained as a cell image. In this specification, "culture medium" refers to the in-plane direction of the bottom surface of the evaluation culture container Pe, and the culture surface is defined as the part of the culture medium of the container where the cells are fixed in the depth direction. An evaluation culture container Pe that becomes a determination object is taken out from the culture unit 4 by the arm 62 of the conveying unit 6, and is conveyed to the subculture timing determination unit 3 by the conveying unit 6. The conveyed evaluation culture container Pe is placed on the photographing table 32 of the subculture timing determination unit 3. The culture medium of the placed evaluation culture container Pe is photographed by the camera 31. After the photographing is completed, the evaluation culture container Pe is recovered from the photographing table 32 by the arm of the conveying unit 6 and is sent back to the culture unit 4 by the conveying unit 6. In the photographing process, the step of further repeating this process and performing the photographing of the cell images on the culture surface for all evaluation culture containers Pe that are determination objects inside the culture unit 4 is also included.

[0083] Figure 2D 31 is a conceptual diagram of an image captured by the camera 31 on the culture surface of the culture medium in the evaluation culture container Pe. Figure 2D As shown in FIG. 1 , the culture surface of the culture medium in a certain evaluation culture container Pe is photographed by the camera 31 and is taken into the storage device of the subculture timing determination unit 3 as a cell image. Figure 2D In the example, the concept of performing 6×6 (36) shots is shown. However, in reality, the shots are performed with finer divisions than this. The shot portion of the culture medium is a portion of the area selected from the entire area of ​​the evaluation culture container Pe, and is a portion of the culture medium of a predetermined plurality of observation portions Op determined by taking the state of the evaluation culture container Pe as a representative in a manner at least suitable for the evaluation of the culture surface of the evaluation culture container Pe. For example, in Figure 3BIn the example of representing the part of the evaluation culture container Pe, the area of ​​the three images of the shaded part is used as the observation part Op. For the observation part Op, the entire area of ​​the culture medium of a certain evaluation culture container Pe is initially photographed, and a part of the image area where the number of cells is sufficient to represent the evaluation culture container Pe is selected, and the part of the image area is determined as the fixed point observation part. The observation part Op is determined by representatively selecting a plurality of parts such as 3 parts and 10 parts. It can be understood that by setting it as a plurality of parts, the judgment accuracy is generally improved. Once the observation part Op is determined, in the process of determining the passage timing for observing the change over time described below, for any evaluation culture container Pe, the same observation part Op is used in the shooting at different times. In the following, any evaluation culture container Pe is used as a representative example for explanation, but it is the same for any of the plurality of evaluation culture containers Pe.

[0084] Figure 2E The concept of executing the photographing process at multiple photographing times is shown. In the photographing process (S11), for any observation part Op of the evaluation culture container Pe, images of cells on the culture surface are photographed at multiple photographing times at a predetermined time interval as the culture proceeds, and are acquired as cell images. At each photographing time, cells on the culture surface are photographed at a predetermined plurality of observation parts Op selected from the culture medium of the evaluation culture container Pe, and cell images are acquired. At all photographing times, the observation part Op is selected at the same part in a manner of fixed-point observation. At each photographing time, the evaluation culture container Pe as the determination object is taken out from the culture unit 4 by the conveying unit 6 and conveyed to the subculture timing determination unit 3, and the evaluation culture container Pe is conveyed to the culture unit 4 after the photographing is completed. As the culture proceeds, when a new photographing time arrives, the photographing process (S11) is performed at the photographing time. In the representative example, each time the photographing process (S11) is performed, the determination data generation process (S12) and the determination process (S13) described below are performed.

[0085] In the first embodiment, only the cell image captured at one of the multiple capturing times is used to determine the timing of the subculture. In this specification, the capturing time used in determining the timing of the subculture is referred to as the focusing capturing time. The focusing capturing time of the first embodiment consists only of the evaluation capturing time. In the first embodiment, only the capturing time selected from the evaluation capturing time is used in determining the timing of the subculture. Figure 2E As described later, in determining the timing of passage, in the second embodiment, cell images captured at two focus shooting moments selected from a plurality of shooting moments are used, and in the third embodiment, cell images captured at three or more focus shooting moments are used. Figure 2E In the figure, as a representative example, regarding the imaging time of interest in the third embodiment, a case where cell images selected from three imaging times of interest are used is shown.

[0086] (Generate judgment data)

[0087] After the imaging step ( S11 ), in the following determination data generating step ( S12 ), determination data for generation determination is generated. Figure 2F The steps constituting the determination data generating step (S12) of the first embodiment are shown. Figure 2F As shown, the determination data generating step (S12) of the first embodiment is composed of a primary information acquiring step (S1201) of acquiring primary information from a cell image acquired by the photographing step (S11), and a statistical indicator value acquiring step (S1202) of calculating a statistical indicator value based on the primary information.

[0088] (Get 1 information)

[0089] In the primary information acquisition process (S1201), the processing device 33 acquires primary information from the image acquired at a selected shooting moment (focus on the shooting moment). The primary information is information related to the cells photographed in each of the cell images of the culture surface captured at each shooting moment. The primary information is, for example, the number of cells, cell density, the area occupied by the cells, the single area of ​​the cells, the relative distance between the cells, etc. The primary information can be acquired from the images captured in each shooting process by using commercially available software, etc. The number of cells in the primary information is the number of cells captured in the captured image, which refers to the number of cells that can be directly acquired. For example, the cell density is the density calculated by dividing the number of cells acquired by the area of ​​the captured part. The area used here can adopt various areas, which is the area of ​​the part that excludes impurities such as bubbles in the culture medium, the part of the image where the shooting failed, etc. that cannot function as information related to the cells, and can be an effective effective area corresponding to the area where the cells can stretch or proliferate. In addition, the total area of ​​the size of the cells occupied by all cells in the area is the occupied area, and the single cell area is the area of ​​each cell captured in the image. The occupied area of ​​the single cell is the size of each cell captured in the image, for example, the relative distance between cells in a cell image is the distance defined by two or more cells in the cells captured in the image. The timing of passage can be determined based on the primary information obtained from the images acquired at each shooting time.

[0090] (Get statistical indicator values)

[0091] In the primary information acquisition process (S1201), after the primary information acquired at one shooting time is acquired, the statistical index value acquisition process (S1202) is performed. Here, the statistical index value corresponding to the shooting time is calculated based on the primary information acquired at one shooting time. As basic data representing the tendency of the state of the primary information, the statistical index value of each primary information is further calculated. The statistical index value is an index value that is limited to a representative value representative of the primary information in order to obtain multiple values ​​in each image, for example, at least one of the deviations such as the sum, the average value, the variance, and the standard deviation. That is, the statistical index value of the primary information is the sum (total value) of the values ​​that can be obtained from each image captured at multiple observation sites Op, the average value of each image, or the variance, the deviation. For example, in the case where the primary information is the number of cells, when the observation site Op at a certain shooting time is 3 sites, the statistical index value of the primary information is the sum of the number of cells in the images of the 3 sites, or any one of the average value, variance, or standard deviation of the number of cells. These primary information and the statistical index values ​​of the primary information are stored as acquired data in a storage unit (not shown) as needed.

[0092] Here, the average value and sum, variance and deviation selected as statistical index values ​​have different technical characteristics as indicators of cell passage according to their definitions. The sum and average value as statistical index values ​​are indicators indicating the increase in the number of cells and the extension or proliferation of cells and their passivation. Regarding the sum and average value, it can be set as the sum and average value of each of the multiple observation sites Op, and as the statistical index value according to the number of observation sites Op, it is set as the sum and average value. Furthermore, it can also be set as a representative value or a representative value of a number less than the number of observation sites Op that is easy to handle by statistical processing such as averaging. On the other hand, the variance or deviation as a statistical index value has the characteristic of showing the uniformity of cells in the container. Regarding the variance or deviation, it can be set as the variance or deviation of each of the multiple observation sites Op, and as the statistical index value of the number of observation sites Op, it is set as the variance or deviation. Furthermore, it can also be set as a representative value or a representative value of a number less than the number of observation sites Op that is easy to handle by statistical processing such as averaging. In the culture of cells, usually, the uniformity of cells deteriorates due to the extension or proliferation of cells. Moreover, before the time of passage brought about by the limit of the number of cells, the time of passage brought about by the heterogeneity of the cells will come earlier. That is, when the sum or mean value is selected as the statistical indicator value, even if the time of passage has not yet arrived, the cells in the culture container will become more uneven. In this case, in a part, there will be a part where the extension or proliferation of cells has reached the limit, which can also be called the appropriate time for passage. The variance or deviation as a statistical indicator value is an indicator of the uniformity of the cells in the culture container, so compared with the case of selecting the sum or mean value as the statistical indicator value, the timing of passage can be determined more appropriately. The characteristics of the sum, mean value, variance and deviation as statistical indicator values ​​are also the same in the second embodiment and the third embodiment described below.

[0093] (Determine the passage)

[0094] After the generation of the judgment data is completed, a judgment process (S13) is performed to judge whether the timing of passage has been reached. In the judgment process (S13), the timing of passage is judged according to the statistical index value generated in the judgment data generation process (S12). The statistical index value quantitatively represents the state of the cells of the culture surface of the specified observation part Op of the specified multiple locations selected from the culture container. Therefore, if the number of cells is taken as an example, the sum of the statistical index values ​​of the positions of the specified observation parts Op is the total number of cells in the region of each specified observation part Op, which becomes the value representing the total number of cells of the entire region of the positions other than the observation part Op of the culture surface in the culture container. If the number of cells is taken as an example, the average value of the statistical index value, for example, can be set to the number of cells per unit area or the number of cells of each observation part Op at one place, which becomes a representative value representing the room for cells to expand or proliferate in the culture container in a manner above the sum.

[0095] In the determination step (S13), the statistical index value calculated in the statistical index value acquisition step (S1202) is compared with a predetermined threshold value. Here, when the determination data reaches the predetermined threshold value, it is determined that the timing of subculturing has arrived. Regarding the predetermined threshold value, a test is performed as a preliminary check under the same conditions as the actual determination of the timing of subculturing, in which the statistical index value at the time of subculturing is acquired and limited to the predetermined threshold value.

[0096] In an ideal culture state where the distance between cells is gradually shortened on average without the deviation of the distance between cells from the state of inoculation of cells, the sum and average value of the statistical index value are very effective in determining the timing of passage. In the case of selecting the sum and average value of the statistical index value, the shooting time corresponding to the moment when the sum or average value of the statistical index value reaches the specified threshold is determined as the moment of reaching the passage timing. As the area portion of the observation part Op, the value of the timing of passage can be uniquely limited as the sum or average value of the original information of the first time. As long as the value is used as the specified threshold. The sum and average value are values ​​that directly represent the extension or proliferation of cells, so theoretically they increase with the passage of time. The specified threshold value can be set to, for example, the actual area in the direction in which cells can extend or proliferate in the culture container, such as the value obtained by multiplying the bottom area by a specified ratio. The specified ratio can be set to about 70% to 90%. In addition, for example, a test as a preliminary inspection is implemented under the same conditions as the actual timing of determining the passage, in which the sum or average value of the statistical index value of the extension or proliferation of cells can no longer be limited to a specified threshold. In this case, it is also possible to obtain in advance the information as the sum or average value of primary information on the timing of passage in a plurality of test culture containers.

[0097] Furthermore, if the number of cells is used as an example, as described above, the variance or deviation as a statistical index value represents the degree of dispersion of cells in the observation site Op, which means that even if the average value is the same, there is a site on the culture surface of the culture container where cells cannot spread or proliferate and reach a state where subculture is required (for example, Figure 5B ). The shooting time corresponding to the moment when the variance or deviation of the statistical index value reaches the specified threshold is determined as the moment when the passage timing is reached. As the area portion of the observation site Op, the value of the timing of passage can be uniquely limited as the variance or deviation of the original one-time information. Just use this value as the specified threshold. In the case of variance or deviation, in the initial state of culture, the extension or proliferation of cells occurs near each cell, so in theory, the variance or deviation is temporarily reduced.

[0098] Then, usually, due to the expansion of cell extension or proliferation, as time passes, it will turn into an increase after reaching a minimum. Then, after the time of passage, the cells cannot stretch or proliferate, and the variance or deviation will turn into a decrease after reaching a maximum. Therefore, the specified threshold can be defined as a maximum value, which is a test implemented as a pre-test under the same conditions as the actual determination of the timing of passage, in which the variance or deviation as a statistical indicator value turns from an increase to a decrease. The maximum value. Further, in special cases, in the initial state of culture, there is also a situation where the cells stretch or proliferate locally and the variance or deviation does not turn into an increase. At this time, the inflection point of the time change of the variance or deviation becomes the time of passage, so the inflection point of the time change can be set as a specified threshold. In this way, in the time of passage, a certain inflection point will appear in the time change as represented by the extreme values ​​such as the minimum value and the maximum value. The moment when such an inflection point of time change appears is set as the threshold as the moment when the time of passage is reached. In particular, when the variance or deviation does not turn to increase, as described above, there is a case where cells locally expand or proliferate rapidly in the initial state of culture, so the value obtained by adding the margin value α to the minimum value can also be set as the threshold. As the margin value α, at least the value obtained by adding the margin value α to the minimum value is set to be smaller than the maximum value. Further, the margin value α is less than 50% of |(maximum value)-(minimum value)|, and preferably the value is 0. This value can also be obtained in advance as the variance or deviation of the primary information on the timing of reaching the subculture in a plurality of culture containers for inspection.

[0099] In the present invention, primary information about cells is acquired from an image obtained by capturing the state of a culture medium containing cells, and its statistical index value is calculated, thereby being able to directly grasp the state of cell expansion or proliferation and appropriately grasp the timing of subculturing.

[0100] [Second embodiment]

[0101] Next, a method and system for determining the timing of subculture according to the second embodiment will be described. The second embodiment is basically the same as the first embodiment but partially modified. The structures of the cell culture system 1 and the subculture timing determination unit 3 are the same as those of the first embodiment. Hereinafter, regarding the second embodiment, the parts that are different from the first embodiment will be described, and the description of the parts that are the same as the first embodiment will be omitted. In the subculture determination process of the second embodiment, the subculture determination process is composed of a photographing step (S11), a determination data generating step (S12), and a determination step (S13). Figure 2B The same as the first embodiment shown. However, in the second embodiment, in the determination data generation step (S12), the generation of the determination data and the threshold value specified in the determination step (S13) are different. In the second embodiment, in the generation of the determination data in the determination data generation step (S12), two shooting moments are selected as the shooting moments of interest from the multiple shooting moments of the shooting step (S11), and the determination data is generated based on the cell images of the two shooting moments and the passage timing is determined, which is different from the first embodiment. That is, in the second embodiment, a time element is added to the determination data.

[0102] (Photographing process of culture surface)

[0103] In the second embodiment, Figure 2E As shown, the cell images are captured at multiple capturing times, which is the same as the first embodiment. The observation site Op is selected at the same site for fixed-point observation at each capturing time, and the capturing process is performed at a new capturing time as the culture progresses.

[0104] (Generate judgment data)

[0105] Figure 3A and Figure 3B This is a diagram showing the flow of the determination data generation step (S12) in the generation determination process of the second embodiment. In the determination data generation step (S12) of the second embodiment, the time-varying index value is calculated in order to add the time-varying element in the first embodiment. Here, Figure 3A 1 is a diagram showing the first example of the second embodiment. In the first example, after the primary information acquisition step (S1211) of acquiring primary information from a cell image and the statistical index value calculation step (S1212) of calculating the index value based on the primary information are completed, the time-varying calculation step (S1213) of calculating the time-varying index value is performed.

[0106] on the other hand, Figure 3B : is a diagram showing the second example of the second embodiment. In the second example, between the primary information acquisition step (S1211) of acquiring primary information from the cell image and the statistical index value calculation step (S1212) of calculating the index value based on the primary information, a time-varying calculation step (S1213) of calculating the time-varying index value is performed. In this specification, in the time-varying calculation step (S1213), the two shooting moments used to calculate the time-varying index value are referred to as the shooting moments of interest, the shooting moment that is earlier in time among the shooting moments of interest is referred to as the reference shooting moment, and the shooting moment that is later in time is referred to as the evaluation shooting moment. The time-varying index value is the value of the change in the statistical index value of the primary information at the shooting moment of interest relative to the statistical index value of the primary information at the reference shooting moment.

[0107] In the second embodiment, in both the first and second examples, two focus imaging times selected from a plurality of imaging times are used to determine the timing of passage. Figure 2E The cell images captured at the first capturing time and the second capturing time in the second embodiment are composed of the reference capturing time and the evaluation capturing time. Typically, the evaluation capturing time is a new capturing time that arrives as the culture progresses. For example, Figure 2E As shown, the first shooting time as the reference shooting time and the second shooting time as the evaluation shooting time are selected. Hereinafter, the shooting time of interest in which the reference shooting time is the first shooting time and the evaluation shooting time is the second shooting time is described. As a method of selecting two shooting times of interest, there are two options, namely the nearest comparison method and the reference comparison method, in both the first and second examples.

[0108] In the most recent comparison method, each time a new imaging time is added, the reference imaging time and the evaluation imaging time are updated to the latest imaging time of interest. That is, when the timing of the generation is determined by the most recent comparison method using an arbitrary imaging time of interest (reference imaging time and evaluation imaging time), when the timing of the generation is determined before the generation, the imaging time of interest (reference imaging time and evaluation imaging time) is updated to determine the generation. For this, refer to Figure 3C Please explain in detail. Figure 3CThe concept of selecting the imaging time of interest by the nearest comparison method of selecting the imaging time of interest (reference imaging time and evaluation imaging time) from a plurality of imaging times (n-3) from imaging time (n-3) to imaging time (n+2) is shown. For example, after selecting imaging time (n-3) as the reference imaging time and imaging time (n-2) as the imaging time of interest as the evaluation imaging time and determining the timing of passage, if it is determined that the timing of passage has not yet arrived, the imaging time (n-2) is selected as the reference imaging time and the imaging time (n-1) is selected as the imaging time of interest as the evaluation imaging time to determine the timing of passage. After that, similarly, while shifting the reference imaging time from imaging time (n-3) to imaging time (n+1) and also shifting the evaluation imaging time from imaging time (n-2) to imaging time (n+2), the timing of passage is determined by repeatedly updating the imaging time of interest until it is determined that the timing of passage has arrived. When updating the reference shooting time and the evaluation shooting time while shifting them, the reference shooting time and the evaluation shooting time must be shifted regularly in order. For example, when the reference shooting time is shifted by one, the evaluation shooting time must also be shifted regularly by only one. An irregular shifting method such as shifting the reference shooting time by only one but shifting the evaluation shooting time by two cannot be used.

[0109] In the reference comparison method, the photographing time of interest consisting of the reference photographing time and the evaluation photographing time is selected as follows. Figure 3EThe concept of selecting the photographing time of interest of the benchmark comparison method for selecting the photographing time of interest (reference photographing time and evaluation photographing time) from a plurality of photographing times (n-3) to a photographing time (n+2) is shown. In the benchmark comparison method, an initial arbitrary photographing time is determined as the reference photographing time from a plurality of photographing times, and an arbitrary photographing time after the reference photographing time is selected as the evaluation photographing time. For example, the photographing time (n-3) is fixed as the reference photographing time and the photographing time (n-2) is first set as the photographing time of interest for determining the timing of generation. After this determination, if it is determined that the timing of generation has not yet arrived, the photographing time of interest is selected by keeping the photographing time (n-2) as the reference photographing time and setting the photographing time (n-1) as the evaluation photographing time to determine the timing of generation. After that, similarly, the reference evaluation shooting time is kept unchanged at the shooting time (n-3), and only the evaluation shooting time is shifted from the shooting time (n-2) to the shooting time (n+2), and the determination of the passage is repeated while the update of the focus shooting time is performed until it is determined that the passage timing has arrived. That is, in the case of the reference comparison method, the reference shooting time is fixedly used in the determination of the timing of each passage, and the new shooting time is changed to the evaluation shooting time, and the selection is performed in a manner that only the evaluation shooting time is updated to the latest shooting time, and this process is repeated in the determination of the timing of each passage. In this case, when updating while shifting the reference shooting time and the evaluation shooting time, it is also necessary to stagger the reference shooting time and the evaluation shooting time in order and regularly.

[0110] In the nearest comparison method, the reference shooting time and the evaluation shooting time move together, so the primary information of the reference shooting time and the evaluation shooting time is relatively compared. On the other hand, in the reference comparison method, the first shooting time as the reference shooting time is fixed, so the primary information of the first shooting time (fixed) of the reference shooting time and the second shooting time (moving) of the evaluation shooting time is absolutely compared. In addition, the time interval between each shooting time can be set arbitrarily, but in the method of selecting two consecutive shooting times as the nearest comparison method, the time interval between each shooting time is preferably set to be constant.

[0111] In recent comparative law, representative examples are Figure 3C As shown in FIG. 1 , the selection is performed in such a way that there is no shooting time between the reference shooting time and the evaluation shooting time, but it may also be performed as follows Figure 3D As shown, selection is performed so as to set an arbitrary number of imaging times not used for evaluation between the reference imaging time and the evaluation imaging time. Figure 3DAn example is shown in which there is a photographing time that is not used for evaluation between the reference photographing time and the evaluation photographing time. Then, as long as the reference photographing time and the evaluation photographing time are selected in sequence with a certain interval between them, it can be used for determining the timing of generation. In addition, in the reference comparison method, the reference photographing time is fixed and the evaluation photographing time is updated, so there is a photographing time between the reference photographing time and the evaluation photographing time.

[0112] In the following, the first and second examples are described respectively using the nearest comparison method as an example, and an example in which a certain photographing time of interest is continuous with a reference photographing time and an evaluation photographing time. In the following, the first photographing time is described as the reference photographing time and the second photographing time is described as the evaluation photographing time. In the case of the nearest comparison method and the reference comparison method in which the reference photographing time and the evaluation photographing time are not continuous, the data of the photographing time other than the photographing time of interest is not used to generate the data for determining the timing of the generation, and therefore the determination process of the generation is the same as in the following example.

[0113] (Get 1 information)

[0114] In the first example and the second example, the primary information acquisition process is the same. In the primary information acquisition process (S1211), the processing device 33 acquires primary information from the image acquired at each shooting time at each shooting time. The primary information acquisition process is the same as that of the first embodiment, for example, the number of cells, cell density, cell occupied area, cell monomer area, cell distance, etc. The description is omitted here. Below, the first example is described, and then the second example is described.

[0115] <Example 1>

[0116] (Get statistical indicator values)

[0117] In the first example, as mentioned above, Figure 3AAs shown, after the primary information of each shooting moment is obtained in the primary information acquisition process (S1211), the statistical index value of each shooting moment is calculated according to the primary information of each shooting moment in the statistical index value acquisition process (S1212). As in the first embodiment, the statistical index value is an index value limited to a representative value that represents the primary information representatively, for example, at least one of the deviations such as the sum, the mean value, the variance, and the standard deviation. The sum is the sum of the number of cells of multiple observation sites, the sum of the occupied area of ​​the cells, the sum of the monomer area of ​​the cells, and the sum of the distance between cells. The mean value is the mean value of the number of cells of multiple observation sites, the mean value of the cell density, the mean value of the occupied area of ​​the number of cells, the mean value of the monomer area of ​​the cells, and the mean value of the distance between cells. The variance and deviation are the variance and deviation of the number of cells of multiple observation sites, the variance and deviation of the cell density, the variance and deviation of the occupied area of ​​the cells, the variance and deviation of the monomer area of ​​the cells, and the variance and deviation of the distance between cells. Here, the variance or deviation as the statistical index value is an index of the uniformity of the cells in the culture container, and thus the timing of subculturing can be determined more appropriately compared to the case where the sum or average value is selected as the statistical index value, which is the same as the first embodiment.

[0118] (Time-dependent change calculation step (obtaining time-dependent change index value))

[0119] Next, in the temporal variation calculation process (S1213), the temporal variation index value is calculated based on the statistical index value of each 1-time information calculated at each shooting moment. One temporal variation index value is generated for the value of the 1-time information of two shooting moments. In the present embodiment, the shooting moments of interest are the same as the number of shooting moments used for determination. 1-time information is obtained for each shooting moment of interest, so the number of shooting moments of interest is the same as the number of 1-time information. Therefore, the number of temporal variation index values ​​is one less than the number of shooting moments of interest. In addition, for the temporal variation index value, the temporal variation index value is calculated according to the number of observation parts Op or according to the number of representative values ​​of multiple observation parts Op when the representative values ​​are reduced to a smaller number.

[0120] The temporal change calculation step (S1213) is a step obtained by adding a step of calculating the temporal change index value to the statistical index value acquisition step (S1212) of calculating the statistical index value of the first embodiment. Figure 3F Provide explanation. Figure 3F This is a diagram showing the concept of changes in values ​​(vertical axis) with respect to time (horizontal axis) for the first and second examples. In the first example, the vertical axis is the statistical index value, and in the second example described later, the vertical axis is the value of primary information. Figure 3FThe example of the closest comparison method is shown as the focus of the shooting time. However, in the case of the aforementioned reference comparison method in which the first shooting time and the second shooting time are not selected as consecutive shooting times, Figure 3F , it is sufficient to replace it with a shooting time not shown between the first shooting time and the second shooting time.

[0121] In the reference comparison method in which the first shooting moment is fixed as the reference shooting moment and the second shooting moment as the evaluation shooting moment is updated in the two shooting moments, the first shooting moment is always fixed. Then, the value of the first shooting moment of the second shooting moment updated relative to the value of the fixed first information is limited, so that the first information increases as the cells expand or proliferate. The time-varying index value is the increase or decrease of the increase ratio of the first information, so according to the increase trend of the first information, the time-varying index value of the statistical index value shows an increase or decrease trend. On the other hand, in the case where the first information is the relative distance between cells, the first information decreases as the cells expand or proliferate. In this case, the time-varying index value is also the increase or decrease of the decrease ratio of the first information, so according to the decrease trend of the first information, the time-varying index value of the statistical index value shows a decrease or increase trend. Moreover, as the time of passage approaches, the degree of change of the increase or decrease slows down, showing a change tendency that the rate of change of increase or decrease is saturated toward a specified value. On the other hand, in the nearest comparison method in which the first shooting time and the second shooting time are selected as the continuous shooting time among the two shooting times, the first shooting time and the second shooting time are always updated, and become the relative comparison of the first shooting time and the second shooting time. Therefore, in the initial stage of cell expansion or proliferation, the primary information simply increases or simply decreases (simply decreases when the primary information is the relative distance between cells), and the time-varying index value of the statistical index value also increases or decreases. However, as the cell expansion or proliferation proceeds, the degree of increase of the primary information at the first shooting time and the degree of increase or decrease of the primary information at the second shooting time change. Therefore, as the timing of passage approaches, the degree of increase or decrease of the time-varying index value tends to slow down. Moreover, when the timing of passage is reached, the time-varying index value that is in an increasing trend decreases with the specified value as the extreme value, and the time-varying index value that is in a decreasing trend increases. Below, an example showing a changing tendency of primary information that increases as cells expand or proliferate is explained here, but in the case of primary information that shows a changing tendency of decreasing as cells expand or proliferate, the example showing a changing tendency of primary information that increases as cells expand or proliferate can be applied by reading the increase and decrease in reverse.

[0122] In calculating the temporal variation index value, two imaging times (the first imaging time and the second imaging time) are selected from a plurality of imaging times. Figure 3F In FIG. 1 , an example is shown in which the two latest consecutive shooting times are selected as the first shooting time (t1) and the second shooting time (t2). Figure 3F , the situation where the statistical index value (a1) at the first imaging time (t1) increases to the statistical index value (a2) at the second imaging time (t2) is shown. Whether the timing of the subculture has arrived is determined at the imaging time. If the timing of the subculture has not arrived, the latest two consecutive imaging times including the new imaging time are selected again to generate the determination data.

[0123] The time-varying index value can be set as the difference amount of the statistical index value of the first information between the first shooting time (reference shooting time) and the second shooting time (evaluation shooting time) with respect to the time interval of the shooting time of interest (the first shooting time and the second shooting time), that is, the differential change amount. In the time-varying index value of the differential change amount, the difference amount of the statistical index value of the first information at the second shooting time relative to the statistical index value of the first information at the first shooting time, that is, the differential change amount (a2-a1). The differential change amount can be said to be an increment. In the example where the first information is set as the number of cells, the differential change amount is the value of the incremental difference of the sum of the number of cells at the first shooting time and the second shooting time.

[0124] In addition, the temporal variation index value can also be set as the proportional change amount of the statistical index value of the first information at the second shooting time (evaluation shooting time) relative to the statistical index value of the first information at the first shooting time (reference shooting time). In the temporal variation index value of the proportional change amount, the proportional change amount of the statistical index value of the first information at the second shooting time relative to the statistical index value of the first information at the first shooting time, i.e. (a2 / a1). In addition, the temporal variation index value can also be set as the ratio of the difference amount of the statistical index value of the first information between the first shooting time (reference shooting time) and the second shooting time (evaluation shooting time) relative to the statistical index value of the first information at the first shooting time (reference shooting time), i.e. the difference proportional change amount. In the difference proportional change amount, the ratio of the increase amount between the statistical index value of the first information at the first shooting time (reference shooting time) and the statistical index value of the first information at the second shooting time (evaluation shooting time) relative to the difference increase amount of the statistical index value of the first information at the first shooting time, i.e. the difference proportional change amount ((a2-a1) / a1). For example, in the example where the 1st information is set as the number of cells, the former proportional change is the value of the ratio of the sum of the number of cells at the second shooting time to the sum of the number of cells at the first shooting time, and the latter differential proportional change is the value of the ratio of the increase in the sum of the number of cells from the first shooting time to the second shooting time to the sum of the number of cells at the first shooting time.

[0125] In addition, the temporal variation index value can also be set to the difference variation, proportional variation, and differential proportional variation of the statistical index value of the first information at the first shooting time and the statistical index value of the first information at the second shooting time relative to the time interval between the two shooting times (the first shooting time and the second shooting time). That is, the difference variation (a2-a1), the proportional variation (a2 / a1), and the differential proportional variation (a2-a1) / a1) can be divided by the time difference (t2-t1) between the first shooting time and the second shooting time to obtain the change rate per unit time, i.e., the temporal variation index value. That is, the statistical index value of the one-time information at two consecutive shooting moments can be set as the unit time change rate of the differential change amount (a2-a1) / (t2-t1), the unit time change rate of the proportional change amount (a2 / [a1·(t2-t1)]), and the unit time change rate of the differential proportional change amount (a2-a1) / [a1·(t2-t1)]). These time-varying index values ​​all refer to the increase rate per unit time of the statistical index value of the one-time information at the shooting moment of interest, and are also the increase speed. In the example where the one-time information is set to the number of cells, it is the increase rate per unit time of the number of cells, that is, the increase speed (proliferation speed). The increase speed (proliferation speed) mentioned here is the speed of the differential value obtained by linearly approximating the statistical index values ​​at the two shooting moments (hereinafter referred to as the simple speed).

[0126] Regarding the rate of increase (proliferation rate), in addition to the examples described above, as long as the time-dependent change of the statistical index value of the primary information can be reflected, it can be defined as the rate of increase relative to the passage of time according to a wide range of definitions. In the proliferation of cells, the cause of the increase is cell division, so the rate of increase (proliferation rate) is not constant. If the conditions are consistent, it is theoretically 2 times, 4 times, 8 times... etc., especially with the characteristic that the multiple increases by 2 times. Usually, in the case of quantitatively expressing the rate of increase (proliferation rate), the specific proliferation rate is used. In the present invention, it goes without saying that in the case where the primary information is the number of cells, even in the case where the primary information is other than the number of cells (the aforementioned cell density, the area occupied by the cells, the monomer area of ​​the cells, the relative distance between the cells, etc.), the fact that the cause of the increase is cell division has not changed, and it is basically increased by 2 times. In fact, even in the case where the primary information is the number of cells, the division timing of each cell is different, so it is rare to increase by exactly 2 times per unit time. Therefore, regardless of whether the primary information is the number of cells, the increase rate per unit time is approximately e. μ The multiplication factor is times. The μ at this time is called the specific growth rate. Therefore, as the increase rate (growth rate), the exponential approximation (e) of the statistical index values ​​at the two shooting times of the reference time and the evaluation time can be used. μt ) and obtain the specific proliferation rate μ.

[0127] Reference Figure 3G Comparison of specific proliferation rate and doubling time t d This example is used as an example of the time-varying index value. That is, the reference time is set as the reference shooting time to the first shooting time t1, and the statistical index value of the single information at this time is set to a1. Then, at any shooting time t i At each time point [i=2 to n], the statistical index value a of the primary information is measured as the evaluation shooting time point. i [i=2 to n] Statistical index value a of primary information at each evaluation shooting time i The relationship between [i=2 to n] and the statistical index value a1 of the primary information at the first imaging time t1 can be approximated as a generalized relationship: i / a1=e [(μi)·(ti-t1)] Here, μ i is the shooting time t i [i=2~n] specific growth rate, μ i =[ln(a i / a1)] / (t i -t1).

[0128] In addition, the doubling time (t ad The doubling time is the time required for the amount at the evaluation time to become twice the amount at the reference time. If we assume that the image is taken at the time t i The doubling time t is doubled at i=k in [i=2~n] ad =t k -t1. In relation to the specific growth rate, if the increase rate per unit time is e μ , then the time becomes e μ·tad = 2. That is, according to the above description, at any shooting time t i At each time point [i=2 to n], if μ is obtained as the time-varying index value of primary information i , then the time-varying index value μ of the primary information at each evaluation shooting time is i [i=2~n] and each evaluation shooting time t i The time-varying index value td of the primary information i As a generalized relationship, e [(μi)·(tdi)] =2 holds true. That is, the doubling time td i =ln2 / μ i =(ln2)·(t i -t1) / ln(a i / a1). Here, as an example for easy understanding, the shooting time t i The statistical index value a of the primary information where i=k[2<k<n] in [2<i<n] k Assume that the statistical index value a1 is twice the first imaging time t1 as the reference imaging time (a k =2a1). At this time, from the first imaging time t1 as the reference imaging time to the imaging time t k The time from [2<k<n] to [2<k<n] is called the doubling time t ad , a k / a1=e (μk·tad) =2 holds true. From here on, at t k At, the specific growth rate μ k =(ln2) / (t k -t1).

[0129] In addition to the doubling time, the time required for the amount at the evaluation time to become a specified multiple of the amount at the reference time can also be used as the increase rate (proliferation rate). In this application, this time is defined as the specified doubling time (tx). The specified doubling time is the time required to become a specified multiple X times. If the increase rate per unit time is e μ , then it is defined as e μ·tx =X. That is, according to the above description, at any shooting time t i At each time point [i=2 to n], if μ is obtained as the time-varying index value of primary information i , then the time-varying index value μ of the primary information at each evaluation shooting time is i [i=2~n] and each evaluation shooting time t i The time-varying index value tx of the primary information i As a generalized relationship, e [(μi)·(txi)] =X holds true. That is, the doubling time tx is specified. i =lnX / μ i =(lnX)·(t i -t1) / ln(a i / a1). The aforementioned shooting time t k The specified doubling time is tx = (lnX)·(t k -t1) / ln2, the aforementioned doubling time is the prescribed doubling time when X=2.

[0130] In the judgment data generation process (S12), the time-varying index value obtained above can be set as judgment data. Similarly, the time-varying index value can also be calculated based on the respective selections of the number of cells, cell density, cell occupied area, cell monomer area, cell distance, etc. as 1-time information, and the respective selections of the sum, mean value, variance, and deviation as statistical index values. The time-varying index value of the first example can be said to be a representative value of the time-varying statistical index value representing 1-time information. Multiple time-varying index values ​​can be used as multiple judgment data of the original number, and multiple time-varying index values ​​can be used as a time-varying index value through an average value, a maximum value, a minimum value, etc., and used as a judgment data.

[0131] (Determine the passage)

[0132] After the generation of the judgment data is completed in the judgment data generation step (S12), a judgment step (S13) is performed to determine whether the time for passage has been reached. In the judgment step (S13), the time for passage is determined based on the judgment data generated in the judgment data generation step (S12). In the first example, the judgment data is a time-varying index value of a statistical index value. The statistical index value quantitatively represents the state of cells on the culture surface of a specified observation site Op at a specified plurality of locations selected from the culture container. Therefore, in the first example, the time-varying index value, which is the sum of the statistical index values ​​at the site of the specified observation site Op, becomes a representative value that can accurately represent the room for cells to expand or proliferate in the culture container compared to the first embodiment.

[0133] In the determination step (S13), the determination data as the time-varying index value calculated in the time-varying calculation step (S1213) is compared with a predetermined threshold value. Here, when the determination data reaches the predetermined threshold value, it is determined that the time of passage has arrived. In the case where two shooting moments are selected in a manner that the first shooting moment is fixed as the reference shooting moment and the second shooting moment is sequentially updated, the predetermined threshold value is the same as in the first embodiment. As in the first embodiment, a test as a preliminary inspection is carried out under the same conditions as the actual determination of the time of passage, in which the statistical index value of the moment of passage is obtained, the time-varying index value is obtained, and it is demarcated as the predetermined threshold value. Even if the element of time-varying is added, the characteristics of the statistical index value in the determination of the time of passage will not disappear, as described in the first embodiment. That is, at the time of passage, a certain inflection point represented by an extreme value such as a minimum value and a maximum value appears in the time change of the time-varying index value. The moment when such an inflection point of time change appears is set as the threshold value for determining the moment of passage arrival.

[0134] On the other hand, when the first imaging time and the second imaging time are selected as consecutive imaging times for two imaging times, the predetermined threshold value is as follows.

[0135] When the information of the first time at the second shooting time increases and the statistical index value also increases, the information of the first time at the first shooting time also increases and the statistical index value also increases. In the initial stage of cell expansion or proliferation, the increase in the statistical index value at the second shooting time is greater than the increase in the statistical index value at the first shooting time, but as the cells expand or proliferate, the respective increases gradually decrease, and at the stage when the time for cell passage is reached, the increase in the statistical index value at the second shooting time is smaller than the increase in the statistical index value at the first shooting time. Therefore, at the stage when the time for cell passage is reached, the statistical index value decreases at an extreme value at an arbitrary threshold. The extreme value is set as a specified threshold. The extreme value is defined by a test that is pre-implemented as a test under the same conditions as the actual timing of determining the passage. In this test, the time-varying index value is obtained based on the statistical index value at the time of passage, and when the time-varying index value takes an extreme value, the extreme value is set as a specified threshold. Furthermore, when the time-varying index value reaches a predetermined threshold, the time for subculturing has been reached, so it can be said that a moment slightly earlier than this moment is the most suitable time for subculturing. Therefore, the predetermined threshold value can be set to "the value obtained by subtracting the margin value α from the extreme value of the time-varying index value ("extreme value of the time-varying index value" - "margin value α")". Thus, it is possible to determine whether cells that have not been observed to slow down in cell extension or proliferation have reached the time for subculturing in a good state at a moment very close to the time for subculturing. The "margin value α" is also set by a test that is a pre-implemented test.

[0136] When the number of determination data is set to 1, a comparison is made to see whether the 1 determination data reaches a predetermined threshold value, and when the predetermined threshold value is reached, it is determined that the time for subculturing has arrived. When the number of determination data is set to plural, a comparison is made to see whether a predetermined number of determination data among the plural determination data reaches a predetermined threshold value, and when the predetermined threshold value is reached, it can be determined that the time for subculturing has arrived.

[0137] Furthermore, when all the determination data in the plurality of determination data reach a predetermined threshold, it may be determined that the time for subculturing has arrived. In this case, since it takes time for all the determination data to reach the predetermined threshold, it will take a long time to determine whether the time for subculturing has arrived.

[0138] On the contrary, it is also possible to determine that the time for subculturing has arrived when at least one of the plurality of determination data reaches a predetermined threshold. In this case, since determination can be made as long as one determination data reaches the predetermined threshold, it is possible to determine whether the time for subculturing has arrived as quickly as possible.

[0139] <Example 2>

[0140] Next, the second example is described. In the first example, after the first information is acquired at each shooting moment in the first information acquisition step (S1211), the statistical index value at each shooting moment is calculated based on the first information at each shooting moment in the statistical index value acquisition step (S1212), and then the temporal change index value is calculated in the temporal change calculation step (S1213) and set as the judgment data. However, in the second example, Figure 3B As shown in the figure, after the primary information of each shooting time is acquired in the primary information acquisition step (S1211), the time change calculation step (S1213) is executed to calculate the time change index value of the primary information, and then the statistical index value of the time change index value is calculated in the statistical index value acquisition step (S1212) and set as the judgment data, which is different from the first example. The primary information and the primary information acquisition step (S1211) are the same as those in the first embodiment and the first example, so the description is omitted.

[0141] (Time-dependent change calculation step (obtaining time-dependent change index value))

[0142] Next, in the temporal change calculation process (S1213), the temporal change index value of each primary information is calculated at each shooting moment. The temporal change index value is the amount of change of the primary information between each shooting moment. The temporal change index value in the second example is the same as that in the first example, which is a differential change amount, a change amount rate, a unit time change rate, a change speed or a change time. In the first example, it is a differential change amount, a change amount rate, a unit time change rate, a change speed or a change time of the statistical index value of the primary information, while in the second example, it is a differential change amount, a change amount rate, a unit time change rate, a change speed or a change time of the primary information, and the only difference is this point. Therefore, in the calculation of the temporal change index value of the aforementioned first example, the temporal change index value is calculated based on the statistical index value of the primary information, but it is sufficient to simply replace the "statistical index value of the primary information" with "primary information" and apply the first example to the second example. Therefore, in the second example, the detailed description of the calculation of the differential change amount, change amount rate, unit time change rate, change speed or change time of the primary information is omitted. In addition, in the second example, as in the first example, the change speed can be applied as the specific multiplication rate, and the change time can be applied as the doubling time.

[0143] Reference Figure 3F The time-dependent change index value of the second example will be described. Figure 3F This is a diagram showing the concept of the change of the primary information (vertical axis) with respect to time (horizontal axis) for the first and second examples. In the second example, the statistical index values ​​in the description of the temporal change calculation step (S1213) of the first example, i.e., a1 and a2 as temporal change index values, are replaced with the values ​​of the primary information acquired in the primary information acquisition step (S1211) of the second example. Here, regarding the temporal change index values, the temporal change index values ​​corresponding to the number of the primary information are obtained. The temporal change index values ​​of the second example can be said to be values ​​that directly represent the temporal change tendency of the primary information.

[0144] (Get statistical indicator values)

[0145] Here, if Figure 3B As shown, after obtaining the time-varying index value of the 1-time information at the first shooting moment and the second shooting moment in the time-varying calculation process (S1213), the statistical index value of the time-varying index value is calculated in the statistical index value acquisition process (S1212). The statistical index value in the second example is an index value limited to a representative value of the time-varying index value representatively representing the 1-time information, for example, at least any one of the deviations such as the mean value, variance, and standard deviation. In the second example, the statistical index value of the time-varying index value is calculated, so it is the mean value, variance, deviation, etc. of the time-varying index value of the 1-time information of each of the multiple observation parts Op. The statistical index value obtained here is set as the judgment data, and the timing of the generation is judged. Multiple statistical index values ​​can be used as multiple judgment data of the original number, or multiple statistical index values ​​can be used as one statistical index value through the average value, maximum value, minimum value, etc., and used as one judgment data.

[0146] (Determine the passage)

[0147] In the judgment data generation step (S12), after the generation of the judgment data is completed, a judgment step (S13) is performed to judge whether the timing of subculturing has arrived. In the judgment step (S13), the timing of subculturing is judged based on the statistical index value generated in the judgment data generation step (S12). The judgment data of the second example is a statistical index value of the time-varying index value, and thus, like the judgment data of the time-varying index value as the statistical index value in the first example, it includes the elements of primary information, time-varying index value, and statistical index value. Therefore, the judgment data as the judgment of the timing of subculturing is unchanged from the first example and is effective in judging the timing of subculturing.

[0148] In the determination step (S13), the statistical index value calculated in the statistical index value acquisition step (S1202) is compared with a predetermined threshold value. The predetermined threshold value is the same as that in the first embodiment. As in the first embodiment, a test is performed as a preliminary check under the same conditions as those for actually determining the timing of the passage, in which the statistical index value at the moment of the passage timing is obtained, the time-varying index value is obtained, and the index value is limited to the predetermined threshold value. Even if the element of the time-varying factor is added, the characteristics of the statistical index value in the determination of the timing of the passage will not disappear, as described in the first embodiment.

[0149] As in the first example, when one representative value is generated for the number of determination data, when the number of determination data is set to one, a comparison is made to see whether the one determination data reaches a predetermined threshold value, and when the predetermined threshold value is reached, it is determined that the time for subculturing has arrived. When the number of determination data is set to a plurality of determination data, a comparison is made to see whether a predetermined number of determination data among the plurality of determination data reaches a predetermined threshold value, and when the predetermined threshold value is reached, it can be determined that the time for subculturing has arrived.

[0150] Furthermore, when all the determination data in the plurality of determination data reach a predetermined threshold, it may be determined that the time for subculturing has arrived. In this case, since it takes time for all the determination data to reach the predetermined threshold, it will take a long time to determine whether the time for subculturing has arrived.

[0151] Conversely, it may be determined that the time for subculturing has arrived when at least one of the plurality of determination data reaches a predetermined threshold value. In this case, determination can be made as long as one determination data reaches the predetermined threshold value, so it is possible to determine whether the time for subculturing has arrived as quickly as possible.

[0152] [Third Embodiment]

[0153] Next, a method and system for determining the timing of subculture according to the third embodiment will be described. The third embodiment is an embodiment in which a portion of the first and second embodiments are modified. In the third embodiment, in the generation of determination data in the determination data generation step (S12), at least three shooting moments are selected as focus shooting moments from a plurality of shooting moments captured in the shooting step (S11), determination data is generated based on the cell images at the focus shooting moments, and the timing of subculture is determined, which is different from the first and second embodiments. In the third embodiment, a time element is also added to the determination data. The structure of the cell culture system 1 and the subculture timing determination unit 3 is the same as that of the first embodiment. Below, with respect to the third embodiment, the parts that are different from the first and second embodiments will be described, and the description of the parts that are the same as the first and second embodiments will be omitted. The subculture determination process of the third embodiment is also the same as that of the first and second embodiments. Figure 2B The generation determination process of the first embodiment shown is the same, and is composed of an imaging step (S11), a determination data generating step (S12), and a determination step (S13).

[0154] (Photographing process of culture surface)

[0155] In the third embodiment, Figure 2E As shown, in the imaging step (S11), cell images are captured at a plurality of imaging times, similarly to the first and second embodiments. At each imaging time, an observation site Op is selected at the same site so as to be a fixed-point observation.

[0156] (Generate judgment data)

[0157] Determine the data generation step (S12) and Figure 3A and Figure 3B In the third embodiment, the generation of the determination data in the determination data generation step (S12) has the same as the first example ( Figure 4A ), Example 2 ( Figure 4B ), Example 3 ( Figure 4C ). In the third embodiment, unlike the second embodiment, the process of calculating the time-varying index value is performed in two stages, namely, the first time-varying process (S1222) and the second time-varying process (S1222). Figure 4A) includes a primary information acquisition step (S1221) for acquiring primary information from a cell image, a statistical index value calculation step (S1222) for calculating a statistical index value based on the primary information, a primary temporal change calculation step (S1223) for calculating a primary temporal change index value of the primary information, and a secondary temporal change calculation step (S1224) for calculating a secondary temporal change index value based on the primary temporal change index value. In the second example ( Figure 4B ) also includes a primary information acquisition step (S1221) for acquiring primary information from a cell image, a primary time change calculation step (S1223) for calculating a primary time information change amount of the primary information, a secondary time change calculation step (S1224) for calculating a secondary time change index value based on the primary time change index value, and a statistical index value calculation step (S1222) for calculating a statistical index value of the secondary time change index value. Furthermore, in the third example ( Figure 4C ) includes a first information acquisition step (S1221) for acquiring first information from a cell image, a first time change calculation step (S1223) for calculating a first time information change amount of the first information, a statistical index value calculation step (S1222) for calculating a statistical index value of a first time change index value, and a second time change calculation step (S1224) for calculating a second time change index value based on the statistical index value of the first time change index value.

[0158] In the third embodiment, in any one of the first to third examples, three or more imaging times are set as imaging times of interest in determining the timing of passage, and the captured cell images are used. As in the second embodiment, the imaging time of interest is composed of the evaluation imaging time and the reference imaging time. When the imaging time of interest is set to three imaging times, for example, the imaging time at the time of passage is used. Figure 2E The cell images captured at the first shooting time, the second shooting time, and the third shooting time are captured. Hereinafter, as a representative example, an example of using cell images captured at three shooting times is described. The case where four or more shooting times are set as the shooting time of interest is the same as the present embodiment, but this will be specifically described as a fourth embodiment later.

[0159] In the third embodiment, the selection of the shooting time of interest is also the same as that of the second embodiment. In any one of the first and second examples, there are two selection methods, namely the most recent comparison method and the reference comparison method. In the most recent comparison method and the reference comparison method of the third embodiment, at least one of the evaluation shooting time and the reference shooting time is set to multiple at the shooting time of interest. First, the following is an explanation of the method of setting the reference shooting time to one moment and the evaluation shooting time to two moments. In this case, for the most recent comparison method, in a representative example, three consecutive shooting moments (the first shooting moment, the second shooting moment, and the third shooting moment) are selected from multiple shooting moments as the shooting moments of interest. Similar to the second embodiment, in the most recent comparison method, as new shooting moments are added, the first shooting moment, the second shooting moment, and the third shooting moment are updated to the latest shooting moments.

[0160] First, refer to Figure 4D The nearest comparison method according to the third embodiment will be described in detail. Figure 4D The concept of selecting the most recent comparison method's focus capture time (reference capture time and evaluation capture time) from any capture time (e.g., from capture time (n-3) to capture time (n+2)) is shown. For example, capture time (n-3) is set as the reference capture time, capture time (n-2) is set as the first evaluation capture time, and capture time (n-1) is set as the second evaluation capture time, and one reference capture time and two evaluation capture times are selected as the focus capture times to determine the timing of generation. Furthermore, if it is determined that the timing of generation has not yet arrived, the focus capture time is selected by setting capture time (n-2) as the reference capture time, setting capture time (n-1) as the first evaluation capture time, and setting capture time n as the second evaluation capture time to determine the timing of generation. After that, similarly, if the shooting time (n-1) is set as the reference shooting time, the shooting time n is set as the first evaluation shooting time and the shooting time n+1 is set as the second evaluation shooting time, and the timing of the generation is determined by repeating the method of updating while shifting the focus shooting time as the new shooting time arrives. In this case, similarly to the second embodiment, when updating while shifting the reference shooting time and the evaluation shooting time, the reference shooting time and the evaluation shooting time must be shifted regularly in order.

[0161] In recent comparative law, representative examples are Figure 4D As shown in FIG. 1 , the selection is performed in such a manner that there is no shooting time between the reference shooting time and the first evaluation shooting time. However, as in the second embodiment, Figure 4EAs shown, the imaging time of interest may be selected so as to set an arbitrary number of imaging times not used for determining the timing of passage between the reference imaging time and the first evaluation imaging time. Figure 4E This example is shown. In addition, in the recent comparison method, the shooting time of interest can also be set in a manner that there are multiple shooting times that are not used for evaluation between the reference shooting time and the first evaluation shooting time. Regardless of whether there are shooting times that are not used for evaluation between the reference shooting time and the first evaluation shooting time, the shooting time of interest can be set in a manner that there are multiple shooting times that are not used for evaluation between the first evaluation shooting time and the second evaluation shooting time. As long as the reference shooting time, the first evaluation shooting time, and the second evaluation shooting time are each separated by a certain interval, and the reference shooting time, the first evaluation shooting time, and the second evaluation shooting time are selected in sequence in the same mode, they can be used as the shooting time of interest in determining the timing of the generation. In addition, in the reference comparison method, the reference shooting time is fixed, and the evaluation shooting time is updated, so there is a shooting time between the reference shooting time and the evaluation shooting time. In this case, regardless of whether there is a shooting time that is not used for evaluation between the baseline shooting time, the first evaluation shooting time, and the second evaluation shooting time, when the baseline shooting time is updated while being staggered from the first evaluation shooting time and the second evaluation shooting time, the baseline shooting time must be staggered from the first evaluation shooting time and the second evaluation shooting time in a regular order.

[0162] Next, refer to Figure 4F The reference comparison method of the third embodiment will be described in detail. In the reference comparison method, the imaging time of interest consisting of the reference imaging time, the first evaluation imaging time, and the second evaluation imaging time is selected as follows. Figure 4FThe concept of selecting the photographing time of interest in the benchmark comparison method of selecting the photographing time of interest (reference photographing time and evaluation photographing time) from a plurality of photographing times from photographing time (n-3) to photographing time (n+2) is shown. In the benchmark comparison method, an initial arbitrary photographing time is determined as the reference photographing time and fixed from a plurality of photographing times, and an arbitrary photographing time after the reference photographing time is selected as the first evaluation photographing time and the second evaluation photographing time. Furthermore, when it is determined that the timing of passage has not yet arrived, the photographing time of interest (reference photographing time and evaluation photographing time) is determined in such a manner that the reference photographing time is not moved but only the first evaluation photographing time and the second evaluation photographing time are shifted. For example, when photographing time (n-3) is set as the reference photographing time, photographing time (n-3) is fixed as the reference photographing time, and the photographing time of interest is selected in such a manner that photographing time (n-2) is set as the first evaluation photographing time and photographing time (n-2) is set as the second evaluation photographing time to determine the timing of passage. Furthermore, after this determination, if it is determined that the timing of the passage has not yet arrived, the imaging time (n-2) is kept unchanged as the reference imaging time, and the imaging time (n-1) is set as the first evaluation imaging time and the imaging time (n-1) is set as the second evaluation imaging time to select the imaging time of interest to determine the timing of the passage. After that, similarly, the reference evaluation imaging time is always the imaging time (n-3), and the imaging time is increased as the timing of the new imaging time arrives, and the imaging time of interest is repeatedly updated while the first evaluation imaging time and the second evaluation imaging time are shifted to determine the passage until it is determined that the timing of the passage has arrived. That is, in the case of the reference comparison method, the reference imaging time is fixedly used in the determination of the timing of each passage, and each time a new imaging time is added, it is updated and selected in a manner that the first evaluation imaging time and the second evaluation imaging time are changed, and this process is repeated in the determination of the timing of each passage. In the case of the benchmark comparison method, as in the case of the most recent comparison method, when updating while shifting the benchmark shooting time from the first evaluation shooting time and the second evaluation shooting time, the benchmark shooting time must be shifted from the first evaluation shooting time and the second evaluation shooting time in a regular order.

[0163] In the case of the reference comparison method, the reference shooting time is fixed, so when a new shooting time arrives and a shooting time is added, the first evaluation shooting time is shifted, so that there must be an arbitrary number of shooting times that are not used for determining the timing of the transfer between the reference shooting time and the first evaluation shooting time. In this case, it can also be set so that there are also an arbitrary number of shooting times that are not used for determining the timing of the transfer between the first evaluation shooting time and the second evaluation shooting time. For example, Figure 4GAn example is shown in which the timing of passage is determined by selecting the photographing time of interest by fixing the photographing time (n-3) as the reference photographing time, setting the photographing time (n-2) as the first evaluation photographing time, and setting the photographing time (n) as the second evaluation photographing time. In this case, the photographing time (n-1) is not selected as the evaluation photographing time and is not used for the timing determination of passage. After this determination, if it is determined that the passage timing has not yet arrived, the photographing time of interest is selected by keeping the photographing time (n-3) as the reference photographing time, setting the photographing time (n-1) as the first evaluation photographing time, and setting the photographing time (n+1) as the second evaluation photographing time to determine the timing of passage. Afterwards, similarly, the reference evaluation photographing time is kept as the photographing time (n-3), and a photographing time is added as the timing of a new photographing time arrives. The photographing time of interest is repeatedly updated while the first evaluation photographing time and the second evaluation photographing time are staggered to determine the passage until the passage timing is determined to have arrived. In this case, as described above, when updating while shifting the reference imaging time from the first evaluation imaging time and the second evaluation imaging time, the reference imaging time must be shifted regularly from the first evaluation imaging time and the second evaluation imaging time in order.

[0164] In the nearest comparison method, the reference shooting time and the evaluation shooting time (the first evaluation shooting time and the second evaluation shooting time) move together, so the primary information of the reference shooting time and the evaluation shooting time are compared relative to each other. On the other hand, in the reference comparison method, the reference shooting time is fixed, so the primary information of the evaluation shooting time (moving with the passage of time) relative to the reference shooting time (fixed regardless of the passage of time) is absolutely compared. The time interval between each shooting time can be set arbitrarily, but in the method of selecting two consecutive shooting times as the nearest comparison method, the time interval between each shooting time is preferably set to be constant.

[0165] Hereinafter, regarding the determination data generation step (S12) of the third embodiment, the first to third examples are described separately. In any one of the first to third examples, the first information acquisition step (S1221) is the same as the first information acquisition step (S1211) of the second embodiment, and therefore the description thereof is omitted.

[0166] <Example 1>

[0167] (Get statistical indicator values)

[0168] In the first example, as mentioned above, Figure 3AAs shown, after the primary information of each shooting moment is obtained in the primary information acquisition process (S1221), the statistical index value of each shooting moment is calculated according to the primary information of each shooting moment in the statistical index value acquisition process (S1222). The statistical index value is an index value limited to a representative value that represents the primary information representatively, as in the first embodiment, such as at least one of the deviations such as the sum, the mean value, the variance, and the standard deviation. The sum is the sum of the number of cells of multiple observation sites, the sum of the occupied area of ​​the cells, the sum of the single area of ​​the cells, and the sum of the distances between cells. The mean value is the mean value of the number of cells of multiple observation sites, the mean value of the cell density, the mean value of the occupied area of ​​the number of cells, the mean value of the single area of ​​the cells, and the mean value of the distances between cells. The variance and deviation are the variance and deviation of the number of cells of multiple observation sites, the variance and deviation of the cell density, the variance and deviation of the occupied area of ​​the cells, the variance and deviation of the single area of ​​the cells, and the variance and deviation of the distances between cells. Here, the variance or deviation as the statistical index value is an index of the uniformity of the cells in the culture container, and thus the timing of subculturing can be determined more appropriately compared to the case where the sum or average value is selected as the statistical index value, which is the same as the first embodiment.

[0169] (Primary Temporal Change Calculation Step (Obtaining Primary Temporal Change Index Value))

[0170] Next, in the 1st time change calculation process (S1223), the 1st time change index value is calculated based on the statistical index value of each 1st information calculated at each shooting time. The 1st time change index value is the increment, that is, the change amount or change rate, of the statistical index value of each 1st information between each shooting time, that is, between the 1st shooting time and the 2nd shooting time and between the 2nd shooting time and the 3rd shooting time. Regarding the 1st time change index value, one 1st time change index value is generated for the values ​​of two 1st information, and as a whole, a 1st time change index value is generated that is one less than the statistical index value of the 1st information. The 1st time change index value is calculated as the number of which is one less than the number of observation parts Op or the number of representative values.

[0171] In this regard, refer to Figure 4H and Fig. 4I Provide explanation. Figure 4H and Fig. 4I This is a diagram showing the concept of changes in values ​​(vertical axis) with respect to time (horizontal axis) for the first and second examples. The vertical axis of the first example is the statistical index value, and the vertical axis of the second example described later is the value of primary information. Figure 4H and Fig. 4IThis is shown as an example of the nearest comparison method of three consecutive shooting times (the first shooting time, the second shooting time, and the third shooting time). In the case of the aforementioned reference comparison method in which the first shooting time, the second shooting time, and the third shooting time are not selected as consecutive shooting times, Figure 4H and Fig. 4I , it is sufficient to replace it with an unillustrated shooting time between the first shooting time, the second shooting time, and the third shooting time. The characteristics of the tendency of increase of the single time-varying index value by the reference comparison method and the latest comparison method are the same as those of the second embodiment.

[0172] In calculating one time-varying index value, two imaging times (the first imaging time and the second imaging time) are selected from a plurality of imaging times. Figure 4H and Fig. 4I , an example is shown in which the two latest consecutive shooting times are selected as the first shooting time (t1), the second shooting time (t2), and the third shooting time (t3). Figure 4H and Fig. 4I , the statistical index value (a1) at the first imaging time (t1) is increased to the statistical index value (a2) at each of the second imaging time (t2) and the third imaging time (t3). At the imaging time, it is determined whether the timing of the passage has arrived. If the timing of the passage has not arrived, the latest three consecutive imaging times including the new imaging time are selected again to generate the determination data.

[0173] In the first example, the calculation of one time-varying index value focuses on the shooting moments between the three shooting moments selected for determination, that is, the two shooting moments selected from the three shooting moments to calculate one time-varying index value. That is, two groups of shooting moments consisting of two shooting moments are extracted from the three shooting moments, and the change in the value between them is calculated as one time-varying index value. In the first example, for example, two shooting moments (the first shooting moment and the second shooting moment) are selected as the first group and two shooting moments (the second shooting moment and the third shooting moment) are selected as the second group, and one time-varying index value between them is calculated. In this specification, the two shooting moments extracted from the shooting moments used for determination are referred to as the shooting moments of interest. In the second embodiment, the combination of the shooting moments of interest is one group, so only one time-varying index value is obtained. However, in this embodiment, one single temporal change index value is obtained from two photographing moments of interest, and thus one single temporal change index value is obtained from each of two groups of photographing moments of interest consisting of two photographing moments, and two single temporal change index values ​​are obtained in total. That is, when there are three photographing moments selected for determination, there are two groups of combinations of photographing moments of interest extracted therefrom, and the number of single temporal change index values ​​is two.

[0174] The method of calculating the one-time temporal variation index value between the two photographing moments of interest is the same as that of the second embodiment. The one-time temporal variation index value can also be set as the difference between the statistical index value of the one-time information of the evaluation photographing moment at the photographing moment of interest and the statistical index value of the one-time information of the photographing moment serving as the reference. Figure 4H As shown in FIG. 1 , the single time-varying index value can be set as the difference amount of the statistical index value of the single information between the first shooting time (reference shooting time) and the second shooting time (evaluation shooting time) and between the second shooting time (reference shooting time) and the third shooting time (evaluation shooting time), which are the combination of the shooting time of interest, that is, the difference change amount. In the time-varying index value of the difference change amount, the difference amount of the statistical index value of the single information at the second shooting time relative to the statistical index value of the single information at the first shooting time, that is, the difference change amount (a2-a1) and (a3-a2) can be set.

[0175] In addition, the one-time temporal change index value can also be set as the proportional change amount of the statistical index value of the one-time information at the evaluation shooting time relative to the statistical index value of the one-time information at the shooting time that serves as the reference among the shooting times of interest. Among the one-time temporal change index values ​​of the proportional change amount, the change rate a2 / a1 of the statistical index value of the one-time information at the second shooting time (evaluation shooting time) relative to the statistical index value of the one-time information at the first shooting time (reference shooting time) and the change rate a3 / a2 of the statistical index value of the one-time information at the third shooting time (evaluation shooting time) relative to the statistical index value of the one-time information at the second shooting time (reference shooting time) are used. These are the values ​​of the ratio of the sum of the number of cells at the second shooting time to the sum of the number of cells at the first shooting time, and the ratio of the sum of the number of cells at the third shooting time to the sum of the number of cells at the second shooting time, respectively.

[0176] In addition, the one-time temporal variation index value can also be set as the difference between the statistical index value of the one-time information of the photographing time at the photographing time of interest and the statistical index value of the one-time information of the photographing time serving as the reference, relative to the difference proportional variation of the statistical index value of the one-time information of the photographing time serving as the reference. In the one-time temporal variation index value of the difference proportional variation, it can be expressed as the ratio of the increase between the statistical index value of the one-time information of the first photographing time and the statistical index value of the one-time information of the second photographing time to the statistical index value of the one-time information of the first photographing time, i.e. (a2-a1) / a1, and the ratio of the increase between the statistical index value of the one-time information of the second photographing time and the statistical index value of the one-time information of the third photographing time to the statistical index value of the one-time information of the second photographing time, i.e. (a3-a2) / a2. For example, in the example where the information is set as the number of cells, it refers to the value of the ratio of the increase in the total number of cells from the first shooting time to the second shooting time to the total number of cells at the first shooting time, and the ratio of the increase in the total number of cells from the second shooting time to the third shooting time to the total number of cells at the second shooting time.

[0177] Furthermore, if Fig. 4IAs shown, the single temporal variation index value can also be set as the difference variation, ratio variation, and difference ratio variation of the statistical index value of the single information at the reference shooting time and the statistical index value of the single information at the evaluation shooting time relative to the time interval of the shooting time of interest. That is, the single temporal variation index value can also be set as the difference variation, ratio variation, and difference ratio variation of the statistical index value of the single information at the first shooting time (reference shooting time) and the statistical index value of the single information at the second shooting time (evaluation shooting time) and the statistical index value of the single information at the second shooting time (reference shooting time) and the statistical index value of the single information at the third shooting time (evaluation shooting time) relative to the time intervals of the two groups of shooting times (the first shooting time and the second shooting time and the second shooting time and the third shooting time). For the first shooting time (reference evaluation time) and the second shooting time (evaluation shooting time), the time-varying index value can be expressed as the unit time change rate of the differential change (a2-a1) / (t2-t1), the unit time change rate of the proportional change (a2 / [a1·(t2-t1)]), and the unit time change rate of the differential proportional change (a2-a1) / [a1·(t2-t1)]). For the second shooting time (reference evaluation time) and the third shooting time (evaluation shooting time), the time-varying index value can be expressed as the unit time change rate of the differential change (a3-a2) / (t3-t2), the unit time change rate of the proportional change (a3 / [a2·(t3-t2)]), and the unit time change rate of the differential proportional change (a3-a2) / [a2·(t3-t2)]). These one-time time-varying index values ​​all refer to the rate of increase per unit time of the statistical index value of the one-time information at the first shooting moment (reference shooting moment) and the second shooting moment (evaluation shooting moment), and the rate of increase per unit time of the statistical index value of the one-time information at the second shooting moment (reference shooting moment) and the third shooting moment (evaluation shooting moment), and are also the rate of increase. In the example where the one-time information is set to the number of cells, it is the rate of increase per unit time of the number of cells, that is, the rate of increase (proliferation rate). The rate of increase (proliferation rate) mentioned here is a simple speed of the difference value obtained by linearly approximating the statistical index values ​​of the two shooting moments.

[0178] As in the second embodiment, as long as the statistical index value of the primary information changes over time, the increase rate (proliferation rate) can be defined according to a wide variety of definitions. Figure 4JAs shown, similarly to the second embodiment, the rate of increase (proliferation rate) of the index value of one time-varying change can also be set to a specific proliferation rate instead of a simple rate limited by a differential amount relative to time. The definition of the specific proliferation rate is the same as that of the second embodiment. Therefore, the description in the second embodiment is applied. To reconfirm the description, it is as follows. Here, the reference time in the calculation of the index value of one time-varying change is set as the reference shooting time and is set to the first shooting time t1, and each evaluation shooting time, that is, the shooting time t i The statistical index value a of the 1st information i The relationship between [i=2 to n] and the statistical index value a1 of the primary information at the first imaging time t1 can be approximated as follows: i / a1=e [(μi)·(ti-t1)] Here, μ i is the shooting time t i [i=2~n] specific growth rate, μ i =[ln(a i / a1)] / (t i -t1).

[0179] Here, when the shooting time t i [2<i<n] where i=k1[2<k1<n] and the statistical index value of the 1st information becomes a k1 When the specific growth rate becomes μ k1 =[ln(a k1 / a1)] / (t k1 -t1). In addition, when the shooting time is t i When i=k2[k1<k2<n] in [i<n], the statistical index value of the information at the time of evaluation shooting is a k2 When the specific growth rate becomes μ k2 =[ln(a k2 / a k1 )] / (t k2 -t k1 ). These specific growth rates μ k2 , μ k1 The second time-varying index value described later is calculated based on the two specific growth rates μ calculated from the values ​​at two moments selected from among the three moments. k2 , μ k1 to calculate.

[0180] Furthermore, similarly to the second embodiment, the doubling time can be used as a primary temporal change index value. iThe statistical index value a of the primary information when i=k1[2<k1<n] in [2<i<n] k1 becomes twice the statistical index value a1 of the first imaging time t1 which is the reference imaging time (a k1 =2a1). At this time, from the first imaging time t1 as the reference imaging time to the k1th imaging time t k1 The time [2<k1<n] is called the doubling time t ad1 (=t k1 -t1). In addition, for reference, at this time a k1 / a1=e (μk1·tad1) =2 holds true, so the doubling time t ad1 The specific growth rate is especially μ k1 =ln2 / t ad1 =ln2 / (t k1 -t1).

[0181] Also, assuming that the shooting time is t i The statistical index value a of the primary information of the evaluation shooting time when i=k2[k1<k2<n] in [i<n] k2 The k1th shooting time t k1 The k1th imaging time t is set as the reference imaging time k1 The statistical index value a k1 2 times (a k2 =2a k1 ). At this time, from the k1th shooting time t as the reference shooting time k1 Until the k2th shooting time t as the evaluation shooting time k2 The time is called the doubling time t ad2 (=t k2 -t k1 ). Here, also for reference, a k2 / a k1 =e (μk2·tad2) =2 holds true, so the doubling time t ad2 The specific growth rate is especially μ k2 =2 / (ln2·t ad2 )=ln2 / (t k2 -t k1 The second temporal variation index value described later as the doubling time is calculated from the two doubling times t calculated from the values ​​at the two time points selected from the three time points. ad1 (=t k1 -t1) and doubling time t ad2 (=t k1 -t1) to find the value.

[0182] Furthermore, similarly to the second embodiment, in addition to the doubling time, a predetermined doubling time (txi) required until the amount at the evaluation time becomes a predetermined multiple of the amount at the reference time can be used as the increase rate (proliferation rate). i The statistical index value a of the primary information when i=k1[2<k1<n] in [2<i<n] k1 The statistical index value a1 of the primary information at the photographing time t1 as the reference time is a predetermined multiplication factor (X times), and the statistical index value a1 of the primary information at the photographing time t1 as the reference time is i=k2[k1<k2<n] k2 The shooting time t is the reference time. k1 The statistical index value a of the 1st information k1 The specified multiplier (X times) is equal to e μi , e [(μi)·(txi)] =X is established, and the specified doubling time tx is obtained when i = k1 and i = k2 respectively. k1 =lnX / μ k1 =(lnX)·(t k1 -t1) / ln(a k1 / a1) and tx k2 =lnX / μ k2 =(lnX)·(t k2 -t k1 ) / ln(a k2 / a k1 ). As in the second embodiment, the doubling time is the predetermined doubling time when X = 2. The secondary temporal variation index values ​​described later using these predetermined doubling times are calculated based on the two predetermined doubling times calculated from the values ​​at two moments selected from among the three moments.

[0183] (Second time change calculation step (first time acquisition of time change index value))

[0184] Next, in the second time-varying change calculation step (S1224), the second time-varying change index value is calculated based on the first time-varying change index value. The second time-varying change index value can be respectively applied as the differential change amount, proportional change amount, and unit time change rate per unit time of the index of the increment of the first time-varying change index value. That is, the differential change amount or proportional change amount of the first time-varying change index value (pre-change value) from the first shooting time to the second shooting time and the first time-varying change index value (post-change value) from the second shooting time to the third shooting time can be applied.

[0185] When the difference change amount is applied to two time-varying index values, for example, Figure 4HAs shown, when the differential change of the statistical indicator value is selected as one time-varying value, and the differential change of the statistical indicator value a2-a1 and a3-a2 are respectively obtained for one time-varying indicator value from the first shooting moment to the second shooting moment (value before change) and one time-varying indicator value from the second shooting moment to the third shooting moment (value after change), the two time-varying indicator values ​​can be set to the differential change obtained by subtracting the value before change from the value after change, (a3-a2)-(a2-a1)=(a3-2a2+a1).

[0186] In addition, in the case of applying the differential change amount to two time-varying index values, when the proportional change amount of the statistical index value is selected as the first time-varying value, and the proportional change amounts a2 / a1 and a3 / a2 of the statistical index value are obtained for the first time-varying index value from the first shooting moment to the second shooting moment (value before change) and the first time-varying index value from the second shooting moment to the third shooting moment (value after change), the two time-varying index values ​​can be set to the differential change amounts of these proportional change amounts, i.e., (a3 / a2)-(a2 / a1).

[0187] When applying the differential change amount to the secondary time-varying index value, when selecting the differential proportional change amount of the statistical index value as the primary time-varying value and obtaining (a2-a1) / a1 and (a3-a2) / a2, the secondary time-varying index value can be set to the differential change amount of these proportional change amounts, i.e. [(a3-a2) / a2-(a2-a1) / a1].

[0188] Furthermore, when the differential change amount is applied to the two time-varying index values, the unit time change rate of the differential change amount is selected as the first time-varying index value, and (a2-a1) / (t2-t1) and (a3-a2) / (t3-t2) are obtained, as shown in Fig. 4I As shown, the two time-varying index values ​​can be respectively set to the difference change amounts of these unit time change rates, that is, [(a3-a2) / (t3-t2)]-[(a2-a1) / (t2-t1)].

[0189] In addition, when the differential change amount is applied to the secondary time-varying index value, when the rate of change per unit time of the proportional change amount is selected as the primary time-varying index value, a3 / [a2·(t3-t2)], a2 / [a1·(t2-t1)] are obtained, as shown in Fig. 4I As shown, the two time-varying index values ​​can be respectively set to the difference change amount of these unit time change rates, that is, a3 / [a2·(t3-t2)]-a2 / [a1·(t2-t1)].

[0190] When the differential change amount is applied to the secondary time-varying index value, the rate of change per unit time of the differential proportional change amount is selected as the primary time-varying index value, and (a2-a1) / [a1·(t2-t1)], (a3-a2) / [a2·(t3-t2)] are obtained, as follows Fig. 4I As shown, the two time-varying index values ​​can also be respectively set to the differential changes of these unit time change rates, that is, (a3-a2) / [a2·(t3-t2)]-(a2-a1) / [a1·(t2-t1)].

[0191] When the differential change amount is applied to the secondary time-varying index value, when the specific growth rate is selected as the primary time-varying index value, as Figure 4J As shown in FIG. 1 , the second time-varying index value is expressed as a differential change amount (μ k2 -μ k1 )=[ln(a k2 / a k1 )] / (t k2 -t k1 )-[ln(a k1 / a1)] / (t k1 -t1).

[0192] When the differential change amount is applied to the second time-varying index value, when the doubling time is selected as the first time-varying index value, as Figure 4J As shown in FIG. 1 , the second time-varying index value is expressed as a differential change amount. ad2 -t ad1 =(t k2 -t k1 )-(t k1 -t1)=t k2 -t1.

[0193] In addition, in the case of applying the proportional change amount to the two time-varying index values, when the differential change amount of the differential value of the statistical index value is selected as the first time-varying index value and (a2-a1) and (a3-a2) are obtained, the ratio of the first time-varying index value from the second shooting time to the third shooting time (value after change) to the first time-varying index value from the first shooting time to the second shooting time (value before change) can be set to (a3-a2) / (a2-a1).

[0194] In the case of applying a proportional change amount to two time-varying index values, when the proportional change amount of the statistical index value is selected as a single time-varying index value and (a2 / a1) and (a3 / a2) are obtained, the ratio of the single time-varying index value (value after change) from the second shooting time to the third shooting time to the single time-varying index value (value before change) from the first shooting time to the second shooting time can be set to (a3 / a2)÷(a2 / a1)=(a1·a3) / a2 2 .

[0195] In the case of applying proportional change to two time-varying index values, when selecting the differential proportional change of the statistical index value as one time-varying index value and obtaining (a2-a1) / a1, (a3-a2) / a2, the ratio of the one time-varying index value (value after change) from the second shooting moment to the third shooting moment to the one time-varying index value (value before change) from the first shooting moment to the second shooting moment can be set to (a3-a2) / a2÷(a2-a1) / a1, =[a1·(a3-a2)] / [a2·(a2-a1)].

[0196] When applying the proportional change amount to the secondary time-varying index value, when selecting the unit time change rate of the differential change amount as the primary time-varying index value and obtaining (a3-a2) / (t3-t2) and (a2-a1) / (t2-t1), it can be set to [(a3-a2) / (t3-t2)] / [(a2-a1) / (t2-t1)]=[(a3-a2)·(t2-t1)] / [(a2-a1) / (t3-t2)].

[0197] When the proportional change amount is applied to the secondary time-varying index value, when the unit time change rate of the proportional change amount is selected as the primary time-varying index value and a2 / [a1·(t2-t1)] and a3 / [a2·(t3-t2)] are obtained, the secondary time-varying index value can be set to [(a3-a2) / (t3-t2)]÷[(a2-a1) / (t2-t1)]=[(a3-a2)·(t2-t1) / (t3-t2)·(a2-a1)].

[0198] When the proportional change amount is applied to the secondary time-varying index value, when the rate of change per unit time of the differential proportional change amount is selected as the primary time-varying index value and (a2-a1) / [a1·(t2-t1)] and (a3-a2) / [a2·(t3-t2)] are obtained, the secondary time-varying index value can also be set to (a3-a2) / [a2·(t3-t2)]÷(a2-a1) / [a1·(t2-t1)].

[0199] When the proportional change amount is applied to the secondary time-varying index value, when the specific growth rate is selected as the primary time-varying index value, as Figure 4J As shown in FIG. 1 , the second time-varying index value is expressed as μ as a proportional change. k2 / μ k1 =(t k1 -t1)·[ln(a k2 / a k1 )] / (t k2 -t k1 )·[ln(a k1 / a1)] / .

[0200] When the proportional change amount is applied to the secondary time-varying index value, when the doubling time is selected as the primary time-varying index value, as Figure 4J As shown in FIG. 1 , the second time-varying index value is expressed as a differential change amount. ad2 / t ad1 =(t k2 -t k1 ) / (t k1 -t1).

[0201] Similarly, when applying the differential proportional change amount to the secondary time-varying index value, when selecting the differential change amount of the differential value of the statistical index value as the primary time-varying index value and obtaining (a2-a1) and (a3-a2), the secondary time-varying index value can be set to [(a3-a2)-(a2-a1)] / (a2-a1)=(a3-2a2+a1) / (a2-a1).

[0202] When applying the differential proportional change amount to the secondary time-varying index value, when selecting the proportional change amount of the statistical index value as the primary time-varying index value and obtaining (a2 / a1) and (a3 / a2), the secondary time-varying index value can be set to [(a3 / a2)-(a2 / a1)] / (a2 / a1).

[0203] When the differential proportional change amount is applied to the secondary time-varying index value, when the differential proportional change amount of the statistical index value is selected as the primary time-varying index value and (a2-a1) / a1 and (a3-a2) / a2 are obtained, the secondary time-varying index value can be set to {[(a3-a2) / a2]-[(a2-a1) / a1]} / (a2-a1) / a1.

[0204] When applying the differential proportional change amount to the secondary time-varying index value, the unit time change rate of the differential change amount is selected as the primary time-varying index value. When obtaining (a3-a2) / (t3-t2) and (a2-a1) / (t2-t1), it can be set to {[(a3-a2) / (t3-t2)]-[(a2-a1) / (t2-t1)]} / (a2-a1) / (t2-t1)={[(a3-a2) / (t3-t2)]-[(a2-a1) / (t2-t1)]}·(t2-t1) / (a2-a1).

[0205] When the differential proportional change amount is applied to the secondary time-varying index value, when the unit time change rate of the proportional change amount is selected as the primary time-varying index value and a2 / [a1·(t2-t1)] and a3 / [a2·(t3-t2)] are obtained, the secondary time-varying index value can be set to {a3 / [a2·(t3-t2)]-a2 / [a1·(t2-t1)]} / a2 / [a1·(t2-t1)]={a3 / [a2·(t3-t2)]-a2 / [a1·(t2-t1)]}·[a1·(t2-t1)] / a2.

[0206] When applying the differential proportional change amount to the secondary time-varying index value, when selecting the rate of change of the differential proportional change amount per unit time as the primary time-varying index value and obtaining (a2-a1) / [a1·(t2-t1)] and (a3-a2) / [a2·(t3-t2)], the secondary time-varying index value can also be set as the differential change amount of these unit time change rates, namely {[(a3-a2) / [a2·(t3-t2)]-[(a2-a1) / [a1·(t2-t1)]} / {(a2-a1) / [a1·(t2-t1)}.

[0207] When the differential proportional change amount is applied to the secondary time-varying index value, when the specific growth rate is selected as the primary time-varying index value, as Figure 4J As shown in FIG. 1 , the second time-varying index value is expressed as a differential change amount (μ k2 -μ k1 ) / μ k1 = = {[ln(a k2 / a k1 )] / (t k2 -t k1 )-[ln(a k1 / a1)] / (t k1 -t1)} / [ln(a k1 / a1)] / (t k1 -t1)}.

[0208] When the differential proportional change amount is applied to the secondary time-varying index value, when the doubling time is selected as the primary time-varying index value, as Figure 4J As shown in FIG. 1 , the second time-varying index value is expressed as a differential change amount (t ad2 -t ad1 ) / t ad1 =[(t k2 -t k1 )-(t k1 -t1)] / (t k1 -t1)=(t k2 -t1) / (t k1 -t1).

[0209] Furthermore, when the specific growth rate and doubling time are selected as the primary time-varying index value, the unit time change rate can also be applied as the secondary time-varying index value. In this case, as in the above, only the secondary time-varying index value needs to be calculated, so the description is omitted here.

[0210] One second time-varying index value is generated for two first time-varying index values, so as a whole, a second time-varying index value is generated that is one less than the number of the first time-varying index value, and a second time-varying index value that is two less than the statistical index value of the first information is generated. Obtain a number of second time-varying index values ​​that is two less than the number of observation parts Op or the number after representative value conversion. The above ends the judgment data generation step (S12), and the second time-varying index value obtained above is set as judgment data. Multiple second time-varying index values ​​can be used as multiple judgment data of the original number, or multiple second time-varying index values ​​can be used as one time-varying index value through an average value, a maximum value, a minimum value, etc., and used as one judgment data.

[0211] Although the 1st time-varying index value is used, the 2nd time-varying index values ​​in the 1st example are the information of the time-varying elements of the 1st information, and quantitatively represent the state of the cells of the culture surface of the specified observation site Op of the specified multiple locations selected from the culture container. The 1st example is the time-varying index value as the sum of the statistical index values ​​of the position of the specified observation site Op, which is a representative value that can accurately represent the room for cells to expand or proliferate in the culture container. The time-varying index value can be calculated according to the respective selections of the number of cells, cell density, cell occupied area, cell monomer area, cell distance, etc. as the 1st information and the respective selections of the sum, mean value, variance, and deviation as the statistical index value. When the sum or mean value is selected as the statistical index value, the judgment data of the 1st example refers to the representative value of the acceleration of the change of the representative value of the 1st information, and when the variance and deviation are selected as the statistical index value, the judgment data of the 1st example refers to the acceleration of the change of the deviation of the 1st information.

[0212] (Determine the passage)

[0213] After the generation of the judgment data is completed in the judgment data generation step (S12), a judgment step (S13) is performed to determine whether the time for passage has been reached. In the judgment step (S13), the time for passage is determined based on the judgment data generated in the judgment data generation step (S12). In the judgment step (S13), the judgment data of the two time-varying index values ​​calculated in the time-varying calculation step (S1213) are compared with a prescribed threshold value. Here, when the judgment data reaches the prescribed threshold value, it is determined that the time for passage has been reached, which is also the same as the second embodiment. In the first example of the third embodiment, a certain inflection point represented by extreme values ​​such as a minimum value and a maximum value will also appear in the time change of the two time-varying index values ​​at the time of passage. The moment when such an inflection point of the time change appears is set as the moment when the time for passage has been reached as the threshold.

[0214] Similar to the second embodiment, when the number of determination data is set to 1, a comparison is made to see whether the 1 determination data reaches a predetermined threshold value, and when the predetermined threshold value is reached, it is determined that the time for subculturing has arrived. When the number of determination data is set to plural, a comparison is made to see whether a predetermined number of determination data among the plural determination data reaches a predetermined threshold value, and when the predetermined threshold value is reached, it can be determined that the time for subculturing has arrived.

[0215] Furthermore, it is also possible to determine that the time for subculturing has arrived when all the determination data among the plurality of determination data reach a predetermined threshold value. In this case, it will be relatively slow to determine whether the time for subculturing has arrived. Conversely, it is possible to determine that the time for subculturing has arrived when at least one determination data among the plurality of determination data reaches a predetermined threshold value. In this case, it is possible to determine whether the time for subculturing has arrived as quickly as possible.

[0216] In the case of the reference comparison method, the predetermined threshold value is the same as that in the first and second embodiments. As in the first embodiment, a test is performed as a preliminary check under the same conditions as those for actually determining the timing of passage, in which the first and second time-varying index values ​​of the statistical index values ​​at the moment when the extension or proliferation of cells no longer occurs and becomes the timing of passage are obtained and set as the predetermined threshold value. Even if the element of time-varying is added, the characteristics of the statistical index value in determining the timing of passage do not disappear, and the method described in the first and second embodiments is the same.

[0217] In addition, regarding the three shooting moments, in the case of the nearest comparison method selected as three consecutive shooting moments, the prescribed threshold value is as follows. As mentioned above, the 2nd time-varying index value is the increment of the 1st time-varying index value. As described in the second embodiment, the 1st time-varying index value is the increment of the statistical index value of the 1st information, so the 1st time-varying index value gradually increases in the initial state, and increases in a manner that the cell stretches or proliferates sharply at a certain moment. Moreover, after that, the increase rate gradually decreases, and when the time of passage is reached, it takes a maximum value and has a tendency to turn into a decrease. Therefore, the 2nd time-varying index value gradually increases with the increase of the tendency of the increase of the 1st time-varying index value in the initial state, takes a positive maximum value, and gradually decreases with the decay of the 2nd time-varying index value. Moreover, when the maximum value of the 1st time-varying index value at the time of passage is reached, the 2nd time-varying index value takes a negative minimum value on the contrary and has a tendency to turn into an increase. Therefore, the minimum value is set as the prescribed threshold value. In addition, similarly to the second embodiment, when the 2nd time-varying index value reaches the prescribed threshold value, the time for subculture has been reached, so it can be said that the time slightly earlier than this time is the most suitable time for subculture. Therefore, the prescribed threshold value can be set to "the value obtained by adding the minimum value of the 2nd time-varying index value to the margin value α ("the minimum value of the 2nd time-varying index value" + "margin value α")". Thus, it is possible to determine whether cells that have not been observed to slow down in cell extension or proliferation have reached the time for subculture in a good state at a time very close to the time for subculture. Similar to the second embodiment, the "margin value α" is also set by a test as a pre-implemented test.

[0218] <Example 2>

[0219] Next, refer to Figure 4B The second example is described below. Figure 4A ), after acquiring the first information at each shooting moment in the first information acquisition step (S1221), the statistical index value of each shooting moment is calculated in the statistical index value acquisition step (S1222) based on the first information at each shooting moment. Then, after calculating the first time-varying index value in the first time-varying calculation step (S1223), the second time-varying index value is calculated in the second time-varying calculation step (S1224) and set as the judgment data. However, in the second example, after acquiring the first information at each shooting moment in the first information acquisition step (S1221), the first time-varying calculation step (S1223) is executed to calculate the time-varying index value of the first information, and then the second time-varying index value is calculated. That is, compared with the first example, the first time-varying calculation step (S1223) and the second time-varying calculation step (S1224) are combined and moved to immediately after the first information acquisition step (S1221), which is different. Then, in the statistical index value acquisition step (S1222), the statistical index value of the secondary temporal change index value is calculated and set as determination data. The primary information and the primary information acquisition step (S1221) are the same as those in the first embodiment and the first example, and thus the description thereof is omitted.

[0220] (First time change calculation step (obtaining time change index value))

[0221] After the 1st information acquisition step (S1221), the 1st time change index value of each 1st information calculated at each shooting moment is calculated in the 1st time change calculation step (S1223). In the 1st time change calculation step (S1223) of the first example, the 1st time change index value of the statistical index value is obtained, and in the 1st information acquisition step (S1221) of the second example, the 1st time change index value of the 1st information is obtained. Therefore, as long as the statistical index value of the 1st time change calculation step (S1223) of the first example is replaced with the 1st information and the 1st time change calculation step (S1223) of the first example is applied, it can become the 1st time change calculation step (S1223) of the second example. The 1st time change index value is the change amount of the 1st information between each shooting moment.

[0222] In addition, the index value of the first time-varying change in the second example is the same as the first example, which is the differential change amount, the rate of change, the rate of change per unit time, the speed of change or the time of change. In the first example, it is the differential change amount, the rate of change, the rate of change per unit time, the speed of change or the time of change of the statistical index value of the first information, while in the second example, it is the differential change amount, the rate of change, the rate of change per unit time, the speed of change or the time of change of the first information, which is the only difference. Therefore, in the calculation of the index value of the first time-varying change in the aforementioned first example, the index value of the first time-varying change is calculated based on the statistical index value of the first information, but it is sufficient to simply replace the "statistical index value of the first information" with "the first information" and apply the first example to the second example. In addition, in the second example, as in the first example, the speed of change can be applied as the specific proliferation rate, and the time of change can be applied as the doubling time.

[0223] Furthermore, refer to Figure 4H and Fig. 4I This is explained. Figure 4H and Fig. 4I This is a diagram showing the concept of a primary change over time, which is a change over time of primary information (vertical axis) with respect to time (horizontal axis) in the first and second examples. Figure 4H It shows the case where the time-varying index value is a change amount or a change rate. Fig. 4I The case where one time-varying index value is a change amount or a change rate per unit time is shown. Figure 4H and Fig. 4I The first time-varying index value calculated in the first time-varying index calculation step (S1223) described here and the second time-varying index value calculated in the second time-varying index calculation step (S1224) described next are shown at the same time. In the second example, the description of the first time-varying index calculation step (S1223) of the first example and Figure 4H and Fig. 4I The a1 and a2 as statistical index values ​​in the first information are replaced by the values ​​of the first information acquired in the first information acquisition step (S1221). Since one first information is obtained from two first information, for the first time change index value, the first time change index value corresponding to the number obtained by subtracting 1 from the number of first information is obtained. The first time change index value of the second example is a value that directly indicates the time change tendency of the first information.

[0224] (Secondary Time-dependent Change Calculation Step (Obtaining Time-dependent Change Index Value))

[0225] After the 1st time change calculation step (S1222), the 2nd time change index value is calculated in the 2nd time change calculation step (S1223). In the 2nd example, the statistical index value of the 1st example is replaced with the 1st information and the same process as the 1st example is applied. The 2nd time change index value is the same as the 1st example, which is the change amount or change rate of the increment of the 1st time change index value. That is, it is the change amount of the 1st time change index value from the 1st shooting time to the 2nd shooting time, and the change amount of each 1st time change index value from the 2nd shooting time to the 3rd shooting time. For this, refer to Figure 4H and Fig. 4I Provide explanation. Figure 4H and Fig. 4I This is a diagram showing the concept of the change of primary information (vertical axis) with respect to time (horizontal axis) for the first and second examples. Figure 4H and Fig. 4I The second time-varying index value calculated in the second time-varying index calculation step (S1224) is shown together with the first time-varying index value. In the second time-varying index calculation step (S1224) of the second example, the second time-varying index value is obtained by the first time-varying index calculation step (S1223), so similarly to the first time-varying index calculation step (S1223), as long as the statistical index value of the first time-varying index calculation step (S1223) of the first example is replaced with the first information and the second time-varying index calculation step (S1224) of the first example is applied, it becomes the second time-varying index calculation step (S1224) of the second example. Since one secondary time-varying index value is generated for two primary time-varying index values, as a whole, one secondary time-varying index value less than the number of primary time-varying index values ​​is generated, and two secondary time-varying index values ​​less than the statistical index value of the primary information are generated, which is also the same as the first example.

[0226] (Get statistical indicator values)

[0227] After calculating the two time-varying index values ​​through the two time-varying calculation steps (S1224), the statistical index value of each shooting moment is calculated in the statistical index value acquisition step (S1222) based on the one-time information of each shooting moment. The statistical index value in the second example is an index value limited to a representative value that represents the two time-varying index values ​​representatively. The statistical index value is at least any one of the deviations such as the mean value, variance, and standard deviation as in the first embodiment and the second embodiment. In the second example, the statistical index value of the two time-varying index values ​​is calculated, so it is the mean value, variance, deviation, etc. of the two time-varying index values ​​of each of the multiple observation parts Op. The statistical index value obtained here is set as the judgment data to determine the timing of the generation. Multiple statistical index values ​​can be used as multiple judgment data of the original number, or multiple statistical index values ​​can be used as one statistical index value through the average value, maximum value, minimum value, etc., and used as one judgment data. The statistical index value of the secondary time-varying index value calculated in the statistical index value acquisition step ( S1222 ) of the second example has the same properties and technical significance as the determination data of the statistical index value of the time-varying index value in the second example of the second embodiment.

[0228] (Determine the passage)

[0229] After the generation of the judgment data is completed in the judgment data generation step (S12), a judgment step (S13) is performed to judge whether the time for the transfer has arrived. In the judgment step (S13), the time for the transfer is judged based on the statistical index value generated in the judgment data generation step (S12). In the judgment data of the second example, the statistical index value as the index value of the second time change index value includes the first information, the elements of the time change as the index value of the first time change index value and the index value of the second time change index value, and the elements of the statistical index value. Therefore, as the judgment data for judging the time for the transfer, it is effective in judging the time for the transfer, just like the second example of the second embodiment and the first example of the present embodiment. In the case of selecting the sum or the average value as the statistical index value, the judgment data of the second example refers to the representative value of the acceleration of the change of the first information, and in the case of selecting the variance or the deviation as the statistical index value, the judgment data of the second example refers to the deviation of the acceleration of the change of the first information.

[0230] In the determination step (S13), the statistical index value calculated in the statistical index value acquisition step (S1222) is compared with a predetermined threshold value. The predetermined threshold value is the same as in the first example. As in the first embodiment, a test is performed as a preliminary check under the same conditions as the actual determination of the timing of the passage, in which the statistical index value at the moment of the passage timing is obtained, the time-varying index value is obtained, and the value is set as the predetermined threshold value. Even if the element of the time-varying factor is added, the characteristics of the statistical index value in the determination of the timing of the passage will not disappear, as described in the first embodiment.

[0231] Regarding the number of determination data, when it is generated as one representative value, when the number of determination data is set to one, a comparison is made as to whether the one determination data reaches a prescribed threshold value, and when the prescribed threshold value is reached, it is determined that the time for passage has arrived. When the number of determination data is set to multiple, a comparison is made as to whether a prescribed number of determination data among the multiple determination data reaches a prescribed threshold value, and when the prescribed threshold value is reached, it can be determined that the time for passage has arrived. In the second example of the third embodiment, at the time of passage, a certain inflection point represented by an extreme value such as a minimum value or a maximum value may appear in the trend of change of the statistical index value. The moment when such an inflection point of the trend of change appears is set as the moment when the time for passage has arrived and is set as the threshold value.

[0232] Furthermore, when all the determination data in the plurality of determination data reach a predetermined threshold, it may be determined that the time for subculturing has arrived. In this case, since it takes time for all the determination data to reach the predetermined threshold, it will take a long time to determine whether the time for subculturing has arrived.

[0233] On the contrary, it is also possible to determine that the time for subculturing has arrived when at least one of the plurality of determination data reaches a predetermined threshold. In this case, since determination can be made as long as one determination data reaches the predetermined threshold, it is possible to determine whether the time for subculturing has arrived as quickly as possible.

[0234] <Example 3>

[0235] Next, refer to Figure 4C The third example is described below. Figure 4C), after acquiring the primary information at each shooting moment in the primary information acquisition step (S1221), the primary time-varying index value is calculated in the primary time-varying calculation step (S1223), and then the statistical index value of each shooting moment is calculated in the statistical index value acquisition step (S1222) based on the primary information at each shooting moment. Then, the secondary time-varying index value of the statistical index value is calculated in the secondary time-varying calculation step (S1224) and set as the judgment data. That is, between the primary time-varying calculation step (S1223) and the secondary time-varying calculation step (S1224), the statistical index value acquisition step (S1222) is executed and the statistical index value of each shooting moment is calculated based on the primary information, and the secondary time-varying index value of the statistical index value is calculated in the secondary time-varying calculation step (S1224). This is described below. The primary information and the primary information acquisition step (S1221) are the same as those in the first embodiment, so the description is omitted.

[0236] (First time change calculation step (obtaining time change index value))

[0237] After the primary information acquisition step (S1221), the primary temporal change index value of each primary information calculated at each shooting time is calculated in the primary temporal change calculation step (S1223). The process of the primary information acquisition step (S1221) and the primary temporal change calculation step (S1223) is the same as that of the second example ( Figure 4B ) is the same, so the detailed description is omitted. In the third example, it is only necessary to repeat the description of the first time change calculation step (S1223) of the first example and Figure 4H and Fig. 4I The statistical index values ​​a1 and a2 in s1222 may be replaced by the values ​​of the primary information acquired in the primary information acquisition step (S1221).

[0238] (Get statistical indicator values)

[0239] After calculating the first time-varying index value of the first information through the first time-varying calculation process (S1223), the statistical index value of the first time-varying index value is calculated in the statistical index value acquisition process (S1222). The statistical index value in the third example is an index value limited to a representative value that represents the first time-varying index value representatively. The statistical index value is at least any one of the deviations such as the mean value, variance, and standard deviation as in the first and second embodiments. In the third example, the statistical index value of the differential change amount, change amount rate, unit time change rate, change speed, or change time of the first time-varying index value is obtained. That is, in the third example, the statistical index values ​​of these first time-varying index values ​​are calculated, so they are the average value, variance, deviation, etc. of the differential change amount, change amount rate, unit time change rate, change speed, or change time of the first information of each of the multiple observation parts Op.

[0240] (Secondary Time-dependent Change Calculation Step (Obtaining Time-dependent Change Index Value))

[0241] After the statistical index value acquisition process (S1222), the 2nd time-varying index value is calculated in the 2nd time-varying calculation process (S1224). In the third example, the 1st time-varying index value of the 2nd example is replaced with the statistical index value, and the 2nd time-varying acquisition process (S1224) is applied in the same process as the 2nd time-varying calculation process (S1224) of the 2nd example. In the third example, the 2nd time-varying index value becomes the differential change amount, change amount rate, unit time change rate, change speed or change time (1st time-varying index value) of the information, the average value, variance, and deviation (statistical index value) of the differential change amount, change amount rate, unit time change rate, change speed or change time (2nd time-varying index value). The statistical index value is set as the judgment data to determine the timing of the generation. Multiple statistical index values ​​can be used as multiple judgment data of the original number, or multiple statistical index values ​​can be used as one statistical index value through the average value, maximum value, minimum value, etc., and used as one judgment data.

[0242] (Determine the passage)

[0243] After the generation of the judgment data is completed in the judgment data generation step (S12), a judgment step (S13) is performed to judge whether the time for subculture has arrived. In the judgment step (S13), the time for subculture is judged based on the statistical index value generated in the judgment data generation step (S12). The judgment data of the third example is the secondary time-varying index value of the statistical index value, so it is the same as the first and second examples, including the primary information, the elements of the time-varying index value of the primary time-varying index value and the secondary time-varying index value, and the elements of the statistical index value. This point has not changed, so as the judgment data for judging the time for subculture, it is effective in judging the time for subculture, just like the second embodiment and the first and second examples of this embodiment. In the case of selecting the sum or average value as the statistical index value, the judgment data of the third example refers to the speed of the representative value of the speed of change of the primary information, and in the case of selecting the variance or deviation as the statistical index value, the judgment data of the third example refers to the speed of the deviation of the speed of change of the primary information.

[0244] In the determination step (S13), the second time-varying index value calculated in the second time-varying calculation step (S1224) is compared with a predetermined threshold value. The predetermined threshold value is the same as in the first example. Figure 4C ), a test as a preliminary check is carried out under the same conditions as those for actually determining the timing of passage, in which a primary time-varying index value of primary information at the moment of passage timing is calculated, a statistical index value is calculated, and then a secondary time-varying index value of the statistical index value is calculated, and the value is set as a predetermined threshold. Even if the element of time-varying is added, the characteristics of the statistical index value in determining the timing of passage do not disappear, as described in the first embodiment.

[0245] When the number of determination data is generated as one representative value, when the number of determination data is set to one, a comparison is made as to whether the one determination data reaches a predetermined threshold value, and when the predetermined threshold value is reached, it is determined that the time for subculturing has arrived. When the number of determination data is set to a plurality of determination data, a comparison is made as to whether a predetermined number of determination data among the plurality of determination data reaches a predetermined threshold value, and when the predetermined threshold value is reached, it can be determined that the time for subculturing has arrived.

[0246] In the third example of the third embodiment, at the time of passage, some inflection points represented by extreme values ​​such as minimum and maximum values ​​appear in the time changes of the two time-varying index values. The time when such an inflection point of the time change appears is set as the time when passage is reached as the threshold.

[0247] Furthermore, when all the determination data in the plurality of determination data reach a predetermined threshold, it may be determined that the time for subculturing has arrived. In this case, since it takes time for all the determination data to reach the predetermined threshold, it will take a long time to determine whether the time for subculturing has arrived.

[0248] On the contrary, it is also possible to determine that the time for subculturing has arrived when at least one of the plurality of determination data reaches a predetermined threshold. In this case, since determination can be made as long as one determination data reaches the predetermined threshold, it is possible to determine whether the time for subculturing has arrived as quickly as possible.

[0249] [Fourth embodiment]

[0250] Next, as a fourth embodiment, referring to FIG. 5A to FIG. 5B A method and system for determining the timing of subculturing will be described. Figure 5A is a graph showing i reference imaging times (i=a natural number from 1 to m) as imaging times of interest in the fourth embodiment and the value R corresponding to these primary information. i (i=1 to m natural numbers), and j evaluation shooting times (j=1 to n natural numbers), and the values ​​V corresponding to these primary information j A conceptual diagram of (j=natural number from 1 to n).

[0251] The third embodiment is a method of calculating the time-varying index value between one reference shooting moment and two or more evaluation shooting moments for the shooting moment of interest. In contrast, in the fourth embodiment, a method of calculating the time-varying index value between two or more reference shooting moments and two or more evaluation shooting moments. The number of reference shooting moments and the number of evaluation shooting moments can be freely set to one or more. That is, Figure 5A As shown, i reference shooting times (i = a natural number from 1 to m) and j evaluation shooting times (j = a natural number from 1 to n) can be freely set as the shooting times of interest. Among them, the example of setting one reference shooting time and one evaluation shooting time (j = a natural number from 1 to n) as the shooting time of interest is the aforementioned second embodiment. In addition, the example of setting one reference shooting time and two evaluation shooting times (j = a natural number from 1 to n) as the shooting times of interest is the aforementioned third embodiment.

[0252] In the fourth embodiment, in particular, the point that a plurality of reference shooting times (i=1 to m natural numbers) are used is different from the third embodiment, but the third embodiment can be applied in the same manner except for the point that a plurality of reference shooting times (i=1 to m natural numbers) are used. There are two methods for applying the third embodiment in the fourth embodiment. One method is a method in which, when executing a time-varying index value (S1223), a statistical index value based on a single information acquired at a plurality of reference shooting times (i=1 to m natural numbers) (the case of the first example) and a single information acquired at a plurality of reference shooting times (i=1 to m natural numbers) (the cases of the second and third examples) are converted into representative values ​​and integrated into a single representative reference value, and the third embodiment (hereinafter referred to as the reference value integration method) is applied. Another method is the following method: for each of the statistical index value based on the one-time information obtained at multiple benchmark shooting times (i=1 to m), and the one-time information obtained at multiple benchmark shooting times (i=1 to m), they are not integrated into one representative value but are set as multiple benchmark values, and the third implementation method (hereinafter referred to as the matrix time-varying method) is applied.

[0253] Figure 5B The concept of the reference value synthesis method is shown. In the reference value synthesis method, as described below, a representative reference value Rrep is generated and the aforementioned third embodiment is applied. In the first example of the third embodiment, in the primary information acquisition step (S1221), the primary information acquired at a plurality of reference shooting times (i = 1 to m) is converted into a representative value by a statistical method such as an average value, sum, or weighted value, and a representative reference value Rrep of the primary information is generated. In addition, in the second and third examples of the third embodiment, in the primary information acquisition step (S1221), the statistical index value of the primary information acquired at a plurality of reference shooting times (i = 1 to m) is converted into a representative value by a statistical method such as an average value, sum, or weighted value, and a representative reference value Rrep of the primary information is generated. In this case, the statistical index value of the primary information is also an average value, sum, or weighted value, so although the statistical processing is continuous, the representative value conversion for generating the representative reference value and the statistical index value of the primary information have different meanings in terms of technology. The former is a process for integrating the values ​​corresponding to a plurality of reference imaging time points (i = a natural number from 1 to m) into a single representative value, while the latter is a process for generating a statistical index value representing the state of the primary information of the cell. iAfter the representative reference value Rrep is integrated, the j evaluation shooting time points (j = 1 to n natural numbers) and the values ​​V corresponding to the primary information are calculated. j (j=natural number from 1 to n) calculates the time-varying index value (Rrep, V1)…(Rrep, V j )…(Rrep,V j ) method, the third implementation method described can be directly applied according to the respective records described in Examples 1 to 3.

[0254] In the matrix time variation method, unlike the benchmark value integration method, the representative value is not converted into a benchmark value, but the third implementation method described in Examples 1 to 3 can be directly applied according to the number of first-order information corresponding to the number of benchmark shooting times (i = a natural number from 1 to m).

[0255] Figure 5C The concept of the matrix temporal change method is shown. That is, the value R corresponding to the primary information of each reference shooting time (i = natural number from 1 to m) is shown. i (i=1 to m natural numbers) and the value V corresponding to the primary information of each evaluation shooting time (j=1 to n natural numbers) j The concept of calculating the temporal change index value once is as follows: (j=natural number from 1 to n).

[0256] In the matrix temporal variation method, unlike the reference value integration method, the representative value is not converted into a representative reference value Rrep, but the third embodiment described above can be directly applied according to the first to third examples, based on the number of primary information corresponding to the number of reference shooting time points (i = a natural number from 1 to m). For example, the value derived from the primary information of each reference shooting time point (i = a natural number from 1 to m) is assumed to be R i (i=a natural number from 1 to m). In the first example of the third embodiment, the value R derived from the primary information i (i=natural number from 1 to m) refers to a statistical index value. In the second and third examples of the third embodiment, the value R derived from the primary information is i (i=1 to m natural numbers) refers to one time-varying index value. In the first to third examples of the third embodiment, the m values ​​R i Each of (i = 1 to m natural numbers) has n values ​​V j Between each of (j=1 to n natural numbers), m×n groups (R1, V1)…(R1, V1) based on R1 are calculated as the primary time-varying index values ​​of the next step. j )…(R1, V n ), based on R i(i = 1 to m) group (R i , V1)…(R i , V j )…(R i , V n ) and R-based m The group (R m , V1)…(R m , V j )…(R m , V n ), the third embodiment is applied in such a way as to calculate the secondary time-varying index values ​​respectively according to these groups. In the third embodiment, there is one reference shooting time, so the primary time-varying index value is generated according to the number of evaluation shooting times, and the secondary time-varying index value which is one less than the number is generated. That is, when the evaluation shooting time is set (i = a natural number from 1 to n), n primary time-varying index values ​​and (n-1) secondary time-varying index values ​​are generated. In the matrix time-varying method of the fourth embodiment, the index values ​​obtained by multiplying them by m, that is, (m×n) primary time-varying index values ​​and m·(n-1) secondary time-varying index values ​​are generated.

[0257] That is, the value derived from the primary information at each evaluation shooting time (j=1 to n natural number) is assumed to be V i (i=1 to m natural number), the value R derived from the primary information at each reference imaging time (i=1 to m natural number) i (i=1 to m natural numbers) and the value V derived from the primary information of each evaluation shooting time (j=1 to n natural numbers) j (j=natural number from 1 to m) The first to third examples of the third embodiment are applied. In the first example of the third embodiment, the value V derived from the primary information j (j=natural number from 1 to n) also refers to a statistical index value. In the second and third examples of the third embodiment, the value V derived from the primary information is j (j=1 to n natural number) also refers to one time-varying index value. That is, the case where the reference shooting time is set to one time (i=1) and the evaluation shooting time is set to three time (j=3) is the third embodiment, and in the case of the reference comparison method, R1=a1, V1=a2 and R1=a1, V2=a3 are set, and in the case of the nearest comparison method, R1=a1, V1=a2 and R2=a2, V2=a3 are set.

[0258] In the matrix temporal variation method, in both the first and third examples of the third embodiment, two temporal variation values ​​can be obtained based on the number of values ​​corresponding to a plurality of reference imaging times. In the determination step (S13) of determining whether the timing of generation has been reached, when setting a predetermined threshold, it can be performed as follows.

[0259] Even in the matrix temporal variation method of the fourth embodiment, the predetermined threshold value is determined by a preliminary test carried out in advance under the same conditions before the actual determination of the timing of the transfer, and in this test, the threshold value is determined based on the result of the time when the transfer is to be made. In this test, the same conditions as those for the determination of the actual timing of the transfer are used, so the number of reference imaging times (i = a natural number from 1 to m) and the number of evaluation imaging times (j = a natural number from 1 to n) are also set to be the same. Therefore, when the first example or the third example of the third embodiment is applied, in this test, as a single temporal variation index value, the groups (R1, V1) ... (R1, V2) based on R1 are respectively calculated. j )…(R1, V n ), based on R i (i = 1 to m) group (R i , V1)…(R i , V j )…(R i , V n ) and R-based m The group (R m , V1)…(R m , V j )…(R m , V n ), and calculate two time-varying index values ​​based on these groups to generate judgment data. Then, judgment data of the moment judged as the time for transfer is generated. Among them, the two time-varying index values ​​(the first and third examples) and the statistical index value of the two time-varying index values ​​(the second example) as judgment data, multiple judgment data can be obtained. However, even in the case where there are multiple reference shooting times, inflection points are observed in the change trends of the two time-varying index values ​​(the first and third examples) and the statistical index value of the two time-varying index values ​​(the second example), and the vicinity thereof is limited to the prescribed threshold value when judging the timing of transfer. In particular, when multiple reference shooting times are set, the group based on R1, the group based on R2, the group based on R3, the group based on R4, the group based on R5, the group based on R6, the group based on R7, the group based on R8, the group based on R9, the group based on R10, the group based on R11, the group based on R12, the group based on R13, the group based on R14, the group based on R15, the group based on R16, the group based on R17, the group based on R18, the group based on R19, the group based on R20, the group based on R21 i (i = 1 to m) and based on R m The change tendency of the group itself also shows a pattern with an inflection point, so it is possible to make an accurate judgment compared with the second and third embodiments having elements that change over time.

[0260] Next, refer to Figure 5DAnother aspect as a modified example of the fourth embodiment will be described. Figure 5D The figure shows the tendency of the change in the value (vertical axis) of the judgment data (○ symbol, ● symbol) as the culture progresses (the passage of "time" as the horizontal axis). Figure 5D In the determination data generation step (S12), when generating determination data, in the stage where the cell expansion or proliferation is in progress before the time of passage, the increase tendency of the value corresponding to the reference imaging time and the increase tendency of the value corresponding to the evaluation imaging time converge within a certain range of the deviation of the increase tendency ( Figure 5D However, when the passage time comes, the increasing tendency of the value corresponding to the reference shooting time and the increasing tendency of the value corresponding to the evaluation shooting time become larger, and the increasing tendency changes ( Figure 5D Therefore, in the generation of the judgment data, Figure 5D The evaluation imaging time at which the value corresponding to the evaluation imaging time of group B begins to show a tendency is determined as the timing of passage. Figure 5D The time when the values ​​of group B corresponding to the evaluation shooting time begin to show a trend is the inflection point of the change trend of the determination data.

[0261] In this case, in the judgment data generating step (S12), the timing of the generation can also be determined based on the stage at which the inflection point of the change tendency of the judgment data appears. This can also be applied to the first embodiment. On the other hand, a preliminary test can be carried out under the same conditions as the actual determination of the timing of the generation, in which the stage at which the inflection point of the change tendency of the judgment data appears is defined as a predetermined threshold, and the first to fourth embodiments are applied.

[0262] [Fifth embodiment]

[0263] Next, refer to Figure 5E A fifth embodiment will be described. Figure 5EThe trend of the change of the value (vertical axis) of the determination data (○ symbol) that changes with the progress of the culture (the passage of "time" as the horizontal axis) is shown. The fifth embodiment is an embodiment that is a modified example of the second to fourth embodiments using the time-varying element in the determination step (S13) for determining the timing of the passage described in the first to fourth embodiments. That is, in the fifth embodiment, the steps of the second to fourth embodiments are directly applied except for the determination step (S13), and the determination step (S13) of the fifth embodiment described below is applied to each of the second to fourth embodiments for the determination step (S13).

[0264] In the determination step (S13) of the fifth embodiment, the determination of the predetermined threshold value is performed by using machine learning or deep learning. The machine learning and deep learning methods themselves are not limited, and all common machine learning or deep learning methods can be widely used. Here, machine learning and deep learning themselves have no characteristics, and by using machine learning and deep learning in the determination of the timing of the passage in the first to fourth embodiments of the present invention, it is possible to further determine the timing of the passage in a timely manner in addition to the second to fourth embodiments.

[0265] Figure 5E The changing tendency of the number of cells as the primary information and the sum of the statistical index values ​​of the first to fourth embodiments is shown as an example. In this case, there is an increasing tendency until the time of passage, and the site where the time of passage arrives has an inflection point of the changing tendency. In addition, a saturation state of the changing tendency in which the increase stops after the time of passage arrives is shown. The changing tendency of the other primary information and other statistical index values ​​of the first to fourth embodiments is different from that shown in FIG5 , but they are the same in that the site where the time of passage arrives has an inflection point of the changing tendency. Below, as an explanation of the fifth embodiment, Figure 5E It is hoped that the change trends shown here will be understood as a common point that an inflection point of the change trend appears at the site where the timing of passage arrives and that the change trends before and after differ.

[0266] The actual time for passage is Figure 5ENear the thick arrow shown by . In this vicinity, the change tendency of the judgment data (○ symbol) becomes an inflection point. However, the timing of the judgment is implemented discretely according to the prescribed time of the culture, and therefore, the timing cannot be determined at a moment that coincides with the inflection point. In the case of the second to fourth embodiments utilizing the time-varying element, each time a prescribed threshold is drawn, a test as a preliminary check is implemented under the same conditions as the actual timing of determining the passage, and the prescribed threshold is determined based on the results of the test. In the fifth embodiment, a test as a preliminary check is also implemented under the same conditions as the actual timing of determining the passage, and machine learning or deep learning is applied to the results of the test to determine the prescribed threshold.

[0267] In the second to fourth embodiments, in determining the results of the test, a preliminary determination is made based on the feelings of so-called experienced users. Figure 5E In the example, the line L1 indicating the trend of change during the ongoing culture stage and the line L2 indicating the trend of change after the culture is progressing and the time for subculturing are reached are judged by feeling, and the time of the inflection point near the intersection of the line L1 indicating the trend of change during the ongoing culture stage is judged as the time for subculturing. However, in the fifth embodiment of applying machine learning or deep learning, it is possible to capture the trend of change that even experienced users cannot recognize. For example, conceptually, Figure 5E The line L indicating the change tendency AI As shown, it is possible to derive a change tendency that even an experienced user cannot recognize, and it is possible to derive the time of the timing of the generation in advance that cannot be obtained without machine learning or deep learning. In the fifth embodiment, this time is defined as a predetermined threshold.

[0268] In the determination step ( S13 ), when the determination data reaches a predetermined threshold value, it is determined that the timing of passage has arrived, which is the same as in the second to fourth embodiments.

[0269] Description of symbols

[0270] 1: Cell culture system; 2: Culture medium unit; 3: Subculture timing determination unit; 4: Culture unit; 5: Cell inoculation device; 6: Transport unit; 7: Centrifugal device; 8: Processing device; 31: Camera (shooting component); 32: Shooting table; 33: Processing component; 61: Moving table; 62: Arm; 63: Actuator; Pe: Evaluation culture container.

Claims

1. A method for determining the timing of subculturing, comprising determining the timing of subculturing cells that are growing or proliferating in a culture medium in a culture container, in, The method for determining the timing of subculturing has the following steps: a photographing step of photographing cells at a plurality of predetermined locations of the culture container at a first photographing time, and acquiring cell images at each of the plurality of predetermined locations at the first photographing time; a primary information acquisition step of acquiring primary information of cells captured in the cell images at the respective points at the first capturing time; A statistical index value acquisition step, calculating the statistical index value of the primary information of the cell; as well as The step of determining is to determine the imaging time corresponding to the time when the statistical index value reaches a predetermined threshold value as the time when the passage timing has arrived.

2. A method for determining the timing of subculturing, which determines the timing of subculturing cells that have expanded or proliferated in a culture medium in a culture container, in, The method for determining the timing of subculturing has the following steps: a photographing step of photographing cells at a plurality of predetermined locations of the culture container at a first photographing time and a second photographing time after the first photographing time, and acquiring cell images at each of the plurality of predetermined locations at each of the first photographing time and the second photographing time; a primary information acquisition step of acquiring primary information of cells captured in the cell images at the respective locations at the first capturing time and the second capturing time; a statistical index value acquisition step of calculating the statistical index value of the first information at each of the first shooting time and the second shooting time; a temporal variation calculation step of calculating a temporal variation index value based on the statistical index values ​​at the first imaging time and the second imaging time; as well as The step of determining is to determine the imaging time corresponding to the time when the temporal change index value reaches a predetermined threshold value as the time when the passage timing has arrived.

3. A method for determining the timing of subculturing, comprising determining the timing of subculturing cells that are growing or proliferating in a culture medium in a culture container, in, The method for determining the timing of subculturing has the following steps: a photographing step of photographing cells at a plurality of predetermined locations of the culture container at a first photographing time and a second photographing time after the first photographing time, and acquiring cell images at each of the plurality of predetermined locations at each of the first photographing time and the second photographing time; a primary information acquisition step of acquiring primary information of cells captured in the cell images at the respective locations at the first capturing time and the second capturing time; a temporal change calculation step of calculating a temporal change index value based on the first information at the first shooting time and the second shooting time; a statistical index value acquisition step of calculating a statistical index value of the temporal variation index value at each of the first imaging time and the second imaging time; as well as The step of determining is to determine the imaging time corresponding to the time when the statistical index value reaches a predetermined threshold value as the time when the passage timing has arrived.

4. A method for determining the timing of subculturing, comprising determining the timing of subculturing cells that are growing or proliferating in a culture medium in a culture container, in, The method for determining the timing of subculturing has the following steps: a photographing step of photographing cells at a plurality of predetermined locations of the culture container at a first photographing time, a second photographing time after the first photographing time, and a third photographing time after the second photographing time, to obtain cell images at each of the plurality of predetermined locations at each of the first photographing time, the second photographing time, and the third photographing time; a primary information acquisition step of acquiring primary information of cells captured in the cell images at the respective locations at the first capturing time, the second capturing time, and the third capturing time; a statistical index value acquisition step of calculating the statistical index value of the first information at each of the first shooting time, the second shooting time, and the third shooting time; a first temporal variation calculation step of calculating a first temporal variation index value of the statistical index value at the first imaging time and the second imaging time, and a first temporal variation index value of the statistical index value at the second imaging time and the third imaging time; a second temporal change calculation step of calculating a second temporal change index value based on the first temporal change index value at the first imaging time and the second imaging time and the first temporal change index value at the second imaging time and the third imaging time; and The step of determining is to determine the imaging time corresponding to the time when the two time-dependent change index values ​​reach a predetermined threshold value as the time when the passage timing has arrived.

5. A method for determining the timing of subculturing, comprising determining the timing of subculturing cells that are expanded or proliferated in a culture medium in a culture container, in, The method for determining the timing of subculturing has the following steps: a photographing step of photographing cells at a plurality of predetermined locations of the culture container at a first photographing time, a second photographing time after the first photographing time, and a third photographing time after the second photographing time, to obtain cell images at each of the plurality of predetermined locations at each of the first photographing time, the second photographing time, and the third photographing time; a primary information acquisition step of acquiring primary information of cells captured in the cell images at the respective locations at the first capturing time, the second capturing time, and the third capturing time; a first temporal change calculation step of calculating a first temporal change index value of the first information at the first shooting time and the second shooting time, and a first temporal change index value of the first information at the second shooting time and the third shooting time; a second temporal change calculation step of calculating a second temporal change index value based on the first temporal change index value at the first imaging time and the second imaging time and the first temporal change index value at the second imaging time and the third imaging time; a statistical index value acquisition step of calculating a statistical index value of the two time-varying index values ​​at each of the first imaging time, the second imaging time, and the third imaging time; and The step of determining is to determine the imaging time corresponding to the time when the statistical index value reaches a predetermined threshold value as the time when the passage timing has arrived.

6. A method for determining the timing of subculturing, comprising determining the timing of subculturing cells that are expanded or proliferated in a culture medium in a culture container, in, The method for determining the timing of subculturing has the following steps: a photographing step of photographing cells at a plurality of predetermined locations of the culture container at a first photographing time, a second photographing time after the first photographing time, and a third photographing time after the second photographing time, to obtain cell images at each of the plurality of predetermined locations at each of the first photographing time, the second photographing time, and the third photographing time; a primary information acquisition step of acquiring primary information of cells captured in the cell images at the respective locations at the first capturing time, the second capturing time, and the third capturing time; a first temporal change calculation step of calculating a first temporal change index value of the first information at the first shooting time and the second shooting time, and a first temporal change index value of the first information at the second shooting time and the third shooting time; a statistical index value acquisition step of calculating a statistical index value of the one time-varying index value at each of the first imaging time, the second imaging time, and the third imaging time; a second temporal variation calculation step of calculating a second temporal variation index value based on the statistical index values ​​at the first imaging time and the second imaging time, and the statistical index values ​​at the second imaging time and the third imaging time; and The step of determining is to determine the imaging time corresponding to the time when the two time-dependent change index values ​​reach a predetermined threshold value as the time when the passage timing has arrived.

7. The method for determining the timing of passage according to claim 2, in, The temporal change index value is a differential change amount, a change amount rate, a change rate per unit time, a change speed, a change time, a specific growth rate, or a predetermined doubling time of the statistical index value.

8. The method for determining the timing of passage according to claim 4, in, The one-time temporal change index value is a differential change amount, a change amount rate, a change rate per unit time, a change speed, a change time, a specific growth rate, or a predetermined doubling time of the statistical index value.

9. The method for determining the timing of passage according to claim 3, in, The temporal change index value is a differential change amount, a change amount rate, a unit time change rate, a change speed, a change time, a specific growth rate, and a predetermined doubling time of the primary information.

10. The method for determining the timing of passage according to claim 5, in, The primary temporal change index value is a differential change amount, a change amount rate, a unit time change rate, a change speed, a change time, a specific growth rate, and a predetermined doubling time of the primary information.

11. The method for determining the timing of passage according to claim 6, in, The primary temporal change index value is a differential change amount, a change amount rate, a unit time change rate, a change speed, a change time, a specific growth rate, and a predetermined doubling time of the primary information.

12. The method for determining the timing of cell culture according to any one of claims 1 to 6, in, The primary information is any one of the number of cells, cell density, cell-occupied area, single cell area, and relative distance between cells in the cell image.

13. The method for determining the timing of cell culture according to any one of claims 1 to 6, in, The statistical indicator value is an average value.

14. The method for determining the timing of cell culture according to any one of claims 1 to 6, in, The statistical indicator value is a sum.

15. The method for determining the timing of cell culture according to any one of claims 1 to 6, in, The statistical indicator value is variance or deviation.

16. The method for determining the timing of cell culture according to any one of claims 2 to 6, in, The predetermined threshold value is defined as a statistical indicator value at which cell extension or proliferation no longer occurs in a preliminary test performed in advance.

17. The method for determining the timing of subculturing according to claim 15, in, The predetermined threshold value is defined as an inflection point in the time variation of the variance or the deviation in a prior test carried out in advance.

18. The method for determining the timing of subculturing according to claim 7, in, The change rate is calculated as a value at any shooting time. The predetermined threshold value is limited to the second time-varying index value at a time when no increasing tendency of the time-varying index value is observed in a preliminary inspection performed in advance.

19. The method for determining the timing of subculturing according to claim 7, in, The change rate is calculated as a ratio relative to the value at the time of the next shot. The predetermined threshold value is defined as an inflection point of the temporal change of the temporal change index value in a preliminary inspection performed in advance.

20. The method for determining the timing of subculturing according to claim 8, in, The change rate is calculated as a value at any shooting time. The predetermined threshold value is limited to the secondary time-varying index value at a time when no increasing tendency of the secondary time-varying index value is observed in a preliminary inspection performed in advance.

21. The method for determining the timing of passage according to claim 8, in, The change rate is calculated as a ratio relative to the value at the time of the next shot. The predetermined threshold value is defined as an inflection point of the time change of the secondary time-varying change index value in a preliminary inspection performed in advance.

22. The method for determining the timing of passage according to claim 9 or 10, in, The change rate is calculated as a value at any shooting time. The predetermined threshold value is limited to the secondary time-varying index value at a time when no increasing tendency of the statistical index value is observed in a preliminary inspection performed in advance.

23. The method for determining the timing of passage according to claim 9 or 10, in, The change rate is calculated as a ratio relative to the value at the time of the next shot. The predetermined threshold value is defined as an inflection point of the temporal change of the statistical index value in a preliminary test performed in advance.

24. The method for determining the timing of subculturing according to claim 11, in, The change rate is calculated as a value at any shooting time. The predetermined threshold value is limited to the secondary time-varying index value at a time when no increasing tendency of the secondary time-varying index value is observed in a preliminary inspection performed in advance.

25. The method for determining the timing of subculturing according to claim 11, in, The change rate is calculated as a ratio relative to the value at the time of the next shot. The predetermined threshold value is defined as an inflection point of the time change of the secondary time change index value in a preliminary inspection performed in advance.

26. The method for determining the timing of cell culture according to any one of claims 4 to 6, in, The time interval between the first imaging time and the second imaging time and the time interval between the second imaging time and the third imaging time are the same time interval.

27. The method for determining the timing of cell culture according to any one of claims 1 to 6, in, The predetermined threshold value is defined by machine learning in a preliminary inspection performed in advance.

28. A subculture timing determination system for determining the subculture timing of cells expanding or proliferating in a culture medium in a culture container, in, The subculture timing determination system has the following features: an imaging unit that captures an image of cells on a culture surface at each of a plurality of predetermined locations of the culture medium in the culture container to acquire a cell image; and a processing device for acquiring primary information of cells captured in the cell image, The imaging means images cells at a plurality of predetermined locations of the culture container at a first imaging time, and acquires cell images at each of the plurality of predetermined locations at the first imaging time. The processing device acquires primary information of the cell captured in the cell image at each point at the first capturing time, and calculates a statistical index value of the primary information of the cell. The processing device determines the imaging time corresponding to the time when the statistical index value reaches a predetermined threshold as the time when the passage timing has arrived.

29. A subculture timing determination system for determining the subculture timing of cells expanding or proliferating in a culture medium in a culture container, in, The subculture timing determination system has the following features: an imaging unit that captures an image of cells on a culture surface at each of a plurality of predetermined locations of the culture medium in the culture container to acquire a cell image; and a processing device for acquiring primary information of cells captured in the cell image, The imaging component images cells at a plurality of predetermined locations of the culture container at a first imaging time and a second imaging time after the first imaging time, and acquires cell images at each of the plurality of predetermined locations at the first imaging time and the second imaging time, respectively. The processing device obtains the first information of the cell captured in the cell image at each location at the first shooting time and the second shooting time, calculates the statistical index value of the first information at the first shooting time and the second shooting time, and calculates the time-varying index value based on the statistical index value at the first shooting time and the second shooting time. The processing device determines the imaging time corresponding to the time when the temporal change index value reaches a predetermined threshold value as the time when the passage timing has arrived.

30. A subculture timing determination system for determining the subculture timing of cells expanding or proliferating in a culture medium in a culture container, in, The subculture timing determination system has the following features: an imaging unit that captures an image of cells on a culture surface at each of a plurality of predetermined locations of the culture medium in the culture container to acquire a cell image; and a processing device for acquiring primary information of cells captured in the cell image, The imaging component images cells at a plurality of predetermined locations of the culture container at a first imaging time and a second imaging time after the first imaging time, and acquires cell images at each of the plurality of predetermined locations at the first imaging time and the second imaging time, respectively. The processing device obtains the first information of the cells captured in the cell images at the respective locations at the first shooting time and the second shooting time, calculates the time-varying index value based on the first information at the first shooting time and the second shooting time, and calculates the statistical index value of the time-varying index value at the respective first shooting time and the second shooting time, The processing device determines the imaging time corresponding to the time when the statistical index value reaches a predetermined threshold as the time when the passage timing has arrived.

31. A subculture timing determination system for determining the subculture timing of cells expanding or proliferating in a culture medium in a culture container, in, The subculture timing determination system has the following features: an imaging unit that captures an image of cells on a culture surface at each of a plurality of predetermined locations of the culture medium in the culture container to acquire a cell image; and a processing device for acquiring primary information of cells captured in the cell image, The photographing component photographs cells at a plurality of predetermined locations of the culture container at a first photographing time, a second photographing time after the first photographing time, and a third photographing time after the second photographing time, and acquires cell images at each of the plurality of predetermined locations at the first photographing time, the second photographing time, and the third photographing time, respectively. The processing device obtains the first information of the cells captured in the cell images at the respective locations at the first shooting time, the second shooting time, and the third shooting time, calculates the statistical index value of the first information at the first shooting time, the second shooting time, and the third shooting time, calculates the first time-varying index value of the statistical index value at the first shooting time and the second shooting time, and the first time-varying index value of the statistical index value at the second shooting time and the third shooting time, and calculates the second time-varying index value based on the first time-varying index value at the first shooting time and the second shooting time, and the first time-varying index value at the second shooting time and the third shooting time. The processing device determines the imaging time corresponding to the time when the second temporal change index value reaches a predetermined threshold value as the time when the passage timing has arrived.

32. A subculture timing determination system for determining the subculture timing of cells expanding or proliferating in a culture medium in a culture container, in, The subculture timing determination system has the following features: an imaging unit that captures an image of cells on a culture surface at each of a plurality of predetermined locations of the culture medium in the culture container to acquire a cell image; and a processing device for acquiring primary information of cells captured in the cell image, The photographing component photographs cells at a plurality of predetermined locations of the culture container at a first photographing time, a second photographing time after the first photographing time, and a third photographing time after the second photographing time, and acquires cell images at each of the plurality of predetermined locations at the first photographing time, the second photographing time, and the third photographing time, respectively. The processing device obtains the first information of the cell captured in the cell image at each location at the first shooting time, the second shooting time, and the third shooting time, calculates the first time change index value of the first information at the first shooting time and the second shooting time, and the first time change index value of the first information at the second shooting time and the third shooting time, and calculates the second time change index value based on the first time change index value at the first shooting time and the second shooting time, and the first time change index value at the second shooting time and the third shooting time, and calculates the statistical index value of the second time change index value at each of the first shooting time, the second shooting time, and the third shooting time, The processing device determines the imaging time corresponding to the time when the statistical index value reaches a predetermined threshold as the time when the passage timing has arrived.

33. A subculture timing determination system for determining the subculture timing of cells expanding or proliferating in a culture medium in a culture container, in, The subculture timing determination system has the following features: an imaging unit that captures an image of cells on a culture surface at each of a plurality of predetermined locations of the culture medium in the culture container to acquire a cell image; and a processing device for acquiring primary information of cells captured in the cell image, The photographing component photographs cells at a plurality of predetermined locations of the culture container at a first photographing time, a second photographing time after the first photographing time, and a third photographing time after the second photographing time, and acquires cell images at each of the plurality of predetermined locations at the first photographing time, the second photographing time, and the third photographing time, respectively. The processing device obtains the first information of the cell captured in the cell image at each location at the first shooting time, the second shooting time, and the third shooting time, calculates the first time change index value of the first information at the first shooting time and the second shooting time, and the first time change index value of the first information at the second shooting time and the third shooting time, calculates the statistical index value of the first time change index value at the first shooting time, the second shooting time, and the third shooting time, and calculates the second time change index value based on the statistical index value at the first shooting time and the second shooting time, and the statistical index value at the second shooting time and the third shooting time. The processing device determines the imaging time corresponding to the time when the second temporal change index value reaches a predetermined threshold value as the time when the passage timing has arrived.

34. The subculture timing determination system according to claim 29, in, The temporal change index value is a differential change amount, a change amount rate, a change rate per unit time, a change speed, a change time, a specific growth rate, or a predetermined doubling time of the statistical index value.

35. The subculture timing determination system according to claim 31, in, The one-time temporal change index value is a differential change amount, a change amount rate, a change rate per unit time, a change speed, a change time, a specific growth rate, or a predetermined doubling time of the statistical index value.

36. The subculture timing determination system according to claim 30, in, The temporal change index value is a differential change amount, a change amount rate, a change rate per unit time, a change speed, a change time, a specific growth rate, or a predetermined doubling time of the primary information.

37. The subculture timing determination system according to claim 32, in, The primary temporal change index value is a differential change amount, a change amount rate, a unit time change rate, a change speed, a change time, a specific growth rate, or a predetermined doubling time of the primary information.

38. The subculture timing determination system according to claim 33, in, The primary temporal change index value is a differential change amount, a change amount rate, a change rate per unit time, a change speed, a change time, a specific growth rate, or a predetermined doubling time of the primary information.

39. The system for determining the timing of cell culture according to any one of claims 28 to 33, in, The primary information is any one of the number of cells, cell density, cell-occupied area, single cell area, and relative distance between cells in the cell image.

40. The system for determining the timing of cell culture according to any one of claims 28 to 33, in, The statistical indicator value is an average value.

41. The system for determining the timing of cell culture according to any one of claims 28 to 33, in, The statistical indicator value is a sum.

42. The system for determining the timing of cell culture according to any one of claims 28 to 33, in, The statistical indicator value is variance or deviation.

43. The system for determining the timing of cell culture according to any one of claims 29 to 33, in, The predetermined threshold value is defined as a statistical indicator value at which cell extension or proliferation no longer occurs in a preliminary test performed in advance.

44. The subculture timing determination system according to claim 42, in, The predetermined threshold value is defined as an inflection point of the temporal change of the variance or the deviation in a prior test performed in advance.

45. The subculture timing determination system according to claim 34, in, The change rate is calculated as a value at any shooting time. The predetermined threshold value is limited to the second time-varying index value at a time when no increasing tendency of the time-varying index value is observed in a preliminary inspection performed in advance.

46. ​​The system for determining the timing of cell culture according to claim 34, in, The change rate is calculated as a ratio relative to the value at the time of the next shot. The predetermined threshold value is defined as an inflection point of the temporal change of the temporal change index value in a preliminary inspection performed in advance.

47. The subculture timing determination system according to claim 35, in, The change rate is calculated as a value at any shooting time. The predetermined threshold value is limited to the secondary time-varying index value at a time when no increasing tendency of the secondary time-varying index value is observed in a preliminary inspection performed in advance.

48. The system for determining the timing of cell culture according to claim 35, in, The change rate is calculated as a ratio relative to the value at the time of the next shot. The predetermined threshold value is defined as an inflection point of the time change of the secondary time-varying change index value in a preliminary inspection performed in advance.

49. The subculture timing determination system according to claim 36 or 37, in, The change rate is limited to the value at any shooting time. The predetermined threshold value is limited to the secondary time-varying index value at a time when no increasing tendency of the statistical index value is observed in a preliminary inspection performed in advance.

50. The subculture timing determination system according to claim 36 or 37, in, The change rate is calculated as a ratio relative to the value at the time of the next shot. The predetermined threshold value is defined as an inflection point of the temporal change of the statistical index value in a preliminary test performed in advance.

51. The subculture timing determination system according to claim 38, in, The change rate is calculated as a value at any shooting time. The predetermined threshold value is limited to the secondary time-varying index value at a time when no increasing tendency of the secondary time-varying index value is observed in a preliminary inspection performed in advance.

52. The subculture timing determination system according to claim 38, in, The change rate is calculated as a ratio relative to the value at the time of the next shot. The predetermined threshold value is defined as an inflection point of the time change of the secondary time-varying change index value in a preliminary inspection performed in advance.

53. The system for determining the timing of cell culture according to any one of claims 31 to 33, in, The time interval between the first imaging time and the second imaging time and the time interval between the second imaging time and the third imaging time are the same time interval.

54. The system for determining the timing of cell culture according to any one of claims 28 to 33, in, The predetermined threshold value is defined by machine learning in a preliminary inspection performed in advance.

55. The system for determining the timing of cell culture according to any one of claims 28 to 33, in, The processing device is configured separately from the imaging means so as to be communicable with the imaging means.

56. A cell culture system, which is a cell culture system of cells expanding or proliferating in a culture medium in a culture container, in, The cell culture system has: The subculture timing determination system according to claim 28; and At least one of a culture medium unit for replacing the culture medium of the culture container, a culture unit for storing the culture container at a predetermined temperature, and a transport unit for transporting the culture container between the subculture timing determination system and the culture unit.

57. A cell culture system, which is a cell culture system of cells expanding or proliferating in a culture medium in a culture container, in, The cell culture system has: The subculture timing determination system according to claim 29; and At least one of a culture medium unit for replacing the culture medium of the culture container, a culture unit for storing the culture container at a predetermined temperature, and a transport unit for transporting the culture container between the subculture timing determination system and the culture unit.

58. A cell culture system, which is a cell culture system of cells expanding or proliferating in a culture medium in a culture container, in, The cell culture system has: The subculture timing determination system according to claim 30; and At least one of a culture medium unit for replacing the culture medium of the culture container, a culture unit for storing the culture container at a predetermined temperature, and a transport unit for transporting the culture container between the subculture timing determination system and the culture unit.

59. A cell culture system, which is a cell culture system of cells expanding or proliferating in a culture medium in a culture container, in, The cell culture system has: The subculture timing determination system according to claim 31; and At least one of a culture medium unit for replacing the culture medium of the culture container, a culture unit for storing the culture container at a predetermined temperature, and a transport unit for transporting the culture container between the subculture timing determination system and the culture unit.

60. A cell culture system, which is a cell culture system of cells expanding or proliferating in a culture medium in a culture container, in, The cell culture system has: The subculture timing determination system according to claim 32; and At least one of a culture medium unit for replacing the culture medium of the culture container, a culture unit for storing the culture container at a predetermined temperature, and a transport unit for transporting the culture container between the subculture timing determination system and the culture unit.

61. A cell culture system, which is a cell culture system of cells expanding or proliferating in a culture medium in a culture container, in, The cell culture system has: The subculture timing determination system according to claim 33; and At least one of a culture medium unit for replacing the culture medium of the culture container, a culture unit for storing the culture container at a predetermined temperature, and a transport unit for transporting the culture container between the subculture timing determination system and the culture unit.

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