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

Abstract

It is demanded to perform subculture at an appropriate timing in accordance with the state of actual cells. The problem is solved by this subculture timing determination method comprising: an imaging step for imaging cells at a plurality of prescribed positions in a culture container at a first imaging time point to obtain cell images at each position of the plurality of the prescribed positions at the first imaging time point; a primary information obtaining step for obtaining primary information of cells imaged in the cell images at each position at the first imaging time point; a statistic index value obtaining step for calculating a statistic index value of the primary information of cells; and a determining step for determining an imaging time point corresponding to the time point when the statistic index value has reached a prescribed threshold value to be a time point when a subculture timing has been reached.

Description

[Technical Field]

[0001] The present invention relates to a method and system for determining the passage timing in cell culture, and a culture system using the same. In particular, the present invention relates to a method and system for determining the passage timing for cells spreading or growing in a medium in a culture vessel, and a culture system using the same. [Background technology]

[0002] In the culture of cells, such as stem cells, cells are seeded into a culture medium placed in a culture vessel such as a dish and cultured in an incubator. The seeded cells adhere to the culture surface at the bottom of the culture medium inside the culture vessel, and after a certain period of time, they spread or proliferate, covering the culture surface. However, as contact between adjacent cells increases, spreading or proliferation ceases. Therefore, to compensate for the lack of space, cells are passaged, i.e., dispersed and reseeded into a new vessel, and the culture is continued. Therefore, optimal passage timing is important for efficient cell spreading or proliferation. Generally, this timing is considered to be when the cell occupancy rate on the culture surface reaches approximately 80% of the confluent state. Traditionally, the passage time has been empirically determined based on the predicted time when this point is reached. [Prior art documents] [Patent documents]

[0003] [Patent Document 1] Japanese Patent Application Laid-Open No. 2005-192485 [Patent Document 2] Japanese Patent Application Laid-Open No. 2001-275659 Summary of the Invention [Problem to be solved by the invention]

[0004] However, cell spreading or proliferation rates vary from cell to cell, and differences in the conditions when cells are inoculated into the culture vessel result in individual differences in the timing of passaging for each culture vessel. Figures 6A and 6B show the distribution of cells within the observation point Op. For example, as shown in Figure 6A, ideal cell culture involves minimal deviation in intercellular distances, with the intercellular distance gradually decreasing on average from the inoculated state without any deviation. In such ideal culture, the optimal time for passaging is when the cell occupancy rate in the culture vessel reaches a predetermined value. An example of this point is when the cell occupancy rate reaches approximately 80% of the confluent state. However, as mentioned above, the timing for passaging is generally determined based on the empirical estimation of the cell occupancy rate, without determining whether the cell occupancy rate has reached a predetermined standard. This is due to the difficulty of easily determining the cell occupancy rate in a short period of time. Patent Documents 1 and 2 disclose, as examples of technical methods to solve this problem, a method for objectively determining the timing of passaging, which was previously determined empirically, based on image data of cells in the culture medium.

[0005] Patent Document 1 discloses a culture state detection device that calculates cell density based on the correlation between the run length of cells on a linear measurement line that intersects image data of the culture medium and the cell density per unit area of ​​the culture medium, and compares this with a predetermined judgment value to determine the timing of passaging. However, although Patent Document 1 uses image data as two-dimensional data, the data is one-dimensional because it uses a linear measurement line that intersects the image data, which reduces the accuracy of accurately capturing the distribution of cells on the culture surface, and this reduced accuracy has a negative impact on determining the accurate timing of passaging.

[0006] On the other hand, Patent Document 2 discloses a culture method and culture device that calculates the cell number, cell concentration, adherent cell occupation area, or adherent cell occupation rate in a culture medium based on image data of cells on the culture surface in the culture medium, and determines the timing of passaging based on the calculation results. However, Patent Document 2 determines the timing of passaging by comparing image data of captured cells with image data of previously captured cells, performing pattern recognition to identify differences, and measuring the degree of cell spreading or proliferation. Furthermore, when predicting the timing of subsequent passaging, the passaging timing is predicted not only from image data but also from medium consumption. That is, Patent Document 2 does not perform fixed-point observation at multiple predetermined points on the culture surface, and further predicts the passaging timing from medium consumption, which increases the computational load for judgment and requires a large system configuration other than the image. Therefore, there is a need for a simple configuration to determine the appropriate passaging timing based only on image data of cells on the culture surface, without using the state of the culture medium.

[0007] Furthermore, as shown in Figure 6B, when there is a large imbalance in intercellular distances within the culture surface, the appropriate time for passaging occurs before the cell occupancy rate in the medium reaches approximately 80%. However, Patent Documents 1 and 2 do not grasp the imbalance of cells within the culture surface and use this information to determine the timing of passaging. Therefore, it is necessary to grasp the imbalance of cells within the culture surface and determine the appropriate timing for passaging.

[0008] Furthermore, even if the start time of culture is the same, the timing of passaging does not necessarily coincide between culture vessels. Therefore, if the timing of passaging is uniformly determined at a fixed time, the optimal timing for passaging will be missed. Therefore, it is necessary to determine the passaging timing based on the changes over time of the cells in the culture vessel at specified intervals, and to extract multiple specified points and perform fixed-point observations as elements for determining the passaging timing, so that the passaging can be determined at the appropriate time depending on the actual state of the cells on the culture surface. [Means for solving the problem]

[0009] One aspect of the present invention is a passaging timing determination method for determining the passaging timing of cells expanding or growing in a medium in a culture vessel, the passaging timing determination method comprising: an imaging step of photographing cells at a plurality of predetermined points in the culture vessel at a first imaging time point and obtaining cell images of each of the predetermined points at the first imaging time point; a primary information acquisition step of acquiring primary information of the cells photographed in the cell images at each of the predetermined points at the first imaging time point; a statistical index value acquisition step of calculating a statistical index value of the primary information of the cells; and a determination step of determining that the imaging time point corresponding to the time point at which the statistical index value reaches a predetermined threshold value is the time point at which the passaging timing has been reached.

[0010] Another aspect of the present invention is a passaging timing determination method for determining the passaging timing of cells expanding or growing in a medium in a culture vessel, the passaging timing determination method comprising: an imaging step of photographing cells at a plurality of predetermined points in the culture vessel at a first photographing time point and a second photographing time point after the first photographing time point, and obtaining cell images of each of the predetermined points at each of the first photographing time point and the second photographing time point; a primary information acquisition step of acquiring primary information of the cells photographed in the cell images of each of the points at each of the first photographing time point and the second photographing time point; a statistical index value acquisition step of calculating statistical index values ​​of the primary information at each of the first photographing time point and the second photographing time point; and a time-course calculation step of calculating a time-course index value from the statistical index values ​​at the first photographing time point and the second photographing time point, wherein the determination step comprises a determination step of determining that the photographing time point corresponding to the time point at which the time-course index value reaches a predetermined threshold is the time point at which the passaging timing has been reached.

[0011] A further aspect of the present invention is a passaging timing determination method for determining the passaging timing of cells expanding or growing in a medium in a culture vessel, the passaging timing determination method comprising: an imaging step of photographing cells at a plurality of predetermined points in the culture vessel at a first photographing time point and a second photographing time point after the first photographing time point, and obtaining cell images of each of the predetermined plurality of points at each of the first photographing time point and the second photographing time point; a primary information acquisition step of acquiring primary information of the cells photographed in the cell images at each of the points at each of the first photographing time point and the second photographing time point; a time-course change calculation step of calculating a time-course change index value from the primary information at the first photographing time point and the second photographing time point; a statistical index value acquisition step of calculating a statistical index value of the time-course change index value at each of the first photographing time point and the second photographing time point; and a determination step of determining that the photographing time point corresponding to the time point at which the statistical index value reaches a predetermined threshold is the time point at which the passaging timing has been reached.

[0012] A further aspect of the present invention is a method for determining the passage timing of cells expanding or growing in a medium in a culture vessel, the method comprising: an imaging step of photographing cells at a plurality of predetermined points in the culture vessel at a first photographing time point, a second photographing time point after the first photographing time point, and a third photographing time point after the second photographing time point, and obtaining cell images of each of the predetermined plurality of points at each of the first photographing time point, the second photographing time point, and the third photographing time point; a primary information acquisition step of acquiring primary information of the cells photographed in the cell images of each of the points at each of the first photographing time point, the second photographing time point, and the third photographing time point; and a primary information acquisition step of acquiring primary information of the cells photographed in the cell images of each of the points at each of the first photographing time point, the second photographing time point, and the third photographing time point. The passaging timing determination method includes: a statistical index value acquisition step of calculating a statistical index value of the primary information at each of the imaging time points; a primary time-course calculation step of calculating a primary time-course change index value of the statistical index value at the first imaging time point and the second imaging time point and a primary time-course change index value of the statistical index value at the second imaging time point and the third imaging time point; a secondary time-course calculation step of calculating a secondary time-course change index value from the primary time-course change index value at the first imaging time point and the second imaging time point and the primary time-course change index value at the second imaging time point and the third imaging time point; and a determination step of determining that the imaging time point corresponding to the time point at which the secondary time-course change index value reaches a predetermined threshold is the time point at which the passaging timing has been reached.

[0013] A further aspect of the present invention is a method for determining the passage timing of cells expanding or growing in a medium in a culture vessel, the method comprising: an imaging step of photographing cells at a plurality of predetermined points in the culture vessel at a first photographing time point, a second photographing time point after the first photographing time point, and a third photographing time point after the second photographing time point, and acquiring cell images of each of the predetermined plurality of points at each of the first photographing time point, the second photographing time point, and the third photographing time point; a primary information acquisition step of acquiring primary information of the cells photographed in the cell images of each of the points at each of the first photographing time point, the second photographing time point, and the third photographing time point; a secondary time-course calculation step of calculating a secondary time-course index value from the primary time-course index values ​​at the first and second imaging time points and the primary time-course index values ​​at the second and third imaging time points; a statistical index value acquisition step of calculating statistical index values ​​of the secondary time-course index values ​​at each of the first, second, and third imaging time points; and a determination step of determining that the imaging time point corresponding to the time point at which the statistical index value reaches a predetermined threshold is the time point at which the passage timing has been reached.

[0014] A further aspect of the present invention is a method for determining the passage timing of cells expanding or growing in a medium in a culture vessel, the method comprising: an imaging step of photographing cells at a plurality of predetermined points in the culture vessel at a first photographing time point, a second photographing time point after the first photographing time point, and a third photographing time point after the second photographing time point, and acquiring cell images of each of the predetermined plurality of points at each of the first photographing time point, the second photographing time point, and the third photographing time point; a primary information acquisition step of acquiring primary information of the cells photographed in the cell images of each of the points at each of the first photographing time point, the second photographing time point, and the third photographing time point; The passaging timing determination method includes a primary time-course change calculation step of calculating a primary time-course change index value of primary information and primary time-course change index values ​​of the primary information at the second and third imaging time points; a statistical index value acquisition step of calculating statistical index values ​​of the primary time-course change index value at each of the first, second, and third imaging time points; a secondary time-course change calculation step of calculating a secondary time-course change index value from the statistical index values ​​at the first and second imaging time points and the statistical index values ​​at the second and third imaging time points; and a determination step of determining that the imaging time point corresponding to the time point at which the secondary time-course change index value reaches a predetermined threshold is the time point at which the passaging timing has been reached.

[0015] A further aspect of the present invention is a passaging timing determination system for determining the passaging timing of cells expanding or growing in a culture medium in a culture vessel, comprising: an imaging means for photographing cells on a culture surface at each of a plurality of predetermined points in the culture medium of the culture vessel and obtaining cell images; and a processing device for obtaining primary information about the cells photographed in the cell images, wherein the imaging means photographs cells at the plurality of predetermined points in the culture vessel at a first photographing time point and obtains cell images of each of the plurality of predetermined points at the first photographing time point; the processing device obtains the primary information about the cells photographed in the cell images at each of the predetermined points at the first photographing time point and calculates a statistical index value of the primary information about the cells; and the processing device determines that the photographing time point corresponding to the time point at which the statistical index value reaches a predetermined threshold is the time point at which the passaging timing has been reached.

[0016] A further aspect of the present invention is a passaging timing determination system for determining the passaging timing of cells expanding or growing in a medium in a culture vessel, comprising: an imaging means for photographing cells on a culture surface at each of a plurality of predetermined points in the medium of the culture vessel and obtaining cell images; and a processing device for obtaining primary information about the cells photographed in the cell images, wherein the imaging means photographs the cells at the plurality of predetermined points in the culture vessel at a first photographing time point and a second photographing time point after the first photographing time point and obtains cell images of each of the predetermined plurality of points at each of the first photographing time point and the second photographing time point; the processing device obtains the primary information about the cells photographed in the cell images at each of the first photographing time point and the second photographing time point and calculates statistical index values ​​of the primary information at each of the first photographing time point and the second photographing time point, and calculates a time-course index value from the statistical index values ​​at the first photographing time point and the second photographing time point; and the processing device determines that the photographing time point corresponding to the time point at which the time-course index value reaches a predetermined threshold is the time point at which the passaging timing has been reached.

[0017] A further aspect of the present invention is a passaging timing determination system for determining the passaging timing of cells expanding or growing in a culture medium in a culture vessel, comprising: an imaging means for photographing cells on a culture surface at each of a plurality of predetermined points in the culture medium in the culture vessel and obtaining cell images; and a processing device for obtaining primary information about the cells photographed in the cell images, wherein the imaging means photographs the cells at the plurality of predetermined points in the culture vessel at a first photographing time point and a second photographing time point after the first photographing time point and obtains cell images of each of the predetermined plurality of points at each of the first photographing time point and the second photographing time point; the processing device obtains primary information about the cells photographed in the cell images at each of the first photographing time point and the second photographing time point, calculates a time-course index value from the primary information at the first photographing time point and the second photographing time point, and calculates statistical index values ​​for the time-course index values ​​at the first photographing time point and the second photographing time point; and the processing device determines that the photographing time point corresponding to the time point at which the statistical index value reaches a predetermined threshold is the time point at which the passaging timing has been reached.

[0018] A further aspect of the present invention is a passaging timing determination system for determining the passaging timing of cells spreading or growing in a medium in a culture vessel, comprising: an imaging means for photographing cells on a culture surface at each of a plurality of predetermined points in the medium of the culture vessel and acquiring them as cell images; and a processing device for acquiring primary information of the cells photographed in the cell images, wherein the imaging means photographs the cells at the plurality of predetermined points in the culture vessel at a first photographing time point, a second photographing time point after the first photographing time point, and a third photographing time point after the second photographing time point, and acquires them as cell images of each of the plurality of predetermined points at the first photographing time point, the second photographing time point, and the third photographing time point, and the processing device a first time-course change index value of the statistical index value at the first time point and the second time point and a first time-course change index value of the statistical index value at the second time point and the third time point; a second time-course change index value from the first time point and the second time point and the first time-course change index value at the second time point and the third time point; and a second time-course change index value from the first time point and the second time point and the first time-course change index value at the second time point and the third time point; and the processing device determines that the photographing time point corresponding to the time point at which the second time-course change index value reaches a predetermined threshold is the time point at which the passaging timing has been reached.

[0019] A further aspect of the present invention is a passaging timing determination system for determining the passaging timing of cells spreading or growing in a medium in a culture vessel, comprising: an imaging means for photographing cells on a culture surface at each of a plurality of predetermined points in the medium of the culture vessel and acquiring the cell images; and a processing device for acquiring primary information of the cells photographed in the cell images, wherein the imaging means photographs the cells at the plurality of predetermined points in the culture vessel at a first photographing time point, a second photographing time point after the first photographing time point, and a third photographing time point after the second photographing time point, and acquires the cell images of each of the plurality of predetermined points at each of the first photographing time point, the second photographing time point, and the third photographing time point, and a first imaging time point and a second imaging time point, and a second imaging time point, and a third imaging time point, respectively; a second imaging time point and a second imaging time point are used to calculate a second imaging time point and a second imaging time point from the first imaging time point and the second imaging time point and the first imaging time point and the second imaging time point, respectively; a second imaging time point and a second imaging time point are used to calculate a second imaging time point and a second imaging time point from the first imaging time point and the second imaging time point and the first imaging time point and the third imaging time point; a second imaging time point and a second imaging time point are used to calculate a second imaging time point and a second imaging time point from the first imaging time point and the second imaging time point and the first imaging time point and the third imaging time point, respectively; and a second imaging time point and a second imaging time point are used to calculate a second imaging time point and a second imaging time point from the second imaging time point and the third imaging time point, respectively; and a second imaging time point and a third ... processing device determines that the imaging time point corresponding to the time when the statistical index value reaches a predetermined threshold is the time when the passage timing has been

[0020] A further aspect of the present invention is a passaging timing determination system for determining the passaging timing of cells spreading or growing in a medium in a culture vessel, comprising: an imaging means for photographing cells on a culture surface at each of a plurality of predetermined points in the medium of the culture vessel and acquiring the cell images; and a processing device for acquiring primary information of the cells photographed in the cell images, wherein the imaging means photographs the cells at the plurality of predetermined points in the culture vessel at a first photographing time point, a second photographing time point after the first photographing time point, and a third photographing time point after the second photographing time point, and acquires the cell images of each of the plurality of predetermined points at the first photographing time point, the second photographing time point, and the third photographing time point, respectively; and the processing device photographs the cells at the first photographing time point, the second photographing time point, and the third photographing time point, and acquires the cell images of each of the plurality of predetermined points at the first photographing time point, the second photographing time point, and the third photographing time point. acquires primary information of the cells photographed in the cell images at each of the points at each of the above; calculates primary time-course change index values ​​of the primary information at the first and second photographing times and primary time-course change index values ​​of the primary information at the second and third photographing times; calculates statistical index values ​​of the primary time-course change index values ​​at each of the first, second, and third photographing times; calculates secondary time-course change index values ​​from the statistical index values ​​at the first and second photographing times and the statistical index values ​​at the second and third photographing times; and the processing device determines that the photographing time corresponding to the time at which the secondary time-course change index value reaches a predetermined threshold is the time at which the passaging timing has been reached. [Effects of the Invention]

[0021] The present invention makes it possible to determine the appropriate timing for passage in cell culture. [Brief explanation of the drawings]

[0022] [Figure 1A] 1 shows a schematic external configuration of a cell culture system of the present invention. [Figure 1B] 1 shows an example of a functional block diagram of a cell culture system of the present invention. [Figure 1C]FIG. 2 shows another example of a functional block diagram of the cell culture system of the present invention. [Figure 1D] FIG. 10 shows yet another example of a functional block diagram of the cell culture system of the present invention. [Figure 2A] 1 shows an outline of the flow of the culture process in the cell culture system of the present invention. [Figure 2B] 1 shows an outline of the overall processing flow of the passage determination process of the present invention. [Figure 2C] 1 shows a schematic external configuration of a passage timing determination unit of the present invention. [Figure 2D] 1 shows the concept of an image captured by the imaging means of the passage timing determination unit of the present invention. [Figure 2E] 1 shows a time-lapse concept of the photographing time points of images acquired in the passage timing determination unit of the present invention. [Figure 2F] 3 shows a flow of a process for generating judgment data in the first embodiment of the present invention. [Figure 3A] 10 shows a flow of a process for generating judgment data in a first example of the second embodiment of the present invention. [Figure 3B] 10 shows a flow of a process for generating judgment data in a second example of the second embodiment of the present invention. [Figure 3C] 10 shows an example of the concept of selecting a target photographing time point in the nearest ratio method according to the second embodiment of the present invention. [Figure 3D] 10 shows another example of the concept of selecting a target photographing time point in the nearest ratio method according to the second embodiment of the present invention. [Figure 3E] 10 shows an example of the concept of selecting a target photographing time point in the reference ratio method according to the second embodiment of the present invention. [Figure 3F] 10 shows a concept of generating an example of a time-varying index value in the second embodiment of the present invention. [Figure 3G] 10 shows the concept of generating specific growth rate and doubling time as time-dependent change index values, which is another example according to the second embodiment of the present invention. [Figure 4A]13 shows a flow of a process for generating judgment data in a first example of the third embodiment of the present invention. [Figure 4B] 13 shows a flow of a process for generating judgment data in a second example of the third embodiment of the present invention. [Figure 4C] 10 shows a flow of a process for generating judgment data in a third example of the third embodiment of the present invention. [Figure 4D] 13 shows an example of the concept of selecting a target photographing time point in the nearest ratio method according to the third embodiment of the present invention. [Figure 4E] 13 shows another example of the concept of selecting a target photographing time point in the nearest ratio method according to the third embodiment of the present invention. [Figure 4F] 13 shows an example of the concept of selecting a target photographing time point in the reference ratio method according to the third embodiment of the present invention. [Figure 4G] 13 shows another example of the concept of selecting a target photographing time point in the reference ratio method according to the third embodiment of the present invention. [Figure 4H] 10 shows a concept of generating a difference change amount and a change amount rate as a primary time-varying index value and a secondary time-varying index value in the third embodiment of the present invention. [Figure 4I] 10 shows the concept of generating a unit time change rate as a primary time change index value and a change rate difference as a secondary time change index value in the third embodiment of the present invention. [Figure 4J] 10 shows the concept of specific growth rate and doubling time as primary time-dependent index values ​​in the third embodiment of the present invention. [Figure 5A] 13 shows an example of a concept of variations in selection of a target photographing time point according to the fourth embodiment of the present invention. [Figure 5B] 10 illustrates the concept of a reference value integration method according to a fourth embodiment of the present invention. [Figure 5C] 10 shows the concept of a matrix time-course method according to a fourth embodiment of the present invention. [Figure 5D] 10 shows the concept of a matrix time-course method according to a fifth embodiment of the present invention. [Figure 5E]10 shows the concept of a matrix time-course method according to a fifth embodiment of the present invention. [Figure 6A] FIG. 1 is a diagram showing a schematic view of the distribution of cells within an observation point Op, showing a case where the distribution of cells is not biased. [Figure 6B] FIG. 1 is a diagram showing a schematic view of the distribution of cells within an observation point Op, showing a case where the distribution of cells is highly biased. DETAILED DESCRIPTION OF THE INVENTION

[0023] [First embodiment] (Cell culture system) A method and system for determining the timing of passaging according to a first embodiment of the present invention will be described with reference to Figures 1A to 2F. The present invention is particularly suited to passaging adherent cells in culture vessels, but is not necessarily limited to adherent cells. The timing of passaging is achieved by a passaging timing determination unit 3, which serves as a passaging timing determination system, and a cell culture system 1 having the same. Below, the passaging timing determination of the first embodiment will be described with reference to the passaging timing determination unit 3 and the cell culture system 1. Figure 1A is a schematic diagram of the appearance of the cell culture system 1. Figure 1B is an example of a functional block diagram showing the electrical connections of the cell culture system 1. Figures 1C and 1D are examples of other functional block diagrams showing the electrical connections of the cell culture system 1. Figure 2A is a diagram showing an outline of the flow of the culture passaging process in the cell culture system 1 of the present invention. Figure 2B is a diagram showing the flow of the passaging determination process according to the first embodiment.

[0024] 1B, the cell culture system 1 includes internal units such as a culture medium unit 2, a passage timing determination unit 3, a culture unit 4 serving as an incubator, a cell inoculation device 5, a transfer unit 6, a centrifuge device 7, and a processing device 8. The cell culture system 1 does not necessarily have to include all of these units, and may include at least some of these units.

[0025] Within the cell culture system 1, the processing device 8 is electrically connected to the group of units that make up the cell culture system 1 (culture medium unit 2, passaging timing determination unit 3, culture unit 4, cell inoculation device 5, transfer unit 6, and centrifuge device 7) so that signals can be exchanged between these units. The culture medium unit 2 is a device that introduces culture medium into the culture vessel, removes and recovers old culture medium for medium replacement, and introduces new culture medium. The passaging timing determination unit 3 is a device that determines the timing of passaging the culture vessel and is equipped with a camera 31 as an imaging means inside. The culture unit 4 is a device that stores the culture vessel in an internal chamber and maintains the chamber at a predetermined temperature. The cell inoculation device 5 is a device that inoculates cells, for example, by dropping a cell suspension into the culture vessel at the initial stage or during passaging. The transfer unit 6 is a device that moves the culture vessel between the group of units. The centrifuge device is a device that separates and recovers cells detached from the culture vessel during passaging.

[0026] 1B, the processing device 8 transmits control signals to each of the culture medium unit 2, the passaging timing determination unit 3, the culture unit 4, the cell inoculation device 5, the transfer unit 6, and the centrifuge device 7 to control their respective operations. The processing device 8 also receives status signals from each of the culture medium unit 2, the passaging timing determination unit 3, the culture unit 4, the cell inoculation device 5, the transfer unit 6, and the centrifuge device 7.

[0027] The processing device 8 controls the group of units constituting the cell culture system 1 of the present invention (culture medium unit 2, passaging timing determination unit 3, culture unit 4, cell inoculation device 5, transfer unit 6, and centrifuge device 7), and receives and transmits the signals required for these. In addition to this, it can also receive signals of data detected in each unit. That is, it can also receive data (such as cell count) detected in the culture medium unit 2, the passaging timing determination unit 3, etc. Furthermore, as will be described later, the processing device 8 can also process data from each unit, particularly the passaging timing determination unit 3, and make necessary decisions.

[0028] Next, the outline of the function of each unit and the culture passage process of the present invention will be described with reference to FIG. 2A. First, in STEP 1, the cell inoculation process, a culture medium containing cells is formed in the evaluation culture vessel Pe. That is, here, a cell suspension containing cells is poured into the evaluation culture vessel Pe using the cell inoculation device 5, or a culture medium is poured into the evaluation culture vessel Pe and the cells are inoculated therein. For example, the evaluation culture vessel Pe is prepared by pouring the cell suspension into a flat culture vessel such as a dish or petri dish using the dispensing tube 21a of the dispenser 21, or by pouring the culture medium and then pouring the cell suspension into the culture medium.

[0029] The evaluation culture vessel Pe inoculated with cells in STEP 1 is placed on shelf 41 in culture unit 4 and stored at a constant temperature for a predetermined period of time as the culture process in STEP 2. During this period, the cells in the evaluation culture vessel Pe adhere to the bottom of the evaluation culture vessel Pe and spread or grow within the evaluation culture vessel Pe containing the medium. When the predetermined period has elapsed, the evaluation culture vessel Pe undergoes the passaging timing determination process in STEP 3. The processing device 8 counts the timing of the predetermined period and sends a control signal to the transfer unit 6 to remove the evaluation culture vessel Pe to be determined from the culture unit 4 and transfer the evaluation culture vessel Pe to the passaging timing determination unit 3.

[0030] In STEP 3, the evaluation culture vessel Pe is removed from the culture unit 4 and transferred to the passaging timing determination unit 3, where the medium in the evaluation culture vessel Pe is photographed to determine whether the time for passaging has arrived. If it is determined that the evaluation culture vessel Pe has not yet arrived at the time for passaging, the process returns to the culture process in STEP 2. STEP 2 and STEP 3 are repeatedly executed until it is determined that the time for passaging has arrived. In the culture process in STEP 2, if it is determined that the time for passaging has not yet arrived but the medium needs to be replaced, the medium is replaced in STEP 4 using the medium unit 2, as necessary, and the process returns to the culture process in STEP 2. Also, if the result of the passaging timing determination indicates that the time for passaging has not yet arrived but the medium needs to be replaced, the medium is replaced in STEP 4 using the medium unit 2, and the process returns to the culture process in STEP 2. On the other hand, if it is determined that the time for passaging has arrived for the evaluation culture vessel Pe in the passaging timing determination process, the process proceeds to the passaging process in STEP 5. The passaging process in STEP 5 can be executed, for example, as follows. First, the cells in the evaluation culture vessel Pe are removed and recovered, a cell suspension containing the removed cells is prepared, and the cell suspension is then introduced into a new evaluation culture vessel Pe using a cell inoculation device 5 to seed the cells. When preparing the cell suspension, a centrifuge device 7 is used to separate the cells if necessary. Once the cells are seeded into the new evaluation culture vessel Pe, the passaging is complete. After passaging, the process returns to the culture process in STEP 2, and STEPs 2 to 5 are repeated as necessary.

[0031] Between each of STEPs from STEP 1 to STEP 5, the transfer of the evaluation culture vessel Pe between the culture medium unit 2, the passage timing determination unit 3, the culture unit 4, and the cell inoculation device 5 is performed by a transfer unit 6. The transfer unit 6 has an extendable arm 62 attached to a moving platform 61 that moves along, for example, an elevation rail, and can move in front of each unit to take the evaluation culture vessel Pe in and out of each unit. The tip of the arm 62 has an actuator 63 that can grip the evaluation culture vessel Pe.

[0032] The processing device 8 is disposed, for example, as shown in FIG. 1B , outside each unit group (such as the culture medium unit 2 and the passaging timing judgment unit 3) constituting the cell culture system 1 so as to provide overall control of the unit groups. However, in addition to or instead of the processing device 8, an internal processor for control and data processing of each unit may be disposed within each unit. In this embodiment, the unit in which the processor is disposed can function independently of the other units. For example, in the case of the passaging timing judgment unit 3, a device for performing this processing may be disposed within the passaging timing judgment unit 3 so as to be specialized for control and data processing of the passaging timing judgment unit 3. The passaging timing judgment unit 3 can be used independently of the other units.

[0033] That is, in this example, the processing device 8 is arranged as the processing device 33, which is an internal processor within the passage timing judgment unit 3, as shown in Figure 1C, and the processing device 33 can be made to control and process data for the passage timing judgment unit 3 instead of the processing device 8, thereby enabling the passage timing judgment unit 3 to function independently of other units.

[0034] Furthermore, as shown in FIG. 1D , the processing device 33 can be located outside the passaging timing judgment device 3a, which is physically and externally recognized as the passaging timing judgment unit 3, and outside the cell culture device 1a, which is physically and externally recognized as the cell culture system 1. In this case, the processing device 33 is communicatively connected to the passaging timing judgment device 3a via a communication means and a network N. For example, the passaging timing judgment device 3a is connected to a camera 31, which serves as an imaging means, via a communication means and the network N. In this case, the network N and the processing device 33 may be configured as a cloud. In this example, even if the processing device 33 appears (physically and externally) to be located outside the passaging timing judgment device 3a or the cell culture device 1a, the passaging timing judgment unit 3 essentially (electrically and system-wise) includes the processing device 33. Furthermore, if the cell culture system 1 includes the passaging timing judgment unit 3, the cell culture system 1 will have the same configuration as that shown in FIG. 1C in terms of its substantial system configuration. This allows the processing device 33 to be located away from the passaging timing determination device 3a in which the camera 31 is located, and allows processing and control in the passaging timing determination device 3a from the outside.

[0035] Next, the passaging timing judgment performed by the passaging timing judgment unit 3 will be described below. Here, an example in which the processing device 33 is disposed within the passaging timing judgment unit 3 (the configuration of FIG. 1C ) will be described, but as described above, the same applies when the processing is performed by a processing device 8 disposed outside the passaging timing judgment unit 3 rather than by disposing the processing device 33 within the unit 3. In this case, the processing device 8 may be provided with the following functions that are performed by the processing device 33.

[0036] (Passaging timing determination unit) 2B, we will now explain the passaging timing determination unit 3 and the passaging determination process in the passaging timing determination method, which is the above-mentioned STEP 3. The passaging determination process first comprises an imaging step (S11) of acquiring a cell image, a determination data creation step (S12) of creating determination data from the cell image, and a determination step (S13) of determining whether the time for passaging has arrived.

[0037] First, in the imaging step (S11), an image of the cells in the evaluation culture vessel Pe is captured. FIG. 2C shows a schematic external configuration of the passaging timing judgment unit 3. The passaging timing judgment unit 3 is equipped with a camera 31 as an imaging means, an imaging stage 32, and a processing device 33. An evaluation culture vessel Pe can be placed on the imaging stage 32, and the camera 31 can capture an image of the medium surface of the evaluation culture vessel Pe. A predetermined location on the medium surface in the evaluation culture vessel Pe transferred by the arm 62 of the transfer unit 6 is captured by the camera 31 and acquired as an image. Data of the acquired image is stored in a storage device (not shown).

[0038] The processing device 33 executes a determination data generation step (S12) and a determination step (S13). The processing device 33 acquires the images acquired in the imaging step (S11) and processes the data obtained from the images to determine the timing of passage. These steps will be specifically described below with reference to Figures 2C to 2F.

[0039] (imaging process) First, in the imaging step (S11), the camera 31 captures images of cells on the culture surface of the medium for all evaluation culture vessels Pe in the culture unit 4 of the cell culture system 1 to obtain cell images. In this specification, the term "medium" refers to the in-plane direction of the bottom surface of the evaluation culture vessel Pe, and the term "culture surface" refers to the portion of the vessel in the depth direction within the medium where cells adhere. One evaluation culture vessel Pe to be evaluated is removed from the culture unit 4 by the arm 62 of the transfer unit 6 and transferred into the passaging timing judgment unit 3 by the transfer unit 6. The transferred evaluation culture vessel Pe is placed on the imaging stage 32 of the passaging timing judgment unit 3. The medium in the placed evaluation culture vessel Pe is imaged by the camera 31. After imaging, the evaluation culture vessel Pe is retrieved from the imaging stage 32 by the arm of the transfer unit 6 and transported back into the culture unit 4 by the transfer unit 6. The imaging step also includes repeating this process to capture images of cells on the culture surface for all evaluation culture vessels Pe to be evaluated within the culture unit 4.

[0040] FIG. 2D is a conceptual diagram of an image captured by the camera 31 of the culture surface of the medium in the evaluation culture vessel Pe. In the imaging process, for example, as shown in FIG. 2D, the culture surface of the medium in a certain evaluation culture vessel Pe is captured by the camera 31, and the captured image is stored in the storage device of the passaging timing determination unit 3 as a cell image. FIG. 2D shows the concept of capturing, for example, 6 x 6 (36) images. However, in reality, images are captured in finer divisions than this. The imaged medium areas are selected from the entire area of ​​the evaluation culture vessel Pe, and are medium areas at a number of predetermined observation points Op determined to represent the state of the evaluation culture vessel Pe so as to be suitable for evaluating at least the culture surface of the evaluation culture vessel Pe. For example, in FIG. 3B, the hatched areas in the three images are designated as observation points Op as examples of areas representative of the evaluation culture vessel Pe. The observation point Op is determined by first photographing the entire area of ​​the medium in a certain evaluation culture vessel Pe, selecting a location in the image area where the number of cells, etc., is sufficient to represent the evaluation culture vessel Pe, and then determining that location as the fixed observation point. Multiple observation points Op are typically selected and determined, such as three or ten locations. It is generally understood that using multiple locations improves the accuracy of determination. Once the observation point Op is determined, the same observation point Op is used for any one evaluation culture vessel Pe, even when photographing at different points in time, in the process of determining the passage timing to observe changes over time, which will be described below. Hereinafter, an arbitrary one evaluation culture vessel Pe will be described as a representative example, but the same applies to any multiple evaluation culture vessels Pe.

[0041] FIG. 2E illustrates the concept of performing an imaging step at multiple imaging time points. In the imaging step (S11), as described above, for an observation point Op of any one evaluation culture vessel Pe, images of cells on the culture surface are captured at multiple imaging time points at predetermined time intervals as the culture progresses, thereby obtaining cell images. At each imaging time point, cells on the culture surface are captured at multiple predetermined observation points Op selected from the medium of the evaluation culture vessel Pe to obtain cell images. The same observation point Op is selected at all imaging time points so that fixed-point observation is performed. At each imaging time point, the evaluation culture vessel Pe to be evaluated is removed from the culture unit 4 by the transfer unit 6 and transferred to the passaging timing determination unit 3. After the imaging is completed, the evaluation culture vessel Pe is transferred back to the culture unit 4. As the culture progresses, when a new imaging time point arrives, the imaging step (S11) is performed at that imaging time point. In a representative example, each time the imaging step (S11) is performed, the determination data creation step (S12) and determination step (S13), described below, are performed.

[0042] In the first embodiment, the timing of passage is determined using only cell images captured at one imaging time point selected from multiple imaging time points. In this specification, the imaging time point used to determine the timing of passage is referred to as the imaging time point of interest. In the first embodiment, the imaging time point of interest consists only of the evaluation imaging time point. In the first embodiment, the timing of passage is determined using only cell images captured at the first imaging time point, which is the imaging time point of interest in Figure 2E. As will be described later, in the second embodiment, cell images captured at two imaging time points of interest selected from multiple imaging time points are used, and in the third embodiment, cell images selected from three or more imaging time points of interest are used. In Figure 2E, the imaging time point of interest in the third embodiment is shown as a representative example in which cell images selected from three imaging time points of interest are used.

[0043] (Creating judgment data) Following the imaging step (S11), determination data for passage determination is created in the determination data creation step (S12). FIG. 2F shows the steps constituting the determination data creation step (S12) in the first embodiment. As shown in FIG. 2F, the determination data creation step (S12) in the first embodiment comprises a primary information acquisition step (S1201) for acquiring primary information from the cell image acquired in the imaging step (S11), and a subsequent statistical index value acquisition step (S1202) for calculating statistical index values ​​from the primary information.

[0044] (Primary information acquisition) In the primary information acquisition step (S1201), the processing device 33 acquires primary information from an image acquired at a selected imaging time point (a target imaging time point). The primary information is information about the cells captured in each cell image of the culture surface captured at each imaging time point. The primary information includes, for example, the cell count, cell density, cell-occupied area, cell area, and relative distance between cells. The primary information can be acquired from the images captured in each imaging step using commercially available or commonly available software. Among the primary information, the cell count refers to the number of cells captured in a captured image, meaning a directly obtainable cell count. For example, the cell density is calculated by dividing the acquired cell count by the area of ​​the imaged location. The area used here can be various, and can be an effective area that excludes impurities such as bubbles in the medium and portions of the image that were not successfully captured, and corresponds to an area where cells can spread or grow. Furthermore, the total area occupied by all cells is the occupied area, and the area of ​​a single cell is the area of ​​each cell captured in the image. The area occupied by a single cell is the size of each cell captured in the image, and for example, the relative distance between cells in a single cell image is the distance defined by two or more cells captured in the image. The timing of passage can be determined from primary information obtained from images captured at each capture time point.

[0045] (obtaining statistical indicator values) In the primary information acquisition step (S1201), primary information acquired at one imaging time point is acquired, followed by a statistical index value acquisition step (S1202). Here, a statistical index value corresponding to one imaging time point is calculated from the primary information acquired at that imaging time point. A statistical index value for each piece of primary information is further calculated as basic data indicating the state trend of the primary information. The statistical index value is an index value defined as a representative value that represents the primary information because it takes multiple values ​​for each image, and is, for example, at least one of a sum, an average value, a variance, and a deviation such as a standard deviation. That is, the statistical index value of the primary information is the sum (total value) of values ​​that can be acquired from each image taken at multiple observation points Op, the average value, or a variance or deviation for each image. For example, if the primary information is the number of cells, and there are three observation points Op at a certain imaging time point, the statistical index value is either the sum of the number of cells in the images at the three points, or the average value, variance, or standard deviation of the number of cells. The primary information and the statistical index value of the primary information are stored as acquired data in a storage means (not shown) as necessary.

[0046] Here, the average value and sum, and the variance and deviation selected as statistical index values ​​have, by definition, different technical characteristics as indicators of cell passage. The sum and average value as statistical index values ​​are indicators of the increase in cell number and cell spreading or proliferation, and its slowdown. The sum and average value can be calculated as the sum and average value at each of multiple observation points Op, and used as statistical index values ​​for the number of observation points Op. Furthermore, these may be averaged or otherwise processed statistically to obtain a representative value of 1 or a number smaller than a manageable number of observation points Op. On the other hand, the variance or deviation as statistical index values ​​has the characteristic of indicating the uniformity of cells within a container. The variance or deviation can be calculated as the variance or deviation at each of multiple observation points Op, and used as statistical index values ​​for the number of observation points Op. Furthermore, these may be averaged or otherwise processed statistically to obtain a representative value of 1 or a number smaller than a manageable number of observation points Op. In cell culture, cell uniformity generally deteriorates as cells spread or proliferate. Furthermore, the timing for passaging due to cell heterogeneity arrives before the timing for passaging due to cell number limitations. In other words, when the sum or mean is selected as the statistical index value, the cells in the culture vessel often become heterogeneous even before the time for passaging has arrived. In this case, some cells have already reached a limit in spreading or proliferation, and this can be said to be an appropriate time for passaging. Since the variance or deviation as a statistical index value is an index of the uniformity of cells in the culture vessel, it is possible to determine the timing for passaging more appropriately than by selecting the sum or mean as the statistical index value. The characteristics of the sum and mean, and the variance and deviation as statistical index values ​​are the same in the second and third embodiments described below.

[0047] (Subculture determination) After the creation of the determination data is completed, a determination step (S13) is performed to determine whether the time for passaging has arrived. In the determination step (S13), the timing for passaging is determined based on the statistical index value created in the determination data creation step (S12). The statistical index value quantitatively indicates the state of cells on the culture surface at predetermined observation points Op at predetermined points selected from within the culture vessel. Therefore, the sum of the statistical index values ​​at the predetermined observation points Op, for example, is the total number of cells within the area of ​​each predetermined observation point Op, and is a value representative of the total number of cells in the entire area of ​​the culture surface in the culture vessel, including points other than the observation point Op. The average statistical index value, for example, can be the number of cells per unit area or the number of cells per observation point Op, and is a representative value indicating the room for cells to spread or grow within the culture vessel beyond the sum.

[0048] In the determination step (S13), the statistical index value calculated in the statistical index value acquisition step (S1202) is compared with a predetermined threshold. Here, when the determination data reaches the predetermined threshold, it is determined that the time for passaging has arrived. The predetermined threshold is determined by conducting a test as a test in advance under the same conditions as those used to actually determine the time for passaging, obtaining a statistical index value at the time of passaging in that test, and defining this as the predetermined threshold.

[0049] The sum and average statistical index values ​​are highly effective in determining the timing of passage under ideal culture conditions in which the intercellular distance gradually decreases on average without any bias from the inoculated state. When the sum and average statistical index values ​​are selected, the passage timing is determined to be reached at the imaging time corresponding to the point at which the sum or average statistical index value reaches a predetermined threshold. The value at the timing when passage is required can be uniquely determined as the sum or average of the primary information as the area portion of the observation point Op. This value can be used as the predetermined threshold. Since the sum and average directly indicate cell spreading or proliferation, they theoretically increase over time. The predetermined threshold can be set, for example, as a value obtained by multiplying the actual area in the direction in which cells can spread or proliferate in the culture vessel, such as the bottom area, by a predetermined percentage. The predetermined percentage can be, for example, approximately 70% to 90%. Alternatively, for example, a test may be performed in advance under the same conditions as those for determining the timing of actual passage, and the sum or average value of the statistical index at which cell spreading or proliferation ceases to be observed in the test may be determined as the predetermined threshold. In this case, the sum or average value of primary information on the cells that have reached the time to passage in a plurality of culture vessels for the test may be obtained in advance.

[0050] Furthermore, taking the cell count as an example, the variance or deviation as a statistical index value indicates the degree of dispersion of cells at the observation point Op, as described above. Even if the average value is the same, it is a representative value indicating that some areas on the culture surface in the culture vessel are no longer able to spread or proliferate, and that passaging is required (e.g., Figure 5B). The photographing time corresponding to the point at which the variance or deviation as a statistical index value reaches a predetermined threshold is determined to be the point at which the passaging timing is reached. As the area portion of the observation point Op, the value at the time when passaging is required can be uniquely determined as the variance or deviation of the primary information. This value can be set as the predetermined threshold. In the case of variance or deviation, in the initial state of culture, cell spreading or proliferation occurs in the vicinity of each cell, so theoretically the variance or deviation will decrease once.

[0051] Typically, as cells spread or proliferate, the variance or deviation reaches a minimum value over time and then begins to increase. After the passage timing, the cells are unable to spread or proliferate, and the variance or deviation reaches a maximum value and then begins to decrease. Therefore, the predetermined threshold can be determined by conducting a preliminary test under the same conditions as those actually used to determine the passage timing, and determining the maximum value at which the variance or deviation as a statistical indicator changes from increasing to decreasing. Furthermore, in unusual cases, cells may rapidly spread or proliferate locally in the initial culture state, preventing the variance or deviation from increasing. In such cases, the inflection point in the time change of the variance or deviation indicates the time for passage, and this inflection point in the time change can be used as the predetermined threshold. Thus, the time for passage will typically involve the appearance of some kind of inflection point in the time change, such as a minimum or maximum. The point at which this inflection point in the time change appears is set as the threshold, indicating the time when the passage timing is reached. In particular, when the variance or deviation does not begin to increase, as described above, this is because cells rapidly spread or proliferate locally in the initial state of culture, and so the threshold value can be set to a value obtained by adding a margin value α to the minimum value. The margin value α is set so that at least the value obtained by adding the margin value α to the minimum value is smaller than the maximum value. Furthermore, the margin value α is preferably 50% or less of |(maximum value)-(minimum value)|, and this value is preferably 0. This value can also be obtained in advance as the variance or deviation of primary information from multiple culture vessels for testing that have reached the time for passage.

[0052] In the present invention, primary information about cells is obtained from photographed images of the state of the culture medium containing the cells, and statistical index values ​​are calculated, thereby making it possible to directly grasp the progress of cell spreading or proliferation, and accurately determine the timing of subculture.

[0053] [Second embodiment] Next, a method and system for determining the timing of passage according to a second embodiment will be described. The second embodiment is basically the same as the first embodiment, but is a modified version. The configurations of the cell culture system 1 and the passage timing determination unit 3 are the same as those of the first embodiment. Below, differences between the second embodiment and the first embodiment will be described, and descriptions of the same parts as the first embodiment will be omitted. The passage determination process of the second embodiment is similar to that of the first embodiment shown in FIG. 2B in that the passage determination process consists of an imaging step (S11), a determination data creation step (S12), and a determination step (S13). However, in the second embodiment, the creation of the determination data in the determination data creation step (S12) and the predetermined threshold in the determination step (S13) are different. The second embodiment differs from the first embodiment in that, in creating the judgment data in the judgment data creating step (S12), two imaging time points are selected as imaging time points of interest from the multiple imaging time points in the imaging step (S11), and judgment data is created from cell images at the two imaging time points to determine the passage timing. That is, in the second embodiment, a time-dependent element is added to the judgment data.

[0054] (Culture surface imaging process) The second embodiment is similar to the first embodiment in that cell images are captured at multiple imaging time points, as shown in Fig. 2E. The same observation point Op is selected at each imaging time point to ensure fixed-point observation, and the imaging process is performed when a new imaging time point arrives as the culture progresses.

[0055] (Creating judgment data) 3A and 3B are diagrams showing the flow of the determination data creation step (S12) in the passage determination process of the second embodiment. The determination data creation step (S12) of the second embodiment calculates a time-course change index value to add a time-course element to the first embodiment. Here, FIG. 3A is a diagram showing a first example of the second embodiment. In the first example, after completing a primary information acquisition step (S1211) for acquiring primary information from a cell image and a statistical index value calculation step (S1212) for calculating an index value from the primary information, a time-course change calculation step (S1213) for calculating a time-course change index value is performed.

[0056] On the other hand, FIG. 3B is a diagram showing a second example of the second embodiment. In the second example, a time-course change calculation step (S1213) of calculating a time-course change index value is performed between a primary information acquisition step (S1211) of acquiring primary information from a cell image and a statistical index value calculation step (S1212) of calculating an index value from the primary information. In this specification, the two imaging time points used to calculate the time-course change index value in the time-course change calculation step (S1213) are referred to as imaging time points of interest, and the earlier imaging time point of the imaging time points of interest is referred to as the reference imaging time point, and the later imaging time point is referred to as the evaluation imaging time point. The time-course change index value is the value of the amount of change in the statistical index value of the primary information at the imaging time point of interest relative to the statistical index value of the primary information at the reference imaging time point.

[0057] In the second embodiment, in both the first and second examples, two imaging time points of interest selected from multiple imaging time points are used to determine the timing of passage. For example, cell images captured at the first and second imaging time points in FIG. 2E are used. In the second embodiment, the imaging time points of interest consist of a reference imaging time point and an evaluation imaging time point. The evaluation imaging time point is typically a new imaging time point that arrives as the culture progresses. For example, as shown in FIG. 2E, the first imaging time point is selected as the reference imaging time point and the second imaging time point is selected as the evaluation imaging time point. Below, we will explain the imaging time points of interest where the reference imaging time point is the first imaging time point and the evaluation imaging time point is the second imaging time point. In both the first and second examples, there are two methods for selecting the two imaging time points of interest: the most recent ratio method and the reference ratio method.

[0058] In the most recent ratio method, both the reference photographing time point and the evaluation photographing time point are updated to the most recent photographing time point of interest each time a new photographing time point is added. That is, when the timing of passage is determined by the most recent ratio method using any photographing time point of interest (reference photographing time point and evaluation photographing time point), if passage has not yet occurred, the photographing time points of interest (reference photographing time point and evaluation photographing time point) are updated to determine passage when the next passage timing is determined. This will be explained in detail with reference to FIG. 3C. FIG. 3C shows the concept of selecting a photographing time point of interest in the most recent ratio method, which selects a photographing time point of interest (reference photographing time point and evaluation photographing time point) from multiple photographing time points (n-3) between photographing time point (n-3) and photographing time point (n+2). For example, if the timing of passage is determined by selecting a time point of interest, with the time point of interest being the reference time point (n-3) and the time point of interest being the evaluation time point (n-2), and then determining that the time for passage has not yet arrived, the time point of interest is selected with the time point of interest being the reference time point (n-2) and the time point of interest being the evaluation time point (n-1), and the time point of interest is determined for passage. Thereafter, similarly, until the time of passage is determined, the reference time point is shifted from the time point of interest (n-3) to the time point of interest (n+1), and the evaluation time point is also shifted from the time point of interest (n-2) to the time point of interest (n+2), and the time point of interest is repeatedly updated to determine the timing of passage. However, when the reference time point and the evaluation time point are updated while being shifted, the reference time point and the evaluation time point must be shifted in a regular order. For example, if the reference time point is shifted by one, the evaluation time point must also be shifted by one. Although the reference shooting time point has been shifted by only one time point, an irregular shift such as shifting the evaluation shooting time point by two times is not possible.

[0059] In the reference ratio method, the imaging time point of interest, consisting of the reference imaging time point and the evaluation imaging time point, is selected as follows. Figure 3E shows the concept of selecting the imaging time point of interest in the reference ratio method, in which imaging time points of interest (reference imaging time point and evaluation imaging time point) are selected from multiple imaging time points (n-3) ranging from imaging time point (n-3) to imaging time point (n+2). In the reference ratio method, an early imaging time point from multiple imaging time points is determined as the reference imaging time point, and any imaging time point after the reference imaging time point is selected as the evaluation imaging time point. For example, imaging time point (n-3) is fixed as the reference imaging time point, and the imaging time point of interest is first selected with imaging time point (n-2) as the evaluation imaging time point to determine the timing of passaging. If, after this determination, it is determined that the time for passaging has not yet been reached, the imaging time point of interest is selected so that imaging time point (n-1) is the evaluation imaging time point, without changing the reference imaging time point (n-2), and the timing of passaging is determined. Thereafter, similarly, until the determination of the passage timing, the reference evaluation photographing time point remains the photographing time point (n-3), and only the evaluation photographing time point is shifted from the photographing time point (n-2) to the photographing time point (n+2), while the photographing time point of interest is repeatedly updated to determine the passage. That is, in the case of the reference ratio method, the reference photographing time point is fixed and used for each determination of the passage timing, and only the evaluation photographing time point is selected to be updated to the latest photographing time point so that a new photographing time point is changed as the evaluation photographing time point, and this is repeated for each passage timing. In this case, too, when the reference photographing time point and the evaluation photographing time point are updated while being shifted, the reference photographing time point and the evaluation photographing time point must be shifted regularly in order.

[0060] In the nearest ratio method, both the reference shooting time point and the evaluation shooting time point move, so a relative comparison of the primary information at each shooting time point between the reference shooting time point and the evaluation shooting time point is made. On the other hand, in the reference ratio, the first shooting time point, which is the reference shooting time point, is fixed, so an absolute comparison of the primary information at the first shooting time point (fixed) of the reference shooting time point and the second shooting time point (moving) of the evaluation shooting time point is made. In addition, the time interval between each shooting time point can be set arbitrarily, but it is preferable to set the time interval between each shooting time point to be constant, especially in the nearest ratio method, which selects two consecutive shooting time points.

[0061] In the most recent ratio method, typically, no imaging time points are selected between the reference imaging time point and the evaluation imaging time point, as shown in FIG. 3C. However, as shown in FIG. 3D, any number of imaging time points not used for evaluation may be selected between the reference imaging time point and the evaluation imaging time point. FIG. 3D shows an example in which one imaging time point is not used for evaluation between the reference imaging time point and the evaluation imaging time point. Subsequently, as long as the interval between the reference imaging time point and the evaluation imaging time point is constant and the reference imaging time point and the evaluation imaging time point are selected in order, they can be used to determine the timing of passage. Furthermore, in the reference ratio method, the reference imaging time point is fixed and the evaluation imaging time point is updated, so imaging time points exist between the reference imaging time point and the evaluation imaging time point.

[0062] Below, for each of the first and second examples, the most recent ratio method is used as an example, and an example will be described in which the reference photographing time point and the evaluation photographing time point are consecutive for a certain photographing time point of interest. Hereinafter, the most recent photographing time point will be described as the reference photographing time point and the second photographing time point as the evaluation photographing time point. Even in the most recent ratio method and the reference ratio method in which the reference photographing time point and the evaluation photographing time point are not consecutive, data from photographing time points other than the most recent photographing time point of interest is simply not used to create data for determining the timing of passage, and therefore the passage determination process is the same as in the following examples.

[0063] (Primary information acquisition) The primary information acquisition process is the same in the first and second examples. In the primary information acquisition process (S1211), the processing device 33 acquires primary information at each imaging time point from the images acquired at each imaging time point. The primary information acquisition process is the same as in the first embodiment, and includes, for example, the number of cells, cell density, cell-occupied area, individual cell area, intercellular distance, etc. A detailed explanation will be omitted here. Below, the first example will be explained, followed by the second example.

[0064] <Example 1> (obtaining statistical indicator values) In the first example, as shown in FIG. 3A, after primary information at each imaging time point is acquired in the primary information acquisition step (S1211), a statistical index value at each imaging time point is calculated from the primary information at each imaging time point in the statistical index value acquisition step (S1212). As in the first embodiment, the statistical index value is an index value defined as a representative value that represents the primary information, and is, for example, at least one of a sum, an average value, a variance, and a deviation such as a standard deviation. The sum is the sum of the cell counts at multiple observation points, the sum of the occupied areas of the cells, the sum of the individual cell areas, and the sum of the intercellular distances. The average values ​​are the average values ​​of the cell counts at multiple observation points, the average values ​​of the cell densities, the average values ​​of the occupied areas of the cell counts, the average values ​​of the individual cell areas, and the average values ​​of the intercellular distances. The variances and deviations are the variances and deviations of the cell counts at multiple observation points, the variances and deviations of the cell densities, the variances and deviations of the occupied areas of the cells, the variances and deviations of the individual cell areas, and the variances and deviations of the intercellular distances. Here, since the variance or deviation as a statistical index value is an index of the uniformity of the cells in the culture vessel, it is possible to determine the timing of passage more appropriately than by selecting the sum or average value as the statistical index value, as in the first embodiment.

[0065] (Time-dependent change calculation process (obtaining time-dependent change index values)) Next, in the time-over-time change calculation step (S1213), a time-over-time change index value is calculated from the statistical index value of each piece of primary information calculated at each shooting time point. One time-over-time change index value is created for each value of primary information at two shooting time points. In this embodiment, the number of shooting time points of interest is the same as the number of shooting time points used for determination. Since primary information is acquired for each shooting time point of interest, the number of shooting time points of interest and the number of pieces of primary information are the same. Therefore, the number of time-over-time change index values ​​is one less than the number of shooting time points of interest. Furthermore, the number of time-over-time change index values ​​is calculated as many times as the number of observation points Op, or if multiple observation points Op are representatively divided into a smaller number, the number of time-over-time change index values ​​is calculated as many times as the number of observation points Op.

[0066] The time-varying change calculation step (S1213) is a step in which a time-varying change index value is calculated after the statistical index value acquisition step (S1212) of the first embodiment, which calculates the statistical index value. This will be described with reference to FIG. 3F. FIG. 3F is a diagram illustrating the concept of change in value (vertical axis) over time (horizontal axis) for the first and second examples. In the first example, the vertical axis represents the statistical index value, while in the second example described below, the vertical axis represents the value of primary information. FIG. 3F is shown as an example of the most recent ratio method of two consecutive shooting time points that are the target shooting time point. However, in the case of the aforementioned reference ratio method in which the first and second shooting time points are not selected as consecutive shooting time points, FIG. 3F can be interpreted as including a shooting time point that is not shown between the first and second shooting time points.

[0067] In the reference ratio method, in which the first imaging time point is fixed as the reference imaging time point for two imaging time points and the second imaging time point, which is the evaluation imaging time point, is selected to be updated, the first imaging time point is always fixed. The value of the primary information at the second imaging time point, which is updated relative to the value of this fixed primary information, is determined, so the primary information increases with cell spreading or proliferation. Because the time-course change index value is an increase or decrease in the rate of increase, the time-course change index value of the statistical index value shows an increasing or decreasing trend in line with the increasing trend of the primary information. On the other hand, when the primary information is the relative distance between cells, the primary information decreases with cell spreading or proliferation. In this case, too, because the time-course change index value is an increase or decrease in the rate of decrease, the time-course change index value of the statistical index value shows an increasing or decreasing trend in line with the decreasing trend of the primary information. As the time for passage approaches, the rate of change in the increase or decrease slows, and the rate of change in the increase or decrease tends to saturate toward a predetermined value. On the other hand, in the most recent ratio method, in which the first and second imaging time points are selected as consecutive imaging time points for two imaging time points, the first and second imaging time points are constantly updated and a relative comparison is made between the first and second imaging time points, so that in the early stages of cell spreading or proliferation, the primary information simply increases or decreases (simple decrease when the primary information is the relative distance between cells) and the time-course index value of the statistical index value also increases or decreases, but as cell spreading or proliferation progresses, the degree of increase in the primary information at the first imaging time point and the degree of increase or decrease in the primary information at the second imaging time point change, so that the rate of increase or decrease in the time-course index value tends to slow as the time for passaging approaches. Then, when the time for passaging is reached, the time-course index value that had been on an increasing trend decreases from a predetermined extreme value, and the time-course index value that had been on a decreasing trend increases conversely. Hereinafter, we will explain an example in which the primary information shows a tendency for an increase as the cells stretch or grow. However, in the case of primary information that shows a tendency for a decrease as the cells stretch or grow, the example in which the primary information shows a tendency for an increase as the cells stretch or grow can be applied by reading the increase and decrease in reverse.

[0068] In calculating the time-course index value, two imaging time points (the first imaging time point and the second imaging time point) are selected from multiple imaging time points. Figure 3F shows an example in which the most recent two consecutive imaging time points are selected as the first imaging time point (t1) and the second imaging time point (t2). Figure 3F also shows that the statistical index value (a1) at the first imaging time point (t1) increases to the statistical index value (a2) at the second imaging time point (t2). It is determined whether the time for passage has arrived at that imaging time point, and if the time for passage has not arrived, the most recent two consecutive imaging time points, including the new imaging time point, are selected again, and determination data is created.

[0069] The time-dependent change index value can be a differential change amount, which is the value of the differential amount of the statistical index value of the primary information between the first imaging time point (reference imaging time point) and the second imaging time point (evaluation imaging time point) for the time interval between the imaging times of interest (first imaging time point and second imaging time point). The time-dependent index value of this differential change amount is a differential change amount (a2-a1), which is the differential amount of the statistical index value of the primary information at the first imaging time point and the statistical index value of the primary information at the second imaging time point. The differential change amount is, so to speak, an incremental amount. In an example where the primary information is the number of cells, this is the value of the incremental difference in the sum of the number of cells at the first imaging time point and the second imaging time point.

[0070] The time-dependent change index value can also be a percentage change in the statistical index value of the primary information at the second shooting time (evaluation shooting time) relative to the statistical index value of the primary information at the first shooting time (reference shooting time). The time-dependent change index value is (a2 / a1), which is the percentage change in the statistical index value of the primary information at the second shooting time relative to the statistical index value of the primary information at the first shooting time. The time-dependent change index value can also be a difference percentage change amount, which is the ratio of the difference in the statistical index value of the primary 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 primary information at the first shooting time (reference shooting time). The difference percentage change amount can be (a2-a1) / a1), which is the ratio of the difference increment between the statistical index value of the primary information at the first shooting time (reference shooting time) and the statistical index value of the primary information at the second shooting time (evaluation shooting time) relative to the statistical index value of the primary information at the first shooting time. For example, in an example where the primary information is the number of cells, the former percentage change amount is the ratio of the total number of cells at the second imaging time to the total number of cells at the first imaging time, and the latter differential percentage change amount is the ratio of the increase in the total number of cells from the first imaging time to the second imaging time to the total number of cells at the first imaging time.

[0071] Furthermore, the time-dependent change index value can also be the difference change amount, percentage change amount, and difference percentage change amount between the statistical index value of the primary information at the first imaging time point and the statistical index value of the primary information at the second imaging time point for the time interval between two imaging times (the first imaging time point and the second imaging time point). That is, the difference change amount (a2-a1), the percentage change amount (a2 / a1), and the difference percentage change amount (a2-a1) / a1) can be divided by the time difference (t2-t1) between the first imaging time point and the second imaging time point to obtain the time-dependent change index value, which is the rate of change per unit time. That is, the statistical index values ​​of the primary information at two consecutive imaging times can be expressed as the unit time change rate of the differential change amount (a2-a1) / (t2-t1), the unit time change rate of the percentage change amount (a2 / [a1·(t2-t1)]), and the unit time change rate of the differential percentage change amount (a2-a1) / [a1·(t2-t1)]). These time-dependent change index values ​​all mean the rate of increase per unit time of the statistical index value of the primary information at the imaging time of interest, and are also the increase speed. In an example where the primary information is the cell count, it is the increase rate per unit time of the cell count, that is, the increase speed (growth speed). Here, the increase speed (growth speed) is a speed (hereinafter referred to as the simple speed) approximated as a difference value obtained by linearly approximating the statistical index values ​​at the two imaging times.

[0072] In addition to the examples mentioned above, the growth rate (proliferation rate) can be defined based on a wide variety of definitions as the rate of increase over time, as long as it can express the change over time in the statistical index value of primary information. In cell growth, the cause of the increase is cell division, so the growth rate (proliferation rate) is not constant. If the conditions are right, the growth rate can theoretically increase by 2x, 4x, 8x, etc., and is characterized by a doubling in the multiplication factor. Generally, the specific growth rate is used to quantitatively express the growth rate (proliferation rate). In the present invention, not only when the primary information is cell number, but also when the primary information is other than cell number (such as the aforementioned cell density, cell-occupied area, individual cell area, relative distance between cells, etc.), the cause of the increase remains cell division, and the increase is approximately doubled. In reality, even when the primary information is cell number, the timing of cell division varies, so it is rare for the growth rate to increase exactly double per unit time. Therefore, regardless of whether the primary information is cell number or not, the growth rate per unit time is e μ The μ at this time is called the specific growth rate. Therefore, the statistical index values ​​at the two imaging points, the base point and the evaluation point, are exponentially approximated (e μt ) can be used.

[0073] Referring to FIG. 3G, the specific growth rate and doubling time t d In other words, the reference time point is set as the first photographing time point t1, and the statistical index value of the primary information at that time is a1. Then, at an arbitrary photographing time point t i At each point in time [i = 2 to n], the statistical index value a of the primary information is used as the evaluation shooting point. i [i=2~n] is measured. The statistical index value a of the primary information at each evaluation shooting point i [i=2~n] is a generalized relationship between the statistical index value of the primary information at the first photographing time t1 and a1. i / a1=e [(μi)·(ti‐t1)] This can be approximated as changing so that the following relationship holds. i is the time of shooting t iIt is the specific growth rate at [i = 2 to n], and μ i = [ln(a i / a1)] / (t i - t1).

[0074] Also, as the growth rate (specific growth rate), the doubling time (tad) can be adopted. The doubling time is the time required for the amount at the evaluation time point to become twice the amount at the reference time point. Assuming that it becomes twice at [i = 2 to n] when i = k, the doubling time t i = t ad - t1. In relation to the specific growth rate, this time is the time when the growth rate per unit time is e k = 2. That is, from the above description, at each arbitrary shooting time point t μ at each time point of [i = 2 to n], if μ μ·tad is obtained as the index value of the change with time of the primary information, the index value of the change with time of the primary information μ i at each evaluation shooting time point [i = 2 to n] has a generalized relationship with the index value of the change with time of the primary information td i at each evaluation shooting time point t i such that the relationship e [(μi)·(tdi)] = 2 holds. That is, the doubling time td i = ln2 / μ i = (ln2)·(t i - t1) / ln(a i / a1). Here, as an easy-to-understand example, assuming that the statistical index value a i of the primary information at i = k [2 < k < n] among the shooting time points t k [2 < i < n] becomes twice the statistical index value a1 of the first shooting time point t1 which is the reference shooting time point (a<00000​​​​​​​​​​​​​​​k -t1).

[0075] In addition to the doubling time, the time required for the amount at the evaluation time to become a predetermined multiple of the amount at the reference time can also be used as the growth rate (growth rate). In this application, this is defined as the predetermined doubling time (tx). The predetermined doubling time is the time required for the amount to become a predetermined multiple X, and the growth rate per unit time is e μ If so, then e μ·tx =X. That is, from the above explanation, at any photographing time t i At each point in time [i=2~n], μ i Once this is obtained, the time-dependent change index value μ of the primary information at each evaluation shooting point is calculated. i [i=2~n] is the time point of each evaluation shot t i The time-varying index value of the primary information is tx i As a generalized relationship between [(μi)·(txi)] =X. That is, the predetermined doubling time tx i =lnX / μ i =(lnX)·(t i ‐t1) / ln(a i / a1) at the time of the above-mentioned photograph t k The predetermined doubling time is tx=(lnX)·(t k -t1) / ln2, and the doubling time mentioned above is the predetermined doubling time when X=2.

[0076] In the judgment data creation step (S12), the time-course change index values ​​obtained above can be used as judgment data. Similarly, the time-course change index values ​​can be calculated according to the selection of the primary information such as the cell count, cell density, cell-occupied area, individual cell area, and intercellular distance, and the selection of the statistical index values ​​such as the sum, average, variance, and deviation. The time-course change index value in the first example can be said to be a representative value that indicates the change over time of the statistical index value of the primary information. The multiple time-course change index values ​​may be used as multiple judgment data, or multiple time-course change index values ​​may be combined into a single time-course change index value based on the average, maximum, minimum, etc., and used as a single judgment data.

[0077] (Subculture determination) After the creation of the judgment data is completed in the judgment data creation step (S12), a judgment step (S13) is executed to determine whether the time for passaging has arrived. In the judgment step (S13), the timing for passaging is determined based on the judgment data created in the judgment data creation step (S12). In the first example, the judgment data is a time-dependent index value. The statistical index value quantitatively indicates the state of cells on the culture surface at a predetermined observation point Op at a predetermined plurality of points selected from within the culture vessel. Therefore, in the first example, the time-dependent index value is the sum of the statistical index values ​​at the predetermined observation point Op, and is a representative value that can more accurately represent the room for cells to spread or grow within the culture vessel than the first embodiment.

[0078] In the determination step (S13), the determination data, which is the time-course change index value calculated in the time-course change calculation step (S1213), is compared with a predetermined threshold. When the determination data reaches the predetermined threshold, it is determined that the time for passage has arrived. The predetermined threshold is the same as in the first embodiment when the first imaging time point is selected so that the first imaging time point is fixed as the reference imaging time point and the second imaging time point is sequentially updated. As in the first embodiment, a test is performed in advance under the same conditions as in the actual determination of the passage timing, and in that test, a statistical index value at the time point corresponding to the time for passage is obtained, and a time-course change index value is calculated and set as the predetermined threshold. Even when a time-course change factor is added, the characteristics of the statistical index value in determining the time for passage are not lost, as described in the first embodiment. That is, at the time of passage, some kind of inflection point, such as a minimum value or a maximum value, appears in the time change of the time-course change index value. The time point at which an inflection point in such a change over time appears is set as a threshold for determining that the time for passage has been reached.

[0079] On the other hand, when selecting the first and second photographing time points as consecutive photographing time points, the predetermined threshold value is as follows: When the primary information at the second imaging time point increases and the statistical index value also increases, the primary information at the first imaging time point also increases and the statistical index value also increases. In the early stages of cell spreading or proliferation, the increase in the statistical index value at the second imaging time point is greater than the increase in the statistical index value at the first imaging time point. However, as the cells spread or proliferate, the respective increases gradually decrease, and at the stage when it is time to passage the cells, the increase in the statistical index value at the second imaging time point becomes smaller than the increase in the statistical index value at the first imaging time point. Therefore, at the stage when it is time to passage the cells, the statistical index value decreases by reaching an extreme value at an arbitrary threshold. This extreme value is set as the predetermined threshold. The extreme value is determined in a test conducted in advance as a test under the same conditions as those actually used to determine the passaging timing. In this test, a time-course index value is calculated from the statistical index value at the time when it is time to passage, and when the time-course index value reaches an extreme value, that extreme value is set as the predetermined threshold. Furthermore, since the time for passaging has already been reached when the time-course change index value reaches a predetermined threshold, a time point slightly before that can be said to be the most suitable time for passaging. Therefore, the predetermined threshold can be set as the value obtained by subtracting margin value α from the extreme value of the time-course change index value ("extreme value of the time-course change index value" - "margin value α"). This makes it possible to determine whether cells that do not yet show a slowdown in cell spreading or proliferation very close to the time for passaging are in a good condition and have reached the time for passaging. The "margin value α" is also set in a test performed in advance as a verification.

[0080] When the number of pieces of judgment data is one, the one piece of judgment data is compared to see if it reaches a predetermined threshold, and if it does, it is determined that the time for passaging has been reached. When the number of pieces of judgment data is multiple, a predetermined number of pieces of judgment data among the multiple pieces of judgment data are compared to see if they reach a predetermined threshold, and if they do, it is determined that the time for passaging has been reached. Furthermore, it may be determined that the time for passaging has been reached when all of the plurality of determination data have reached a predetermined threshold value. In this case, since it takes time for all of the determination data to reach the predetermined threshold value, whether the time for passaging has been reached will be determined relatively late. Conversely, it may be determined that the time for passaging has been reached when at least one of the plurality of determination data reaches a predetermined threshold value. In this case, since the determination can be made when one determination data reaches a predetermined threshold value, it is possible to determine early whether the time for passaging has been reached.

[0081] <Example 2> Next, a second example will be described. In the first example, after primary information is acquired at each shooting time point in the primary information acquisition step (S1211), a statistical index value at each shooting time point is calculated from the primary information at each shooting time point in the statistical index value acquisition step (S1212), and then a time-over-time index value is calculated in the time-over-time change calculation step (S1213) to obtain the determination data. However, in the second example, as shown in FIG. 3B, after primary information at each shooting time point is acquired in the primary information acquisition step (S1211), a time-over-time change calculation step (S1213) is executed to calculate a time-over-time index value of the primary information, and then a statistical index value of the time-over-time index value is calculated in the statistical index value acquisition step (S1212) to obtain the determination data, which is different. The primary information and the primary information acquisition step (S1211) are the same as those in the first embodiment and first example, and therefore will not be described here.

[0082] (Time-dependent change calculation process (obtaining time-dependent change index value)) Next, in the time-varying calculation step (S1213), a time-varying index value for each piece of primary information calculated at each imaging time point is calculated. The time-varying index value is the amount of change in the primary information between each imaging time point. The time-varying index value in the second example is the differential amount of change, the rate of change, the rate of change per unit time, the rate of change, or the change time, as in the first example. The first example differs only in that the differential amount of change, the rate of change, the rate of change per unit time, the rate of change, or the change time of the statistical index value of the primary information is used, whereas the second example differs in that the differential amount of change, the rate of change, the rate of change per unit time, the rate of change, or the change time of the primary information is used. Therefore, in the calculation of the time-varying index value in the first example described above, when the time-varying index value is calculated from the statistical index value of the primary information, the "statistical index value of the primary information" can simply be read as "primary information," and the first example can be applied to the second example. Therefore, a detailed description of the calculation of the differential amount of change, the rate of change, the rate of change per unit time, the rate of change, or the change time of the primary information will be omitted in the second example. In the second example, as in the first example, the rate of change can be applied as the specific growth rate, and the change time can be applied as the doubling time.

[0083] The time-over-time index value of the second example will be described with reference to FIG. 3F. FIG. 3F is a diagram illustrating the concept of change in primary information (vertical axis) over time (horizontal axis) for the first and second examples. In the second example, the statistical index values ​​in the explanation of the time-over-time calculation step (S1213) of the first example, i.e., a1 and a2 as time-over-time index values, can be read as the values ​​of the primary information acquired in the primary information acquisition step (S1211) of the second example. Here, the number of time-over-time index values ​​obtained corresponds to the number of pieces of primary information. The time-over-time index value of the second example can be said to be a value that directly indicates the time-over-time trend of the primary information.

[0084] (obtaining statistical indicator values) In the second example, as shown in FIG. 3B, a time-course change index value of the primary information at the first and second imaging times is acquired in a time-course change calculation step (S1213), and then a statistical index value of the time-course change index value is calculated in a statistical index value acquisition step (S1212). The statistical index value in the second example is an index value defined as a representative value that represents the time-course change index value of the primary information, and is, for example, at least one of a mean value, a variance, a deviation such as a standard deviation, etc. In the second example, a statistical index value of the time-course change index value is calculated, so that the statistical index value is the mean value, variance, deviation, etc. of the time-course change index value of the primary information at each of multiple observation points Op. The statistical index value calculated here is used as judgment data to determine the timing of passage. The multiple statistical index values ​​may be used as multiple judgment data as they are, or multiple statistical index values ​​may be combined into a single statistical index value based on the mean value, maximum value, minimum value, etc., and used as a single judgment data.

[0085] (Subculture determination) After the creation of the judgment data in the judgment data creation step (S12) is completed, a judgment step (S13) is executed to determine whether the timing for passaging has arrived. In the judgment step (S13), the timing for passaging is determined based on the statistical index value created in the judgment data creation step (S12). The judgment data in the second example is a statistical index value of the time-course change index value, and therefore, like the judgment data that is the time-course change index value of the statistical index value in the first example, it contains elements of primary information, change over time, and statistical index value, and therefore is no different from the first example as judgment data for determining the timing of passaging and is effective in determining the timing of passaging.

[0086] 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 in the first embodiment. As in the first embodiment, a test is conducted in advance as a test under the same conditions as those for actually determining the timing of passaging, and in that test, a statistical index value at the time point when passaging is to be obtained is obtained, and a time-course index value is determined, which is then set as the predetermined threshold value. Even if an element of time-course change is added, the characteristics of the statistical index value in determining the timing of passaging are not lost, as explained in the first embodiment. As in the first example, when the number of pieces of judgment data is created as one representative value, if the number of pieces of judgment data is one, that one piece of judgment data is compared to see if it reaches a predetermined threshold, and if it does, it is determined that the time for passaging has been reached. If the number of pieces of judgment data is multiple, a predetermined number of pieces of judgment data among the multiple pieces of judgment data are compared to see if they reach a predetermined threshold, and if they do, it is determined that the time for passaging has been reached. Furthermore, it may be determined that the time for passaging has been reached when all of the plurality of determination data have reached a predetermined threshold value. In this case, since it takes time for all of the determination data to reach the predetermined threshold value, whether the time for passaging has been reached will be determined relatively late. Conversely, it may be determined that the time for passaging has been reached when at least one of the plurality of determination data reaches a predetermined threshold value. In this case, since the determination can be made when one determination data reaches a predetermined threshold value, it is possible to determine early whether the time for passaging has been reached.

[0087] [Third embodiment] Next, a method and system for determining the timing of passage according to a third embodiment will be described. The third embodiment is an embodiment that is a partial modification of the first and second embodiments. The third embodiment differs from the first and second embodiments in that, in creating the determination data in the determination data creation step (S12), at least three imaging time points are selected as target imaging time points from the multiple imaging time points captured in the imaging step (S11), and determination data is created from cell images at the target imaging time points to determine the passage timing. In the third embodiment, a time-dependent element is also added to the determination data. The configurations of the cell culture system 1 and the passage timing determination unit 3 are the same as those in the first embodiment. Below, the third embodiment will be described in terms of differences from the first and second embodiments, and a description of the same parts as the first and second embodiments will be omitted. The passage determination process of the third embodiment also comprises an imaging step (S11), a determination data creation step (S12), and a determination step (S13), similar to the passage determination process of the first embodiment shown in FIG. 2B.

[0088] (Culture surface imaging process) In the third embodiment, as shown in Fig. 2E, cell images are captured at multiple imaging time points in the imaging step (S11), similarly to the first and second embodiments. At each imaging time point, the same observation point Op is selected so as to perform fixed-point observation.

[0089] (Creating judgment data) The judgment data generation step (S12) is similar to the passage judgment process of the second embodiment shown in Figures 3A and 3B. In the third embodiment, the generation of judgment data in the judgment data generation step (S12) includes a first example (Figure 4A), a second example (Figure 4B), and a third example (Figure 4C). In the third embodiment, unlike the second embodiment, the process of calculating the time-course index value is performed in two stages: a primary time-course change step (S1222) and a secondary time-course change step (S1222). A first example of this embodiment (FIG. 4A) includes a primary information acquisition step (S1221) of acquiring primary information from a cell image, followed by a statistical index value calculation step (S1222) of calculating a statistical index value from the primary information, a primary time-course change calculation step (S1223) of calculating a primary time-course change index value of the primary information, and a secondary time-course change calculation step (S1224) of calculating a secondary time-course change index value from the primary time-course change index value. A second example of this embodiment (FIG. 4B) also includes a primary information acquisition step (S1221) of acquiring primary information from a cell image, a primary time-course change calculation step (S1223) of calculating a primary time-course information change amount of the primary information, and a secondary time-course change calculation step (S1224) of calculating a secondary time-course change index value from the primary time-course change index value, followed by a statistical index value calculation step (S1222) of calculating a statistical index value of the secondary time-course change index value. Furthermore, a third example of this embodiment (Figure 4C) includes a primary information acquisition step (S1221) for acquiring primary information from a cell image, a primary time-course change calculation step (S1223) for calculating the amount of change in primary time-course information of the primary information, a statistical index value calculation step (S1222) for calculating a statistical index value of the primary time-course change index value, and a secondary time-course change calculation step (S1224) for calculating a secondary time-course change index value from the statistical index value of the primary time-course change index value.

[0090] In the third embodiment, in all of the first to third examples, cell images taken at three or more imaging time points are used as imaging time points of interest in determining the timing of passage. Similar to the second embodiment, the imaging time points of interest consist of an evaluation imaging time point and a reference imaging time point. When there are three imaging time points of interest, for example, cell images taken at the first, second, and third imaging time points in FIG. 2E are used. Below, as a representative example, an example in which cell images taken at three imaging time points are used will be described. The case in which four or more imaging time points are used as imaging time points of interest is similar to this embodiment, but this will be specifically described later as the fourth embodiment.

[0091] In the third embodiment, as in the second embodiment, the selection of the photographing time point of interest is performed in both the first and second examples using two selection methods: the most recent ratio method and the reference ratio method. The most recent ratio method and the reference ratio method of the third embodiment are configured such that at least one of the evaluation photographing time point and the reference photographing time point is set to a plurality of times at the photographing time point of interest. First, the following description will be given assuming that there is one reference photographing time point and two evaluation photographing time points. In this case, in a typical example, the most recent ratio method selects three consecutive photographing time points (the first photographing time point, the second photographing time point, and the third photographing time point) from the plurality of photographing time points as the photographing time point of interest. As in the second embodiment, the most recent ratio method adds new photographing time points and updates the first photographing time point, the second photographing time point, and the third photographing time point as the most recent photographing time points.

[0092] First, the nearest ratio method of the third embodiment will be specifically described with reference to FIG. 4D. FIG. 4D illustrates the concept of selecting target photographing time points (reference photographing time point and evaluation photographing time point) for the nearest ratio method from any photographing time point, for example, from photographing time points between photographing time point (n-3) and photographing time point (n+2). For example, one reference photographing time point and two evaluation photographing time points are selected as target photographing time points, such as photographing time point (n-3) as the reference photographing time point, photographing time point (n-2) as the first evaluation photographing time point, and photographing time point (n-1) as the second evaluation photographing time point, and the timing of passage is determined. If it is subsequently determined that the time for passage has not yet arrived, the target photographing time points are selected as follows: photographing time point (n-2) as the reference photographing time point, photographing time point (n-1) as the first evaluation photographing time point, and photographing time point n as the second evaluation photographing time point, and the timing of passage is determined. Thereafter, similarly, if the photographing time point (n-1) is the reference photographing time point, the photographing time point n is the first evaluation photographing time point, the photographing time point n+1 is the second evaluation photographing time point, and so on, and the timing of passage is determined by repeatedly updating the photographing time point of interest while shifting it as new photographing time points arrive.In this case, as in the second embodiment, when updating the reference photographing time point and the evaluation photographing time point while shifting them, the reference photographing time point and the evaluation photographing time point must be shifted regularly in order.

[0093] In the nearest ratio method, typically, as shown in FIG. 4D, a photographing time point of interest is selected so that there is no photographing time point between the reference photographing time point and the first evaluation photographing time point. However, as in the second embodiment, as shown in FIG. 4E, a photographing time point of interest may be selected so that any number of photographing time points not used for determining the timing of passage are set between the reference photographing time point and the first evaluation photographing time point. FIG. 4E shows an example of this. In addition, in the nearest ratio method, a photographing time point of interest may be set so that there are multiple photographing time points not used for evaluation between the reference photographing time point and the first evaluation photographing time point. Alternatively, regardless of whether there are photographing time points not used for evaluation between the reference photographing time point and the first evaluation photographing time point, a photographing time point of interest may be set so that there are multiple photographing time points not used for evaluation between the first evaluation photographing time point and the second evaluation photographing time point. As long as there is a constant interval between the reference photographing time point, the first evaluation photographing time point, and the second evaluation photographing time point are selected in the same order, the photographing time point of interest can be used to determine the timing of passage. In the reference ratio method, the reference shooting time point is fixed and the evaluation shooting time point is updated, so there are shooting time points between the reference shooting time point and the evaluation shooting time point. In this case, too, regardless of whether there are shooting time points not used for evaluation between the reference shooting time point and the first evaluation shooting time point and the second evaluation shooting time point, when updating while shifting the reference shooting time point and the first evaluation shooting time point and the second evaluation shooting time point, the reference shooting time point and the first evaluation shooting time point and the second evaluation shooting time point must be shifted regularly in order.

[0094] Next, the reference ratio method of the third embodiment will be described in detail with reference to FIG. 4F. In the reference ratio method, a photographing time point of interest consisting of a reference photographing time point, a first evaluation photographing time point, and a second evaluation photographing time point is selected as follows. FIG. 4F shows the concept of selecting a photographing time point of interest in the reference ratio method, which selects a photographing time point of interest (reference photographing time point and evaluation photographing time point) from multiple photographing time points from photographing time point (n-3) to photographing time point (n+2). In the reference ratio method, an early arbitrary photographing time point from the multiple photographing time points is determined and fixed as the reference photographing time point, and arbitrary photographing time points after the reference photographing time point are selected as the first evaluation photographing time point and the second evaluation photographing time point. Then, if it is determined that the time for passage has not yet been reached, the photographing time points of interest (reference photographing time point and evaluation photographing time point) are determined so as to shift only the first evaluation photographing time point and the second evaluation photographing time point without moving the reference photographing time point. For example, if the photographing time point (n-3) is set as the reference photographing time point, the photographing time point (n-3) is fixed as the reference photographing time point, and the photographing time point (n-2) is selected as the first evaluation photographing time point and the photographing time point (n-2) is selected as the second evaluation photographing time point to determine the timing of passage. Furthermore, if it is determined after this determination that the time for passage has not yet arrived, the photographing time point (n-2) is left as the reference photographing time point, and the photographing time point (n-1) is selected as the first evaluation photographing time point and the photographing time point (n-1) is selected as the second evaluation photographing time point to determine the timing of passage. Thereafter, similarly, until the timing of passage is determined, the reference evaluation photographing time point remains the photographing time point (n-3), and as new photographing time points arrive and photographing time points are added, the first evaluation photographing time point and the second evaluation photographing time point are shifted, and the photographing time point of interest is repeatedly updated to determine passage. That is, in the case of the reference ratio method, the reference photographing time point is fixed and used for each determination of the passage timing, and the first evaluation photographing time point and the second evaluation photographing time point are updated and selected each time a new photographing time point is added, and this is repeated for each passage timing. In the case of the reference ratio method, as in the case of the most recent ratio method, when updating while shifting the reference photographing time point from the first evaluation photographing time point and the second evaluation photographing time point, the reference photographing time point and the first evaluation photographing time point and the second evaluation photographing time point must each be shifted regularly in order.

[0095] In the case of the reference ratio method, the reference imaging time point is fixed. Therefore, by shifting the first evaluation imaging time point when a new imaging time point arrives and is added, there will always be any number of imaging time points between the reference imaging time point and the first evaluation imaging time point that will not be used to determine the timing of passage. In this case, it is also possible to set any number of imaging time points between the first evaluation imaging time point and the second evaluation imaging time point that will not be used to determine the timing of passage. For example, Figure 4G shows an example in which imaging time point (n-3) is fixed as the reference imaging time point, imaging time point (n-2) is selected as the first evaluation imaging time point, and imaging time point (n) is selected as the second evaluation imaging time point to determine the timing of passage. In this case, imaging time point (n-1) is not selected as the evaluation imaging time point and will not be used to determine the timing of passage. If, after this determination, it is determined that the time for passage has not yet been reached, the photographing time (n-3) is left as the reference photographing time, and the photographing time (n-1) is selected as the first evaluation photographing time, and the photographing time (n+1) is selected as the second evaluation photographing time, and the time for passage is determined. Thereafter, similarly, until the time for passage is determined, the reference evaluation photographing time remains the photographing time (n-3), and as new photographing time points arrive and photographing time points are added, the first evaluation photographing time and the second evaluation photographing time are shifted, and the time for passage is determined by repeatedly updating the reference photographing time. In this case, too, as described above, when the reference photographing time and the first evaluation photographing time and the second evaluation photographing time are shifted and updated, the reference photographing time and the first evaluation photographing time and the second evaluation photographing time must be shifted regularly in order.

[0096] In the nearest ratio method, both the reference shooting time point and the evaluation shooting time point (first evaluation shooting time point and second evaluation shooting time point) move, so a relative comparison of the primary information at each shooting time point between the reference shooting time point and the evaluation shooting time point is made. On the other hand, in the reference ratio, the reference shooting time point is fixed, so an absolute comparison of the primary information as the evaluation shooting time point (which moves with the passage of time) is made with respect to the reference shooting time point (which is fixed regardless of the passage of time). The time interval between each shooting time point can be set arbitrarily, but it is preferable to set the time interval between each shooting time point to be constant, especially in the nearest ratio method, which selects two consecutive shooting time points.

[0097] The judgment data creation step (S12) of the third embodiment will be described below in separate examples 1 to 3. In all of the first to third examples, the primary information acquisition step (S1221) is the same as the primary information acquisition step (S1211) of the second embodiment, and therefore will not be described again.

[0098] <Example 1> (obtaining statistical indicator values) In the first example, as shown in FIG. 3A, after primary information at each imaging time point is acquired in the primary information acquisition step (S1221), a statistical index value at each imaging time point is calculated from the primary information at each imaging time point in the statistical index value acquisition step (S1222). As in the first embodiment, the statistical index value is an index value defined as a representative value that represents the primary information, and is, for example, at least one of a sum, an average value, a variance, and a deviation such as a standard deviation. The sum is the sum of the cell counts at multiple observation points, the sum of the occupied areas of the cells, the sum of the individual cell areas, and the sum of the intercellular distances. The average values ​​are the average values ​​of the cell counts at multiple observation points, the average values ​​of the cell densities, the average values ​​of the occupied areas of the cells, the average values ​​of the individual cell areas, and the average values ​​of the intercellular distances. The variances and deviations are the variances and deviations of the cell counts at multiple observation points, the variances and deviations of the cell densities, the variances and deviations of the occupied areas of the cells, the variances and deviations of the individual cell areas, and the variances and deviations of the intercellular distances. Here, since the variance or deviation as a statistical index value is an index of the uniformity of the cells in the culture vessel, it is possible to determine the timing of passage more appropriately than by selecting the sum or average value as the statistical index value, as in the first embodiment.

[0099] (First-order time-course change calculation process (obtaining first-order time-course change index value)) Next, in a primary time-varying calculation step (S1223), a primary time-varying index value is calculated from the statistical index value of each piece of primary information calculated at each photographing time point. The primary time-varying index value is the amount of change or rate of change that is the increment of the statistical index value of each piece of primary information between each photographing time point, i.e., between the first photographing time point and the second photographing time point and between the second photographing time point and the third photographing time point. One primary time-varying index value is created for each value of two pieces of primary information, and a primary time-varying index value that is one less than the statistical index value of the primary information overall is created. The number of primary time-varying index values ​​calculated is the number of observation locations Op, or one less than the number of representative values.

[0100] This will be explained with reference to FIGS. 4H and 4I. FIGS. 4H and 4I are diagrams illustrating the concept of change in value (vertical axis) over time (horizontal axis) for the first and second examples. In the first example, the vertical axis represents the statistical index value, while in the second example, described later, the vertical axis represents the value of the primary information. FIGS. 4H and 4I are shown as an example of the most recent ratio method for three consecutive imaging time points (the first imaging time point, the second imaging time point, and the third imaging time point). In the case of the aforementioned reference ratio method, in which the first imaging time point, the second imaging time point, and the third imaging time point are not selected as consecutive imaging time points, FIGS. 4H and 4I can be interpreted as if there were an imaging time point not shown between the first imaging time point, the second imaging time point, and the third imaging time point. The characteristics of the increase tendency of the primary time-change index value for the reference ratio method and the most recent ratio method are the same as those in the second embodiment.

[0101] In calculating the primary time-course index value, two imaging time points (the first and second imaging time points) are selected from multiple imaging time points. Figures 4H and 4I show an example in which the most recent two consecutive imaging time points are selected as the first imaging time point (t1), the second imaging time point (t2), and the third imaging time point (t3). Figures 4H and 4I also show that the statistical index value (a1) at the first imaging time point (t1) increases to the statistical index value (a2) at each of the second imaging time point (t2) and the third imaging time point (t3). It is determined whether the time for passage has arrived at that imaging time point. If the time for passage has not arrived, the most recent three consecutive imaging time points, including the new imaging time point, are selected again, and the determination data is generated.

[0102] In the first example, the calculation of the primary time-change index value focuses on the interval between the three shooting time points selected for use in the judgment, i.e., two shooting time points selected from the three shooting time points. That is, two sets of two shooting time points are extracted from the three shooting time points, and the change in value between them is calculated as the primary time-change index value. In the first example, for example, two shooting time points as the first set (the first shooting time point and the second shooting time point) and two shooting time points as the second set (the second shooting time point and the third shooting time point) are selected, and the primary time-change index value between them is calculated. In this specification, the two shooting time points extracted from the shooting time points used for the judgment are referred to as the target shooting time points. In the second embodiment, there is only one combination of target shooting time points, so only one time-change index value is obtained. However, in this embodiment, one primary time-change index value is obtained from two photographing time points of interest, so a total of two primary time-change index values ​​are obtained, one from each of two sets of photographing time points of interest. In other words, if three photographing time points are selected for use in determination, two combinations of photographing time points of interest will be extracted, and the number of primary time-change index values ​​will be two.

[0103] The method of calculating the primary time-varying index value between each of two photographing time points of interest is the same as in the second embodiment. The primary time-varying index value can also be, for example, the difference between the statistical index value of the primary information at the evaluation photographing time point among the photographing time points of interest and the statistical index value of the primary information at the reference photographing time point. That is, as shown in FIG. 4H, the primary time-varying index value can be a differential change amount, which is the value of the differential amount of the statistical index value of the primary information between the first photographing time point (reference photographing time point) and the second photographing time point (evaluation photographing time point), and between the second photographing time point (reference photographing time point) and the third photographing time point (evaluation photographing time point), which are combinations of photographing time points of interest. The time-varying index value of this differential change amount can be the differential change amount (a2-a1), (a3-a2), which is the difference between the statistical index value of the primary information at the second photographing time point and the statistical index value of the primary information at the first photographing time point.

[0104] The primary time-dependent change index value can also be the rate of change in the statistical index value of the primary information at the evaluation imaging time point relative to the statistical index value of the primary information at a reference imaging time point among the imaging times of interest. The primary time-dependent change index value is the rate of change a2 / a1 of the statistical index value of the primary information at the second imaging time point (evaluation imaging time point) relative to the statistical index value of the primary information at the first imaging time point (reference imaging time point), and the rate of change a3 / a2 of the statistical index value of the primary information at the third imaging time point (evaluation imaging time point) relative to the statistical index value of the primary information at the second imaging time point (reference imaging time point). These mean the rate of the sum of the cell counts at the second imaging time point to the sum of the cell counts at the first imaging time point, and the rate of the sum of the cell counts at the third imaging time point to the sum of the cell counts at the second imaging time point to the sum of the cell counts at the first imaging time point, respectively.

[0105] Furthermore, the primary time-varying index value can also be a difference percentage change amount relative to the statistical index value of the primary information at a shooting time point that serves as a reference for the difference amount between the statistical index value of the primary information at the evaluation shooting time point among the shooting times of interest and the statistical index value of the primary information at the reference shooting time point. The primary time-varying index value of the difference percentage change amount can be expressed as (a2-a1) / a1, which is the ratio of the increase amount between the statistical index value of the primary information at the first shooting time point and the statistical index value of the primary information at the second shooting time point to the statistical index value of the primary information at the first shooting time point, and (a3-a2) / a2, which is the ratio of the increase amount between the statistical index value of the primary information at the second shooting time point and the statistical index value of the primary information at the third shooting time point to the statistical index value of the primary information at the second shooting time point. For example, in an example where the primary information is the number of cells, it means the ratio of the increase in the total number of cells from the first imaging time to the second imaging time to the total number of cells at the first imaging time, and the ratio of the increase in the total number of cells from the second imaging time to the third imaging time to the total number of cells at the second imaging time.

[0106] 4I, the primary time-varying index value can also be the difference change amount, percentage change amount, and difference percentage change amount between the statistical index value of the primary information at the reference shooting time point and the statistical index value of the primary information at the evaluation shooting time point, for a time interval between the target shooting time point. That is, the primary time-varying index value can also be the difference change amount, percentage change amount, and difference percentage change amount between the statistical index value of the primary information at the first shooting time point (reference shooting time point) and the statistical index value of the primary information at the second shooting time point (evaluation shooting time point), and between the statistical index value of the primary information at the second shooting time point (reference shooting time point) and the statistical index value of the primary information at the third shooting time point (evaluation shooting time point), for each time interval between two sets of shooting time points (first and second shooting time points, and second and third shooting time points). The primary time-varying index values ​​can be expressed as the unit time change rate of the difference change (a2-a1) / (t2-t1), the unit time change rate of the percentage change (a2 / [a1·(t2-t1)]), and the unit time change rate of the difference percentage change (a2-a1) / [a1·(t2-t1)]) for the first imaging time (reference evaluation time) and the second imaging time (evaluation imaging time).The primary time-varying index values ​​can be expressed as the unit time change rate of the difference change (a3-a2) / (t3-t2), the unit time change rate of the percentage change (a3 / [a2·(t3-t2)]), and the unit time change rate of the difference percentage change (a3-a2) / [a2·(t3-t2)]) for the second imaging time (reference evaluation time) and the third imaging time (evaluation imaging time). These primary time-course change index values ​​all mean the rate of increase per unit time of the statistical index value of the primary information between the first imaging time point (reference imaging time point) and the second imaging time point (evaluation imaging time point), and the rate of increase per unit time of the statistical index value of the primary information between the second imaging time point (reference imaging time point) and the third imaging time point (evaluation imaging time point), and are also the growth rate. In an example where the primary information is the number of cells, it is the rate of increase per unit time of the number of cells, that is, the growth rate (growth rate). Here, the growth rate (growth rate) is a simple rate approximated as the difference value obtained by linearly approximating the statistical index values ​​at the two imaging times.

[0107] The acceleration of increase (growth rate) can be defined based on a wide variety of definitions as long as it represents the change over time of the statistical index value of the primary information, similar to the second embodiment. That is, as shown in FIG. 4J, the acceleration of increase (growth rate) as the primary change-over-time index value can be the specific growth rate instead of the simple speed defined by the differential component with respect to time, similar to the second embodiment. The definition of the specific growth rate is the same as that in the second embodiment. Therefore, the explanation in the second embodiment is applicable. To explain again for confirmation, it is as follows. Here, the reference time point in the calculation of the primary change-over-time index value is set as the first imaging time point t1, which is the reference imaging time point, and the statistical index value a of the primary information at each evaluation imaging time point, which is the evaluation time point in the calculation of the primary change-over-time index value, at the imaging time point t i of the primary information i [i = 2 to n] is approximated as a generalized relationship between the statistical index value of the primary information at the first imaging time point t1 and a1, and a i / a1 = e [(μi)·(ti‐t1)] The relationship can be approximated as changing so that it holds. Here, μ i is the specific growth rate at the imaging time point t i [i = 2 to n], and μ i = [ln(a i / a1)] / (t i −t1).

[0108] Here, among the imaging time points t i [2 < i < n], when the statistical index value of the primary information becomes a k1 at i = k1 [2 < k1 < n], the specific growth rate is μ k1 = [ln(a k1 / a1)] / (t k1 −t1). Also, when the statistical index value of the primary information becomes a i at the evaluation imaging time point when i = k2 [k1 < k2 < n] among the imaging time points t k2 [i < n], the specific growth rate is μ[[ID=3​​​​​​​​​​​k1 The secondary time-dependent change index value described below using k1 is the two specific growth rates μ calculated from the values at two specific points selected from among these three points k2 , μ k1 are calculated from.

[0109] Furthermore, similar to the second embodiment, the doubling time can be used as the primary time-dependent change index value. That is, for the shooting time t i [2 < i < n], assuming that the statistical index value a of the primary information at i = k1 [2 < k1 < n] k1 becomes twice the statistical index value a1 of the first shooting time t1 which is the reference shooting time (a k1 = 2a1). At this time, the time from the first shooting time t1 which is the reference shooting time to the k1-th shooting time t k1 [2 < k1 < n] which is the evaluation shooting time is the so-called doubling time t ad1 (= t k1 - t1). For reference, at this time, since the relationship a k1 / a1 = e (μk1·tad1) = 2 holds, the specific growth rate at the doubling time t ad1 is specifically μ k1 = ln2 / t ad1 = ln2 / (t k1 - t1).

[0110] And similarly, for the shooting time t i [i < n], assuming that the statistical index value a of the primary information at the evaluation shooting time when i = k2 [k1 < k2 < n] k2 becomes twice the statistical index value a k1 of the k1-th shooting time t k1 when the k1-th shooting time t k1 is used as the reference shooting time (a k2 = 2a k1 ). At this time, the time from the k1-th shooting time t k1 which is the reference shooting time to the k2-th shooting time t k2 which is the evaluation shooting time is the so-called doubling time t ad2 (= t k2 - t k1 ). Here too, for reference, a k2 / a k1 =e (μk2·tad2) Since the relationship of = 2 holds, the doubling time t ad2 The specific specific growth rate at is μ k2 = 2 / (ln2 · t ad2 ) = ln2 / (t k2 -t k1 ) is obtained. The secondary time-dependent change index value described later as the doubling time is calculated from the two doubling times t ad1 (= t k1 -t1) and the doubling time t ad2 (= t k1 -t1), which are selected from between these three time points.

[0111] Furthermore, similar to the second embodiment, in addition to the doubling time, as the growth rate (specific growth rate), the predetermined doubling time (txi) required until the amount at the evaluation time point becomes a predetermined multiple of the amount at the reference time point can be adopted. That is, in the above example, assuming that, among the shooting time points t i [2 < i < n], the statistical index value a k1 of the primary information at i = k1 [2 < k1 < n] becomes a predetermined multiple (X times) of the statistical index value a1 of the primary information at the shooting time point t1 serving as the reference time point, and the statistical index value a k2 of the primary information at the evaluation shooting time point when i = k2 [k1 < k2 < n] becomes a predetermined multiple (X times) of the statistical index value a k1 of the primary information at the shooting time point t k1 serving as the reference time point, then if the doubling rate per unit time is e μi , the relationship of e [(μi)·(txi)] = X holds, and at each of i = k1 and i = k2, the predetermined doubling time tx 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) is obtained. As in the second embodiment, the doubling time is the predetermined doubling time when X=2. A secondary time-course change index value, which will be described later using these predetermined doubling times, is calculated from two predetermined doubling times calculated from values ​​at two time points selected from among the three time points.

[0112] (Secondary time-course change calculation process (obtaining primary time-course change index value)) Next, in a secondary time-course change calculation step (S1224), a secondary time-course change index value is calculated from the primary time-course change index value. The secondary time-course change index value can be calculated using a differential change amount, a percentage change amount, or a rate of change per unit time as an index of the increment of the primary time-course change index value. That is, the differential change amount or percentage change amount between the primary time-course change index value (before-change value) from the first imaging time point to the second imaging time point and each primary time-course change index value (after-change value) from the second imaging time point to the third imaging time point can be used.

[0113] When applying the differential change amount to the secondary time-course change index value, for example, as shown in Figure 4H, when the differential change amount of the statistical index value is selected as the primary time-course change value, and the primary time-course change index value (before-change value) from the first shooting time to the second shooting time and the primary time-course change index value (after-change value) from the second shooting time to the third shooting time are respectively obtained as differential change amounts a2-a1 and a3-a2 of the statistical index values, the secondary time-course change index value can be the differential change amount (a3-a2)-(a2-a1)=(a3-2a2+a1), which is the after-change value minus the before-change value. Furthermore, when applying the differential change amount to the secondary time-course change index value, the percentage change amount of the statistical index value is selected as the primary time-course change value, and when the primary time-course change index value (before-change value) from the first shooting time to the second shooting time and the primary time-course change index value (after-change value) from the second shooting time to the third shooting time are obtained as percentage changes a2 / a1 and a3 / a2 of the statistical index values, the secondary time-course change index value can be the differential change amount of those percentage changes, (a3 / a2)-(a2 / a1). When applying the differential change amount to the secondary time-varying index value, if the differential percentage change amount of the statistical index value is selected as the primary time-varying value and (a2-a1) / a1, (a3-a2) / a2 are obtained, the secondary time-varying index value can be the differential change amount of those percentage changes [(a3-a2) / a2-(a2-a1) / a1]. Furthermore, when applying the differential change amount to the secondary time-dependent change index value, if the unit time change rate of the differential change amount is selected as the primary time-dependent change index value and (a2-a1) / (t2-t1) and (a3-a2) / (t3-t2) are obtained, the secondary time-dependent change index value can be the differential change amount of those unit time change rates, [(a3-a2) / (t3-t2)]-[(a2-a1) / (t2-t1)], as shown in Figure 4I. Furthermore, when applying the differential change amount to the secondary time-course change index value, if the change rate per unit time of the percentage change amount is selected as the primary time-course change index value and a3 / [a2·(t3-t2)], a2 / [a1·(t2-t1)] are obtained, the secondary time-course change index value can also be a3 / [a2·(t3-t2)]-a2 / [a1·(t2-t1)], which is the differential change amount of these unit time change rates, as shown in Figure 4I. When applying the differential change amount to the secondary time-course change index value, if the change rate per unit time of the differential percentage change amount is selected as the primary time-course change index value and (a2-a1) / [a1·(t2-t1)] or (a3-a2) / [a2·(t3-t2)] is obtained, the secondary time-course change index value can also be the differential change amount of these unit time change rates, (a3-a2) / [a2·(t3-t2)]-(a2-a1) / [a1·(t2-t1)], as shown in Figure 4I. When the differential change amount is applied to the secondary time-course index value, if the specific growth rate is selected as the primary time-course index value, the secondary time-course index value is calculated as the differential change amount (μ k2 -μ k1 )=[ln(a k2 / a k1 )] / (t k2 -t k1 )-[ln(a k1 / a1)] / (tk1 -t1). When the differential change amount is applied to the secondary time-course index value, if the doubling time is selected as the primary time-course index value, the secondary time-course index value is calculated as the differential change amount, as shown in Figure 4J. ad2 -t ad1 =(t k2 -t k1 )-(t k1 -t1)=t k2 It becomes -t1.

[0114] Furthermore, when applying a percentage change to a secondary time-course change index value, if a differential change, which is a difference value between statistical index values, is selected as the primary time-course change index value and (a2-a1), (a3-a2) are obtained, the ratio of the primary time-course change index value (post-change value) from the second shooting time point to the primary time-course change index value (pre-change value) from the first shooting time point to the second shooting time point can be (a3-a2) / (a2-a1). When applying the percentage change to the secondary time-course index value, if the percentage change of the statistical index value is selected as the primary time-course index value and (a2 / a1) and (a3 / a2) are obtained, the ratio of the primary time-course index value (before change) from the first to the second imaging time point to the primary time-course index value (after change) from the second to the third imaging time point is calculated as (a3 / a2) ÷ (a2 / a1) = (a1·a3) / a2. 2 It can be said that: When applying the percentage change to the secondary time-course index value, if the differential percentage change of the statistical index value is selected as the primary time-course index value and (a2-a1) / a1, (a3-a2) / a2 are obtained, the ratio of the primary time-course index value (post-change value) from the second shooting time to the third shooting time to the primary time-course index value (pre-change value) from the first shooting time to the second shooting time can be calculated as (a3-a2) / a2÷(a2-a1) / a1, = [a1·(a3-a2)] / [a2·(a2-a1)]. When applying the percentage change to the secondary time-varying index value, if the unit time change rate of the differential change is selected as the primary time-varying index value and (a3-a2) / (t3-t2), (a2-a1) / (t2-t1) are obtained, then [(a3-a2) / (t3-t2)] / [(a2-a1) / (t2-t1)]=[(a3-a2)·(t2-t1)] / [(a2-a1) / (t3-t2)] can be obtained. When applying the percentage change to the secondary time-course index value, if the unit time change rate of the percentage change is selected as the primary time-course index value to obtain a2 / [a1·(t2-t1)], a3 / [a2·(t3-t2)], the secondary time-course index value can be calculated as [(a3-a2) / (t3-t2)]÷[(a2-a1) / (t2-t1)]=[(a3-a2)·(t2-t1) / (t3-t2)·(a2-a1)]. When applying the percentage change to the secondary time-course index value, if the rate of change per unit time of the differential percentage change is selected as the primary time-course index value and (a2-a1) / [a1·(t2-t1)], (a3-a2) / [a2·(t3-t2)] is obtained, the secondary time-course index value can also be (a3-a2) / [a2·(t3-t2)]÷(a2-a1) / [a1·(t2-t1)]. When the rate of change is applied to the secondary time-course index value, if the specific growth rate is selected as the primary time-course index value, the secondary time-course index value is calculated as the rate of change, as shown in Figure 4J. k2 / μ k1 =(t k1 ‐t1)·[ln(a k2 / a k1 )] / (t k2 -t k1 )·[ln(a k1 / a1)] / . When the percentage change is applied to the secondary time-course index, if the doubling time is selected as the primary time-course index, the secondary time-course index is calculated as the differential change, t ad2 / t ad1 =(t k2 -t k1 ) / (t k1 -t1).

[0115] Similarly, when applying the differential percentage change to the secondary time-varying index value, if the differential change, which is the difference value of the statistical index value, is selected as the primary time-varying index value and (a2-a1), (a3-a2) are obtained, the secondary time-varying index value can be [(a3-a2)-(a2-a1)] / (a2-a1)=(a3-2a2+a1) / (a2-a1). When applying the differential percentage change to the secondary time-varying index value, if the percentage change of the statistical index value is selected as the primary time-varying index value and (a2 / a1), (a3 / a2) are obtained, the secondary time-varying index value can be [(a3 / a2)-(a2 / a1)] / (a2 / a1). When applying the differential percentage change to the secondary time-varying index value, if the differential percentage change of the statistical index value is selected as the primary time-varying index value and (a2-a1) / a1, (a3-a2) / a2 are obtained, the secondary time-varying index value can be {[(a3-a2) / a2]-[(a2-a1) / a1]} / (a2-a1) / a1. When applying the differential percentage change to the secondary time-course index value, if the unit time change rate of the differential change is selected as the primary time-course index value and (a3-a2) / (t3-t2), (a2-a1) / (t2-t1) are obtained, then {[(a3-a2) / (t3-t2)]―[(a2-a1) / (t2-t1)]} / (a2-a1) / (t2-t1)={[(a3-a2) / (t3-t2)]―[(a2-a1) / (t2-t1)]}·(t2-t1) / (a2-a1). When applying the differential percentage change to the secondary time-course index value, if the unit time change rate of the percentage change is selected as the primary time-course index value and a2 / [a1·(t2-t1)], a3 / [a2·(t3-t2)] is obtained, the secondary time-course index value can be {a3 / [a2·(t3-t2)]-a2 / [a1·(t2-t1)]} / a2 / [a1·(t2-t1)]={a3 / [a2·(t3-t2)]-a2 / [a1·(t2-t1)]}·[a1·(t2-t1)] / a2. When applying the differential percentage change to the secondary time-course index value, if the rate of change per unit time of the differential percentage change is selected as the primary time-course index value and (a2-a1) / [a1·(t2-t1)], (a3-a2) / [a2·(t3-t2)] is obtained, the secondary time-course index value can also be the differential change of those unit time change rates, {[(a3-a2) / [a2·(t3-t2)]-[(a2-a1) / [a1·(t2-t1)]} / {(a2-a1) / [a1·(t2-t1)}. When the differential rate of change is applied to the secondary time-course index value, if the specific growth rate is selected as the primary time-course index value, the secondary time-course index value is calculated as the differential rate of change, as shown in Figure 4J. 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)}. When the differential percentage change is applied to the secondary time-course index, if the doubling time is selected as the primary time-course index, the secondary time-course index is calculated as the differential change (t ad2 -t ad1 ) / t ad1 =[(t k2 -t k1 )-(t k1 -t1)] / (t k1 -t1)=(t k2 -t1) / (t k1 -t1).

[0116] Furthermore, when the specific growth rate and doubling time are selected as the primary time-course index, the rate of change per unit time can be applied to the secondary time-course index. In this case, the secondary time-course index can be calculated in the same manner as above, so the details are omitted here.

[0117] Since one secondary time-course change index value is created for two primary time-course change index values, overall, one less secondary time-course change index value is created than the number of primary time-course change index values, and two less secondary time-course change index values ​​are created than the statistical index values ​​of the primary information. A number of secondary time-course change index values ​​that is two less than the number of observation locations Op or the number of representative values ​​is acquired. This completes the judgment data creation step (S12), and the secondary time-course change index values ​​obtained above are used as judgment data. The multiple secondary time-course change index values ​​may be used as multiple judgment data without changing the number, or multiple secondary time-course change index values ​​may be used as a single time-course change index value by using an average value, maximum value, minimum value, etc., and used as a single judgment data.

[0118] Although the secondary time-course index value in the first example is calculated via the primary time-course index value, as in the second embodiment, it represents the time-course element of the primary information and quantitatively indicates the state of cells on the culture surface at a predetermined observation point Op at a plurality of predetermined points selected within the culture vessel. In the first example, the secondary time-course index value is the sum of statistical index values ​​at the predetermined observation point Op, and is a representative value that can accurately represent the room for cell spreading or proliferation within the culture vessel. The time-course index value can be calculated based on the selection of primary information such as cell number, cell density, cell-occupied area, cell-single area, and cell-to-cell distance, and the selection of statistical index values ​​such as sum, mean, variance, and deviation. In the first example, the judgment data represents a representative value of the acceleration of change in the representative value of the primary information when the sum or mean is selected as the statistical index value, and represents the acceleration of change in the variance of the primary information when the variance or deviation is selected as the statistical index value.

[0119] (Subculture determination) After the creation of the judgment data in the judgment data creation step (S12) is completed, a judgment step (S13) is executed to determine whether the time for passaging has arrived. In the judgment step (S13), the timing for passaging is determined based on the judgment data created in the judgment data creation step (S12). In the judgment step (S13), the judgment data, which is the secondary time-course change index value calculated in the time-course change calculation step (S1213), is compared with a predetermined threshold. Here, similar to the second embodiment, it is determined that the time for passaging has arrived when the judgment data reaches the predetermined threshold. In the first example of the third embodiment, at the time for passaging, some kind of inflection point, represented by an extreme value such as a minimum or maximum value, appears in the time change of the secondary time-course change index value. The time when such an inflection point appears in the time change is set as the threshold, indicating that the time for passaging has arrived.

[0120] As in the second embodiment, when the number of pieces of judgment data is one, the one piece of judgment data is compared to see if it reaches a predetermined threshold, and if it does, it is determined that the time for passaging has arrived. When the number of pieces of judgment data is multiple, a predetermined number of pieces of judgment data among the multiple pieces of judgment data are compared to see if they reach a predetermined threshold, and if they do, it is determined that the time for passaging has arrived. Furthermore, when all of the plurality of determination data reach a predetermined threshold, it may be determined that the time for passaging has arrived. In this case, it is possible to determine earlier whether the time for passaging has arrived. In this case, it is determined relatively late whether the time for passaging has arrived. Conversely, if at least one of the plurality of determination data reaches a predetermined threshold, it may be determined that the time for passaging has arrived, in which case it is possible to determine earlier whether the time for passaging has arrived.

[0121] In the case of the reference ratio method, the predetermined threshold is the same as in the first and second embodiments. As in the first embodiment, a test is conducted in advance as a verification under the same conditions as those for actually determining the timing of passaging, and in that test, a primary time-course change index value and a secondary time-course change index value are obtained for the statistical index value at the time point at which the passage timing is reached when cell spreading or proliferation no longer appears, and these are defined as the predetermined threshold. Even if a time-course change element is added, the characteristics of the statistical index value in determining the timing of passaging are not lost, and this is the same as the aspects described in the first and second embodiments.

[0122] Furthermore, in the case of the most recent ratio method, in which three imaging time points are selected as three consecutive imaging time points, the predetermined threshold is as follows: As described above, the secondary time-course index value is the increment of the primary time-course index value. As described in the second embodiment, the primary time-course index value is the increment of the statistical index value of the primary information. Therefore, the primary time-course index value gradually increases in the initial state and then increases at a certain point, as if the cell spreading or proliferation suddenly increases. Thereafter, the rate of increase gradually decreases, and at the time of passaging, it tends to reach a maximum value and then start decreasing. Therefore, the secondary time-course index value gradually increases in line with the increasing trend of the primary time-course index value in the initial state, reaches a positive maximum value, and then gradually decreases in line with the attenuation of the secondary time-course index value. Then, at the time of passaging, when the primary time-course index value reaches its maximum value, the secondary time-course index tends to conversely reach a negative minimum value and start increasing. Therefore, this minimum value is set as the predetermined threshold. Furthermore, as in the second embodiment, when the secondary time-course change index value reaches a predetermined threshold, the time for passaging has already been reached, and therefore a time point slightly before that can be said to be the most suitable time for passaging. Therefore, the predetermined threshold can be set as the value obtained by adding a margin value α to the minimum value of the secondary time-course change index value ("minimum value of the secondary time-course change index value" + "margin value α"). This makes it possible to determine whether cells that do not yet show a slowdown in cell spreading or proliferation very close to the time for passaging are in a good condition and have reached the time for passaging. As in the second embodiment, the "margin value α" is also set in a test performed in advance as a verification.

[0123] <Example 2> Next, a second example will be described with reference to FIG. 4B. In the first example (FIG. 4A), after primary information is acquired at each imaging time point in the primary information acquisition step (S1221), a statistical index value at each imaging time point is calculated from the primary information at each imaging time point in the statistical index value acquisition step (S1222). Then, a primary time-course change index value is calculated in the primary time-course change calculation step (S1223), and then a secondary time-course change index value is calculated in the secondary time-course change calculation step (S1224) to be used as determination data. However, in the second example, after primary information at each imaging time point is acquired in the primary information acquisition step (S1221), a primary time-course change index value is calculated by executing the primary time-course change calculation step (S1223), and then a secondary time-course change index value is calculated. That is, compared to the first example, the difference is that the primary time-varying change calculation step (S1223) and the secondary time-varying change calculation step (S1224) are combined and moved to immediately after the primary information acquisition step (S1221). Then, in the statistical index value acquisition step (S1222), the statistical index value of the secondary time-varying change index value is calculated and used as judgment data. The primary information and the primary information acquisition step (S1221) are the same as those in the first embodiment and first example, so a description thereof will be omitted.

[0124] (First time-course change calculation process (obtaining time-course change index value)) Following the primary information acquisition step (S1221), a primary time-course change calculation step (S1223) calculates a primary time-course change index value for each piece of primary information calculated at each imaging time point. While the primary time-course change calculation step (S1223) of the first example acquires a primary time-course change index value of a statistical index value, the primary information acquisition step (S1221) of the second example acquires a primary time-course change index value of the primary information. Therefore, if the statistical index value of the primary time-course change calculation step (S1223) of the first example is read as primary information and the primary time-course change calculation step (S1223) of the first example is applied, it becomes the primary time-course change calculation step (S1223) of the second example. The primary time-course change index value is the amount of change in the primary information between each imaging time point. Furthermore, the primary time-varying index value in the second example is a differential change amount, a rate of change, a rate of change per unit time, a rate of change, or a change time, as in the first example. The only difference is that in the first example, the primary information statistical index value is a differential change amount, a rate of change, a rate of change per unit time, a rate of change, or a change time, whereas in the second example, the primary information statistical index value is a differential change amount, a rate of change, a rate of change per unit time, a rate of change, or a change time. Therefore, in the calculation of the primary time-varying index value in the first example, where the primary time-varying index value is calculated from the primary information statistical index value, the "primary information statistical index value" can simply be read as "primary information," and the first example can be applied to the second example. Also, in the second example, as in the first example, the rate of change can be applied as a specific growth rate, and the change time can be applied as a doubling time. This will be further explained with reference to Figures 4H and 4I. Figures 4H and 4I are diagrams illustrating the concept of primary time-course change, which is the change in primary information (vertical axis) over time (horizontal axis) for the first and second examples. Figure 4H illustrates a case where the primary time-course change index value is a change amount or rate of change, and Figure 4I illustrates a case where the primary time-course change index value is a change amount or rate of change per unit time. Both Figures 4H and 4I simultaneously illustrate the primary time-course change index value calculated in the primary time-course change calculation step (S1223) described here and the secondary time-course change index value calculated in the secondary time-course change calculation step (S1224) described next. In the second example, the statistical index values ​​a1 and a2 in the description of the primary time-course change calculation step (S1223) of the first example and in Figures 4H and 4I can be interpreted as the values ​​of the primary information acquired in the primary information acquisition step (S1221). Since one piece of primary information is obtained from two pieces of primary information, the number of primary time-dependent change index values ​​obtained corresponds to the number of pieces of primary information minus 1. The primary time-dependent change index value in the second example is a value that directly indicates the time-dependent change tendency of the primary information.

[0125] (Secondary time-dependent change calculation process (obtaining time-dependent change index values)) Following the primary time-course change calculation step (S1222), a secondary time-course change index value is calculated in a secondary time-course change calculation step (S1223). In the second example, the statistical index value in the first example is replaced with primary information, and a process similar to that in the first example is applied. As in the first example, the secondary time-course change index value is a change amount or rate of change that is an increment of the primary time-course change index value. That is, it is the primary time-course change index value from the first imaging time point to the second imaging time point, and the change amount of each primary time-course change index value from the second imaging time point to the third imaging time point. This will be explained with reference to Figures 4H and 4I. Figures 4H and 4I are diagrams illustrating the concept of change in primary information (vertical axis) with respect to time (horizontal axis) for the first and second examples. Both Figures 4H and 4I show the secondary time-course change index value calculated in the secondary time-course change calculation step (S1224) along with the primary time-course change index value. In the secondary time-course change calculation step (S1224) of the second example, secondary time-course change index values ​​are obtained by the primary time-course change calculation step (S1223), and so, similarly to the primary time-course change calculation step (S1223), if the statistical index values ​​in the primary time-course change calculation step (S1223) of the first example are replaced with primary information and the secondary time-course change calculation step (S1224) of the first example is applied, the result is the secondary time-course change calculation step (S1224) of the second example. Since one secondary time-course change index value is created for two primary time-course change index values, overall, one secondary time-course change index value less than the number of primary time-course change index values ​​is created, and two secondary time-course change index values ​​less than the statistical index values ​​of the primary information are created, which is also similar to the first example.

[0126] (obtaining statistical indicator values) After calculating the secondary time-course change index value in the secondary time-course change calculation step (S1224), the statistical index value at each imaging time point is calculated from the primary information at each imaging time point in the statistical index value acquisition step (S1222). The statistical index value in the second example is an index value defined as a representative value that represents the secondary time-course change index value. As in the first and second embodiments, the statistical index value is at least one of the mean value, variance, and deviation such as standard deviation. In the second example, the statistical index value of the secondary time-course change index value is calculated, so it is the mean value, variance, deviation, etc. of the secondary time-course change index value at each of the multiple observation points Op. The statistical index value calculated here is used as judgment data to determine the timing of passage. The multiple statistical index values ​​may be used as multiple judgment data as they are, or multiple statistical index values ​​may be combined into a single statistical index value based on the mean value, maximum value, minimum value, etc., and used as a single judgment data. The statistical index value of the secondary time-varying index value calculated in the statistical index value acquisition process (S1222) of the second example has the same properties and technical significance as the judgment data, which is the statistical index value of the time-varying index value in the second example of the second embodiment.

[0127] (Subculture determination) After the creation of the judgment data in the judgment data creation step (S12) is completed, a judgment step (S13) is executed to determine whether the timing for passaging has arrived. In the judgment step (S13), the timing of passaging is determined based on the statistical index value created in the judgment data creation step (S12). The judgment data of the second example includes, as the statistical index value of the secondary time-course change index value, primary information, elements of time-course change, which are the primary time-course change index value and the secondary time-course change index value, and elements of the statistical index value. Therefore, as judgment data for determining the timing of passaging, it is no different from the second example of the second embodiment and the first example of this embodiment, and is effective in determining the timing of passaging. The judgment data of the second example means a representative value of the acceleration of change in the primary information when the sum or average is selected as the statistical index value, and means the variation in the acceleration of change in the primary information when the variance or deviation is selected as the statistical index value.

[0128] 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 conducted in advance as a test under the same conditions as those actually used to determine the timing of passaging, and in that test, a statistical index value at the time point when passaging is to be obtained is obtained, and a time-course index value is determined, which is then set as the predetermined threshold value. Even if an element of time-course change is added, the characteristics of the statistical index value in determining the timing of passaging are not lost, as explained in the first embodiment. When the number of judgment data is created as a representative value of 1, if the number of judgment data is one, that one judgment data is compared to see if it reaches a predetermined threshold, and if it does, it is determined that the time for passaging has been reached. When the number of judgment data is multiple, a predetermined number of the judgment data among the multiple judgment data are compared to see if they reach a predetermined threshold, and if they do, it is determined that the time for passaging has been reached. In the second example of the third embodiment, too, at the time of passaging, some kind of inflection point, represented by an extreme value such as a minimum or maximum value, appears in the trend of change in the statistical index value. The point at which such an inflection point in the trend of change appears is set as the threshold, indicating that the time for passaging has been reached. Furthermore, it may be determined that the time for passaging has been reached when all of the plurality of determination data have reached a predetermined threshold value. In this case, since it takes time for all of the determination data to reach the predetermined threshold value, whether the time for passaging has been reached will be determined relatively late. Conversely, it may be determined that the time for passaging has been reached when at least one of the plurality of determination data reaches a predetermined threshold value. In this case, since the determination can be made when one determination data reaches a predetermined threshold value, it is possible to determine early whether the time for passaging has been reached.

[0129] <Example 3> Next, a third example will be described with reference to FIG. 4C. In the third example (FIG. 4C), after primary information is acquired at each imaging time point in the primary information acquisition step (S1221), a primary time-dependent change index value is calculated in the primary time-dependent change calculation step (S1223), and then a statistical index value at each imaging time point is calculated from the primary information at each imaging time point in the statistical index value acquisition step (S1222). Then, a secondary time-dependent change index value of the statistical index value is calculated in the secondary time-dependent change calculation step (S1224) to be used as determination data. That is, between the primary time-dependent change calculation step (S1223) and the secondary time-dependent change calculation step (S1224), the statistical index value acquisition step (S1222) is executed to calculate a statistical index value at each imaging time point from the primary information, and then the secondary time-dependent change index value of the statistical index value is calculated in the secondary time-dependent change calculation step (S1224). This will be described below. The primary information and the primary information acquisition step (S1221) are the same as those in the first embodiment, and therefore will not be described here.

[0130] (First time-course change calculation process (obtaining time-course change index value)) Following the primary information acquisition step (S1221), a primary time-course change calculation step (S1223) calculates the primary time-course change index value of each piece of primary information calculated at each imaging time point. The processes of the primary information acquisition step (S1221) and the primary time-course change calculation step (S1223) are the same as those in the second example (FIG. 4B), so a detailed description will be omitted. In the third example, the statistical index values ​​a1 and a2 in the description of the primary time-course change calculation step (S1223) in the first example and in FIGS. 4H and 4I can be read as the values ​​of the primary information acquired in the primary information acquisition step (S1221).

[0131] (obtaining statistical indicator values) After calculating the primary time-dependent change index value of the primary information in the primary time-dependent change calculation step (S1223), a statistical index value of the primary time-dependent change index value is calculated in the statistical index value acquisition step (S1222). The statistical index value in the third example is an index value defined as a representative value that represents the primary time-dependent change index value. As in the first and second embodiments, the statistical index value is at least one of deviations such as the average value, variance, and standard deviation. In the third example, statistical index values ​​of the primary time-dependent change index value, such as the difference change amount, change amount rate, unit time change rate, change speed, or change time, are calculated. That is, in the third example, statistical index values ​​of these primary time-dependent change index values ​​are calculated, and therefore are the average value, variance, deviation, etc. of the difference change amount, change amount rate, unit time change rate, change speed, or change time of the primary information at each of the multiple observation locations Op.

[0132] (Secondary time-dependent change calculation process (obtaining time-dependent change index values)) Following the statistical index value acquisition step (S1222), a secondary time-course change index value is calculated in a secondary time-course change calculation step (S1224). In the third example, the primary time-course change index value in the second example is replaced with a statistical index value, and the secondary time-course change acquisition step (S1224) is performed using a process similar to that of the secondary time-course change calculation step (S1224) in the second example. In the third example, the secondary time-course change index value is the differential change amount, rate of change, rate of change per unit time, rate of change, or time of change (secondary time-course change index value) of the average value, variance, or deviation (statistical index value) of the differential change amount, rate of change, rate of change per unit time, rate of change, or time of change (primary time-course change index value). This statistical index value is used as judgment data to determine the timing of passage. The multiple statistical index values ​​may be used as multiple judgment data as they are, or multiple statistical index values ​​may be combined into a single statistical index value based on the average value, maximum value, minimum value, etc., and used as a single judgment data.

[0133] (Subculture determination) After the creation of the judgment data in the judgment data creation step (S12) is completed, a judgment step (S13) is performed to determine whether the time for passaging has arrived. In the judgment step (S13), the timing of passaging is determined based on the statistical index value created in the judgment data creation step (S12). The judgment data in the third example is a secondary time-course change index value of the statistical index value. As in the first and second examples, the judgment data includes primary information, time-course change elements (primary time-course change index value and secondary time-course change index value), and statistical index value elements. Therefore, as judgment data for determining the timing of passaging, it is the same as the second embodiment and the first and second examples of this embodiment, and is effective for determining the timing of passaging. The judgment data in the third example means the rate of a representative value of the rate of change of the primary information when the sum or average value is selected as the statistical index value, and means the rate of variation in the rate of change of the primary information when the variance or deviation is selected as the statistical index value.

[0134] In the determination step (S13), the secondary time-course change index value calculated in the secondary time-course change calculation step (S1224) 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 actually conducted in advance under the same conditions as the process of the third example (FIG. 4C) for determining the timing of passage. In the test, the primary time-course change index value of the primary information at the time of passage is calculated, a statistical index value is calculated, and a secondary time-course change index value of the statistical index value is calculated, and this is defined as the predetermined threshold value. Even if a time-course element is added, the characteristics of the statistical index value in determining the timing of passage are not lost, as explained in the first embodiment. When the number of pieces of judgment data is created as one representative value, if the number of pieces of judgment data is one, that one piece of judgment data is compared to see if it reaches a predetermined threshold, and if it does, it is determined that the time for passaging has been reached. When the number of pieces of judgment data is multiple, a predetermined number of pieces of judgment data among the multiple pieces of judgment data are compared to see if they reach a predetermined threshold, and if they do, it can be determined that the time for passaging has been reached. In the third example of the third embodiment, at the time of passage, some inflection point, such as a minimum value, a maximum value, or other extreme value, appears in the time change of the secondary time change index value. The time point at which such an inflection point appears in the time change is set as the threshold value, indicating that the time of passage has been reached. Furthermore, it may be determined that the time for passaging has been reached when all of the plurality of determination data have reached a predetermined threshold value. In this case, since it takes time for all of the determination data to reach the predetermined threshold value, whether the time for passaging has been reached will be determined relatively late. Conversely, it may be determined that the time for passaging has been reached when at least one of the plurality of determination data reaches a predetermined threshold value. In this case, since the determination can be made when one determination data reaches a predetermined threshold value, it is possible to determine early whether the time for passaging has been reached.

[0135] [Fourth embodiment] Next, as a fourth embodiment, a method and system for determining the timing of passage will be described with reference to Figures 5A and 5B. Figure 5A is a diagram showing the concept of i reference imaging time points (i = a natural number from 1 to m) that are imaging time points of interest in the fourth embodiment, and values ​​Ri (i = a natural number from 1 to m) corresponding to the primary information thereof, and j evaluation imaging time points (j = a natural number from 1 to n) and values ​​Vj (j = a natural number from 1 to n) corresponding to the primary information thereof.

[0136] In the third embodiment, a time-lapse index value is calculated for a photographing time point of interest between one reference photographing time point and two or more evaluation photographing time points. In contrast, in the fourth embodiment, a time-lapse index value is calculated for a photographing time point of interest between two or more reference photographing time points and two or more evaluation photographing time points. The number of reference photographing time points and the number of evaluation photographing time points can each be freely set to one or more. That is, as shown in FIG. 5A , i reference photographing time points (i = a natural number from 1 to m) and j evaluation photographing time points (j = a natural number from 1 to n) can be freely set as photographing time points of interest. Of these, the second embodiment described above is an example in which one reference photographing time point and one evaluation photographing time point (j = a natural number from 1 to n) are set as photographing time points of interest. The third embodiment described above is an example in which one reference photographing time point and two evaluation photographing time points (j = a natural number from 1 to n) are set as photographing time points of interest.

[0137] The fourth embodiment differs from the third embodiment in that a plurality of reference imaging time points (i = a natural number from 1 to m) is used, but the third embodiment can be applied in the same manner except for the use of a plurality of reference imaging time points (i = a natural number from 1 to m). There are two methods for applying the third embodiment to the fourth embodiment. One is a method (hereinafter referred to as the reference value integration method) in which, when executing the primary time-varying index value (S1223), the statistical index value (in the first example) based on primary information acquired at a plurality of reference imaging time points (i = a natural number from 1 to m) and the primary information (in the second and third examples) acquired at a plurality of reference imaging time points (i = a natural number from 1 to m) are integrated into a single representative reference value by performing representative value integration for each case, and then the third embodiment is applied. The other is a method (hereinafter referred to as the matrix time-course method) in which the third embodiment is applied to each of the cases of statistical index values ​​based on primary information acquired at multiple reference shooting times (i = a natural number from 1 to m) (in the case of the first example) and primary information acquired at multiple reference shooting times (i = a natural number from 1 to m) (in the cases of the second and third examples) as multiple reference values ​​without integrating them into a single representative value.

[0138] FIG. 5B illustrates the concept of the reference value integration method. In the reference value integration method, a representative reference value Rrep is created as follows, and the third embodiment described above is applied. In a first example of the third embodiment, in the primary information acquisition step (S1221), a representative value is created using a statistical method such as averaging, summing, or weighting the primary information acquired at multiple reference imaging times (i = a natural number from 1 to m) to create a representative reference value Rrep for one piece of primary information. In addition, in the second and third examples of the third embodiment, in the primary information acquisition step (S1221), a representative value is created using a statistical method such as averaging, summing, or weighting the statistical index values ​​of the primary information acquired at multiple reference imaging times (i = a natural number from 1 to m) to create a representative reference value Rrep for one piece of primary information. At this time, because the statistical index values ​​of the primary information are also averages, sums, or weighting values, statistical processing is continuous, but the representative value creation for creating a representative reference value and the statistical index values ​​of the primary information have different technical significance. The former is a process for integrating numerical values ​​corresponding to multiple reference imaging time points (i = a natural number from 1 to m) into a single representative value, while the latter is a process for creating a statistical index value representing the state of the primary information of the cell. After integrating values ​​Ri (i = a natural number from 1 to m) based on multiple pieces of primary information acquired at multiple reference imaging time points (i = a natural number from 1 to m) into a single representative reference value Rrep, the third embodiment can be applied as is in accordance with the above descriptions of the first to third examples so as to calculate the primary time-course index value (Rrep,V1) (Rrep,Vj) (Rrep,Vj) between j evaluation imaging time points (j = a natural number from 1 to n) and values ​​Vj (j = a natural number from 1 to n) corresponding to the primary information.

[0139] Unlike the reference value integration method, the matrix time-course method does not involve representativeizing to a single reference value, but rather simply applies the third embodiment described above to each of the first to third examples based on the number of primary information corresponding to the number of reference shooting time points (i = natural numbers from 1 to m).

[0140] 5C illustrates the concept of the matrix time-course method, that is, the concept of calculating a primary time-course index value from values ​​Ri (i = a natural number from 1 to m) corresponding to the primary information at each reference imaging time point (i = a natural number from 1 to m) and values ​​Vj (j = a natural number from 1 to n) corresponding to the primary information at each evaluation imaging time point (j = a natural number from 1 to n).

[0141] Unlike the reference value integration method, the matrix time-course method does not require the data to be represented by a single representative reference value Rrep, but rather applies the third embodiment to each of the first to third examples based on the number of primary information items corresponding to the number of reference imaging time points (i = a natural number from 1 to m). For example, assume that a value derived from the primary information at each reference imaging time point (i = a natural number from 1 to m) is Ri (i = a natural number from 1 to m). This value Ri (i = a natural number from 1 to m) derived from the primary information represents a statistical index value in the first example of the third embodiment, and a primary time-course index value in the second and third examples of the third embodiment. In the first to third examples of the third embodiment, for each of the m values ​​Ri (i = a natural number from 1 to m), and each of the n values ​​Vj (j = a natural number from 1 to n), the following m × n primary time-varying index values ​​are calculated for the next step: a set based on R1 (R1,V1)...(R1,Vj)...(R1,Vn), a set based on Ri (i = 1 to m), (Ri,V1)...(Ri,Vj)...(Ri,Vn), and a set based on Rm (Rm,V1)...(Rm,Vj)...(Rm,Vn), and secondary time-varying index values ​​are calculated from each of these. In the third embodiment, since there was only one reference imaging time point, primary time-varying index values ​​were generated for the number of evaluation imaging time points, and a secondary time-varying index value that was one less than that was generated. That is, when the evaluation shooting time point is set (i=a natural number from 1 to n), n primary time-course index values ​​and (n-1) secondary time-course index values ​​are generated. In the matrix time-course method of the fourth embodiment, these are multiplied by m to generate index values, i.e., (m×n) primary time-course index values ​​and m·(n-1) secondary time-course index values.

[0142] That is, assuming that the value derived from the primary information at each evaluation shooting time point (j = a natural number from 1 to n) is Vi (i = a natural number from 1 to m), the first to third examples of the third embodiment are applied between the value Ri (i = a natural number from 1 to m) derived from the primary information at each reference shooting time point (i = a natural number from 1 to m) and the value Vj (j = a natural number from 1 to m) derived from the primary information at each evaluation shooting time point (j = a natural number from 1 to n). The value Vj (j = a natural number from 1 to n) derived from the primary information also means a statistical index value in the first example of the third embodiment, and a primary time-dependent change index value in the second and third examples of the third embodiment. That is, in the third embodiment, the reference shooting time point is one point (i=1) and the evaluation shooting time point is three points (j=3), and in the case of the reference ratio method, R1=a1, V1=a2 and R1=a1, V2=a3, and in the case of the most recent ratio method, R1=a1, V1=a2 and R2=a2, V2=a3.

[0143] In the matrix time-course method, in either the first or third example of the third embodiment, a secondary time-course value is obtained based on the number of values ​​corresponding to a plurality of reference imaging time points. In the determination step (S13) of determining whether the time for passage has arrived, a predetermined threshold can be set as follows.

[0144] Even in the matrix time-course method of the fourth embodiment, a preliminary test is performed under the same conditions before the actual passaging timing is determined, and the predetermined threshold is determined from the results of that test at the time point when the passaging timing is reached. Since this test is performed under the same conditions as the actual passaging timing determination, the number of reference imaging time points (i = a natural number from 1 to m) and the number of evaluation imaging time points (j = a natural number from 1 to n) are also set to the same. Therefore, when applying the first or third example of the third embodiment, in the test, the set (R1,V1)···(R1,Vj)···(R1,Vn) based on R1, the set (Ri,V1)···(Ri,Vj)···(Ri,Vn) based on Ri (i = 1 to m), and the set (Rm,V1)···(Rm,Vj)···(Rm,Vn) based on Rm are calculated as primary time-course index values, and secondary time-course index values ​​are calculated from each of these to create judgment data. Then, judgment data is created at the time when it is determined that the time for subculture has arrived. In this case, multiple judgment data are generated for each of the secondary time-course index values ​​(first and third examples) and the statistical index value of the secondary time-course index values ​​(second example) as judgment data. However, even when multiple reference imaging time points are used, inflection points are observed in the change trends of the secondary time-course change index values ​​(Example 1 and Example 3) and the statistical index values ​​of the secondary time-course change index values ​​(Example 2), and these points are defined as predetermined thresholds for determining the timing of passage. In particular, when multiple reference imaging time points are used, patterns having inflection points also appear in the change trends themselves of the set based on R1, the set based on Ri (i = 1 to m), and the set based on Rm, making it possible to make more accurate determinations than in the second and third embodiments, which have time-course change elements.

[0145] Next, with reference to FIG. 5D , another embodiment, which is a modification of the fourth embodiment, will be described. FIG. 5D shows the tendency of change in the values ​​(vertical axis) of the determination data (circles and black dots) that change with the progress of culture (the passage of time, which is the horizontal axis). In FIG. 5D , the circles represent values ​​corresponding to the reference imaging time point, and the black dots represent values ​​corresponding to the evaluation imaging time point. When creating the determination data in the determination data creation step (S12), during the stage in which cell spreading or proliferation is progressing before the time for passaging arrives, the increasing trend of the values ​​corresponding to the reference imaging time point and the increasing trend of the values ​​corresponding to the evaluation imaging time point fall within a certain range of variation in the increasing trend (Group A of values ​​corresponding to the evaluation imaging time point in FIG. 5D ). However, when the time for passaging arrives, a significant change in the increasing trend of the values ​​corresponding to the reference imaging time point and the evaluation imaging time point appears (Group B of values ​​corresponding to the evaluation imaging time point in FIG. 5D ). Therefore, in creating the determination data, the evaluation imaging time point at which the trend of Group B of values ​​corresponding to the evaluation imaging time point in FIG. 5D begins to appear is determined to be the time for passaging. The point in time when the tendency of the group B of values ​​corresponding to the evaluation photographing time in FIG. 5D begins to appear is the inflection point of the change tendency of the judgment data.

[0146] In this case, in the judgment data generation step (S12), the timing of passaging can be determined based on the stage at which an inflection point appears in the change trend of the judgment data. This can be applied to the first embodiment. Alternatively, a test as a verification test may be performed in advance under the same conditions as those actually used to determine the timing of passaging, and the stage at which an inflection point appears in the change trend of the judgment data in the verification test may be defined as the predetermined threshold, and the first to fourth embodiments may be applied.

[0147] [Fifth embodiment] Next, a fifth embodiment will be described with reference to FIG. 5E. FIG. 5E shows the tendency of change in the values ​​(vertical axis) of the determination data (circles) that change with the progress of culture (the passage of time on the horizontal axis). The fifth embodiment is a variation of the second to fourth embodiments, which utilizes time-varying factors in the determination step (S13) of determining the passage timing described in the first to fourth embodiments. That is, the fifth embodiment applies the steps of the second to fourth embodiments as they are, except for the determination step (S13). However, the determination step (S13) of the fifth embodiment, which is the present embodiment described below, is applied to each of the second to fourth embodiments.

[0148] In the determination step (S13) of the fifth embodiment, machine learning or deep learning is used to determine the predetermined threshold value. There are no limitations on the type of machine learning or deep learning, and any commonly used machine learning or deep learning can be used. Here, the machine learning or deep learning itself is not unique, and by using machine learning or deep learning to determine the timing of passaging in the first to fourth embodiments of the present invention, it is possible to determine the timing of passaging more timely than in the second to fourth embodiments.

[0149] FIG. 5E shows an example of the change trends of the number of cells as primary information and the sum as statistical index values ​​in the first to fourth embodiments. In this case, the change trend increases until the time of passaging, and an inflection point of the change trend is reached at the time of passaging. After the time of passaging arrives, the increase stops and the change trend reaches a saturation state. The change trends of other primary information and other statistical index values ​​in the first to fourth embodiments differ from those shown in FIG. 5, but they are the same in that they have an inflection point of the change trend at the time of passaging. In the following explanation of the fifth embodiment, the change trend shown in FIG. 5E appears at the time of passaging, and it should be understood as a common point where a difference in the change trend appears before and after that point.

[0150] The actual time for passaging is near the point indicated by the thick arrow in Figure 5E. Around this point, the trend of change in the judgment data (circles) reaches an inflection point. However, because the judgment is performed discretely at predetermined time intervals during the progression of the culture, it is not possible to perform the judgment at a time that coincides with an inflection point. In the second to fourth embodiments, which utilize time-dependent change factors, a test is performed in advance as a test under the same conditions as those actually used to determine the timing of passaging, and the predetermined threshold is determined from the test results. In the fifth embodiment, a test is also performed in advance as a test under the same conditions as those actually used to determine the timing of passaging, and machine learning or deep learning is applied to the test results to determine the predetermined threshold.

[0151] In the second to fourth embodiments, the results of the certification test are determined in advance based on the intuition of an experienced user. In this case, in FIG. 5E, a line L1 showing the change trend during the culture stage and a line L2 showing the change trend after the culture has progressed and the time for passage has arrived are intuitively judged, and the time for passage is determined to be the inflection point around the intersection of line L1 showing the change trend during the culture stage. However, in the fifth embodiment, which applies machine learning or deep learning, it is possible to capture change trends that even experienced users cannot recognize. For example, conceptually, line L2 showing the change trend in FIG. 5E is judged to be the time for passage. AI As described above, it is possible to derive a change trend that even an experienced user cannot recognize, and to derive a time point for advance passage timing that cannot be obtained without using machine learning or deep learning. In the fifth embodiment, this time point is defined as a predetermined threshold.

[0152] In the determination step (S13), when the determination data reaches a predetermined threshold, it is determined that the time for passage has arrived, as in the second to fourth embodiments. [Explanation of symbols]

[0153] 1. Cell culture system 2 Culture medium units 3. Passage Timing Determination Unit 4. Culture Unit 5 Cell inoculation device 6 Transport Unit 7. Centrifugal device 8 Processing equipment 31 Camera (imaging means) 32 Shooting Stage 33 Processing means 61 Mobile stand 62 Arm 63 Actuator Pe Evaluation Culture Vessel

Claims

1. A method for determining the passage timing of cells that are spreading or growing in a medium in a culture vessel, comprising: an imaging step of photographing cells at a plurality of observation points, which are partial regions selected from the entire area of ​​the culture vessel, at a first imaging time point and a second imaging time point after the first imaging time point, and acquiring cell images of the plurality of observation points at each of the first imaging time point and the second imaging time point; a primary information acquisition step of acquiring primary information about any one of the number of cells, the density of the number of cells, the area occupied by a cell, the area of ​​a single cell, and the relative distance between cells, captured in the cell images of the plurality of observation points at each of the first and second photographing points; a statistical index value acquisition step of calculating statistical index values ​​of the primary information of the cells being photographed at the plurality of observation points at each of the first photographing time point and the second photographing time point; a time-varying change calculation step of calculating a time-varying change index value from the statistical index values ​​at the first imaging time point and the second imaging time point, and determining that the time point at which the photographing was performed corresponding to the time point at which the time-course index value reached a predetermined threshold value is the time point at which the passaging timing has been reached.

2. A method for determining the passage timing of cells that are spreading or growing in a medium in a culture vessel, comprising: an imaging step of photographing cells at a plurality of observation points, which are partial regions selected from the entire area of ​​the culture vessel, at a first imaging time point and a second imaging time point after the first imaging time point, and acquiring cell images of the plurality of observation points at each of the first imaging time point and the second imaging time point; a primary information acquisition step of acquiring primary information about any one of the number of cells, cell density, cell number density, cell occupied area, cell individual area, and relative distance between cells captured in the cell images of the plurality of observation points at each of the first and second photographing points; a time-course change calculation step of calculating a time-course change index value from the primary information of the cells being photographed at the plurality of observation points at the first photographing time point and the second photographing time point; a statistical index value acquisition step of calculating statistical index values ​​of the time-varying index values ​​of the plurality of observation locations at each of the first imaging time point and the second imaging time point; and determining that the photographing time point corresponding to the time point at which the statistical index value reaches a predetermined threshold value is the time point at which the passaging timing has been reached.

3. A method for determining the passage timing of cells that are spreading or growing in a medium in a culture vessel, comprising: an imaging step of photographing cells at a plurality of observation points that are partial regions selected from the entire area of ​​the culture vessel at a first imaging time point, a second imaging time point after the first imaging time point, and a third imaging time point after the second imaging time point, and acquiring cell images of the plurality of observation points at the first imaging time point, the second imaging time point, and the third imaging time point, respectively; a primary information acquisition step of acquiring primary information about any one of the number of cells, the density of the number of cells, the area occupied by cells, the area of ​​a single cell, and the relative distance between cells, captured in the cell images of the plurality of observation points at each of the first, second, and third photographing points; a statistical index value acquisition step of calculating statistical index values ​​of the primary information of the cells being photographed at the plurality of observation points at each of the first photographing time point, the second photographing time point, and the third photographing time point; a primary time-varying calculation step of calculating a primary time-varying index value of the statistical index value at the first imaging time point and the second imaging time point, and a primary time-varying index value of the statistical index value at the second imaging time point and the third imaging time point; a secondary time-varying calculation step of calculating a secondary time-varying index value from the primary time-varying index values ​​at the first imaging time point and the second imaging time point and the primary time-varying index values ​​at the second imaging time point and the third imaging time point; and determining that the time point at which the secondary time-course change index value reaches a predetermined threshold is the time point at which the passage timing has been reached.

4. A method for determining the passage timing of cells that are spreading or growing in a medium in a culture vessel, comprising: an imaging step of photographing cells at a plurality of observation points that are partial regions selected from the entire area of ​​the culture vessel at a first imaging time point, a second imaging time point after the first imaging time point, and a third imaging time point after the second imaging time point, and acquiring cell images of the plurality of observation points at the first imaging time point, the second imaging time point, and the third imaging time point, respectively; a primary information acquisition step of acquiring primary information about any one of the number of cells, the density of the number of cells, the area occupied by cells, the area of ​​a single cell, and the relative distance between cells, captured in the cell images of the plurality of observation points at each of the first, second, and third photographing points; a primary time-course change calculation step of calculating primary time-course change index values ​​of the primary information of the photographed cells at the plurality of observation points at the first photographing time point and the second photographing time point, and primary time-course change index values ​​of the primary information of the photographed cells at the second photographing time point and the third photographing time point; a secondary time-varying calculation step of calculating a secondary time-varying index value from the primary time-varying index values ​​of the plurality of observation locations at the first photographing time point and the second photographing time point and the primary time-varying index values ​​of the plurality of observation locations at the second photographing time point and the third photographing time point; a statistical index value acquisition step of calculating statistical index values ​​of the secondary time-varying index values ​​of the plurality of observation locations at each of the first photographing time point, the second photographing time point, and the third photographing time point; and determining that the photographing time point corresponding to the time point at which the statistical index value reaches a predetermined threshold value is the time point at which the passaging timing has been reached.

5. A method for determining the passage timing of cells that are spreading or growing in a medium in a culture vessel, comprising: an imaging step of photographing cells at a plurality of observation points that are partial regions selected from the entire area of ​​the culture vessel at a first imaging time point, a second imaging time point after the first imaging time point, and a third imaging time point after the second imaging time point, and acquiring cell images of the plurality of observation points at the first imaging time point, the second imaging time point, and the third imaging time point, respectively; a primary information acquisition step of acquiring primary information about any one of the number of cells, the density of the number of cells, the area occupied by cells, the area of ​​a single cell, and the relative distance between cells, captured in the cell images of the plurality of observation points at each of the first, second, and third photographing points; a primary time-course change calculation step of calculating primary time-course change index values ​​of the primary information of the photographed cells at the plurality of observation locations at the first photographing time point and the second photographing time point, and primary time-course change index values ​​of the primary information of the photographed cells at the plurality of observation locations at the second photographing time point and the third photographing time point; a statistical index value acquisition step of calculating statistical index values ​​of the primary time-varying index values ​​of the plurality of observation locations at each of the first photographing time point, the second photographing time point, and the third photographing time point; a secondary time-varying calculation step of calculating a secondary time-varying index value from the statistical index values ​​of the plurality of observation locations at the first imaging time point and the second imaging time point and the statistical index values ​​of the plurality of observation locations at the second imaging time point and the third imaging time point; and determining that the time point at which the secondary time-course change index value reaches a predetermined threshold is the time point at which the passage timing has been reached.

6. The method for determining the passage timing according to claim 1, A method for determining the timing of passage, wherein the time-course change index value is a differential change amount, a rate of change amount, a rate of change per unit time, a rate of change, a change time, a specific growth rate, or a predetermined doubling time of the statistical index value.

7. The method for determining the passage timing according to claim 2, The method for determining the passage timing, wherein the time-course change index value is a differential change amount, a rate of change amount, a rate of change per unit time, a rate of change, a change time, a specific growth rate, or a predetermined doubling time of the primary information.

8. The method for determining the passage timing according to claim 3, A method for determining the timing of passage, wherein the primary time-course change index value is a differential change amount, a rate of change amount, a rate of change per unit time, a rate of change, a change time, a specific growth rate, or a predetermined doubling time of the statistical index value.

9. The method for determining the passage timing according to claim 4, A method for determining passaging timing, wherein the primary time-course change index value is a differential change amount, a rate of change amount, a rate of change per unit time, a rate of change, a change time, a specific growth rate, or a predetermined doubling time of the primary information.

10. The method for determining the passage timing according to claim 5, A method for determining passaging timing, wherein the primary time-course change index value is a differential change amount, a rate of change amount, a rate of change per unit time, a rate of change, a change time, a specific growth rate, or a predetermined doubling time of the primary information.

11. The method for determining the passage timing according to any one of claims 1 to 5, The method for determining the passaging timing, wherein the statistical index value is an average value.

12. The method for determining the passage timing according to any one of claims 1 to 5, The method for determining the passaging timing, wherein the statistical index value is a sum.

13. The method for determining the passage timing according to any one of claims 1 to 5, The method for determining the timing of passage, wherein the statistical index value is a variance or deviation.

14. The method for determining the passage timing according to any one of claims 1 to 5, A method for determining the timing of passage, wherein the predetermined threshold value is determined as a statistical index value at which cell spreading or proliferation no longer appears in a preliminary test.

15. The method for determining the passage timing according to claim 13, A method for determining the timing of passage, wherein the predetermined threshold is determined in a preliminary test as an inflection point in the time change of the variance or the deviation.

16. The method for determining the passage timing according to claim 6, The rate of change is calculated as a value at any one photographing time point, A method for determining the timing of passage, wherein the predetermined threshold is determined as a time point at which a preliminary test is performed and the time-course index value no longer shows an increasing trend.

17. The method for determining the passage timing according to claim 6, The rate of change is calculated as a ratio to the value at the time of immediately preceding imaging, A method for determining the timing of passage, wherein the predetermined threshold is determined as an inflection point in the time change of the time-course index value in a preliminary test.

18. The method for determining the passage timing according to claim 8, The rate of change is calculated as a value at any one photographing time point, A method for determining the timing of passage, wherein the predetermined threshold is determined as a time point at which a preliminary test is performed and an increasing trend of the secondary time-course change index value is no longer observed in the test.

19. The method for determining the passage timing according to claim 8, The rate of change is calculated as a ratio to the value at the time of immediately preceding imaging, A method for determining the passage timing, wherein the predetermined threshold is determined as an inflection point in the time change of the secondary time-course index value in a preliminary test.

20. The method for determining the passage timing according to claim 7 or 9, The rate of change is calculated as a value at any one photographing time point, A method for determining the timing of passage, wherein the predetermined threshold is determined as a time point at which a preliminary test is performed and an increasing trend of the statistical index value is no longer observed in the test.

21. The method for determining the passage timing according to claim 7 or 9, The rate of change is calculated as a ratio to the value at the time of immediately preceding imaging, A method for determining the timing of passage, wherein the predetermined threshold is determined as an inflection point in the change over time of the statistical index value in a preliminary test.

22. The method for determining the passage timing according to claim 10, The rate of change is calculated as a value at any one photographing time point, A method for determining the timing of passage, in which the predetermined threshold is determined by conducting a preliminary test in advance and determining the secondary time-course change index value as the point at which an increasing trend in the secondary time-course change index value is no longer observed in the test.

23. The method for determining the passage timing according to claim 10, The rate of change is calculated as a ratio to the value at the time of immediately preceding imaging, A method for determining the passage timing, wherein the predetermined threshold is determined as an inflection point in the time change of the secondary time-course index value in a preliminary test.

24. The method for determining the passage timing according to any one of claims 3 to 5, A method for determining passaging timing, wherein the time interval between the first photographing time point and the second photographing time point and the time interval between the second photographing time point and the third photographing time point are the same time interval.

25. The method for determining the passage timing according to any one of claims 1 to 5, A method for determining the passage timing, wherein the predetermined threshold is determined by machine learning in a preliminary test.

26. A passage timing determination system for determining the passage timing of cells expanding or growing in a medium in a culture vessel, comprising: an imaging means for capturing images of cells at a plurality of observation points, which are selected from the entire area of ​​the culture medium in the culture vessel, and acquiring the images as cell images; a processing device that acquires primary information about any one of the number of cells captured in the cell image, the density of the number of cells, the area occupied by the cells, the area of ​​each cell, and the relative distance between the cells; the imaging means photographs cells at the plurality of observation points at a first imaging time point and a second imaging time point after the first imaging time point, and acquires cell images of the plurality of observation points at the first imaging time point and the second imaging time point, respectively; the processing device acquires primary information about any one of the number of cells, the density of the number of cells, the area occupied by a cell, the area of ​​a single cell, and the relative distance between cells, captured in the cell images of the plurality of observation points at each of the first and second imaging points, and calculates statistical index values ​​of the primary information about the cells captured at the plurality of observation points at each of the first and second imaging points, and calculates a time-course change index value from the statistical index values ​​at the first and second imaging points, The processing device is a passaging timing determination system that determines that the passaging timing has been reached at the photographing time point corresponding to the time point at which the time-course change index value has reached a predetermined threshold value.

27. A passage timing determination system for determining the passage timing of cells expanding or growing in a medium in a culture vessel, comprising: an imaging means for capturing images of cells at a plurality of observation points, which are selected from the entire area of ​​the culture medium in the culture vessel, and acquiring the images as cell images; a processing device that acquires primary information about any one of the number of cells captured in the cell image, the density of the number of cells, the area occupied by the cells, the area of ​​each cell, and the relative distance between the cells; the imaging means photographs cells at the plurality of observation points at a first imaging time point and a second imaging time point after the first imaging time point, and acquires cell images of the plurality of observation points at the first imaging time point and the second imaging time point, respectively; the processing device acquires primary information about any one of the number of cells, the density of the number of cells, the area occupied by a cell, the area of ​​a single cell, and the relative distance between cells, captured in the cell images of the plurality of observation locations at each of the first and second photographing times, calculates a time-course change index value from the primary information about the cells captured at the plurality of observation locations at each of the first and second photographing times, and calculates a statistical index value for the time-course change index value of the plurality of observation locations at each of the first and second photographing times, The processing device is a passaging timing determination system that determines that the passaging timing has been reached at the photographing time point corresponding to the time point at which the statistical index value has reached a predetermined threshold value.

28. A passage timing determination system for determining the passage timing of cells expanding or growing in a medium in a culture vessel, comprising: an imaging means for capturing images of cells at a plurality of observation points, which are selected from the entire area of ​​the culture medium in the culture vessel, and acquiring the images as cell images; a processing device that acquires primary information about any one of the number of cells captured in the cell image, the density of the number of cells, the area occupied by the cells, the area of ​​each cell, and the relative distance between the cells; the imaging means photographs cells at the plurality of observation points at a first imaging time point, a second imaging time point after the first imaging time point, and a third imaging time point after the second imaging time point, and acquires cell images of the plurality of observation points at the first imaging time point, the second imaging time point, and the third imaging time point, respectively; the processing device acquires primary information about any one of the number of cells, the density of the number of cells, the area occupied by a cell, the area of ​​an individual cell, and the relative distance between cells, captured in the cell images of the plurality of observation points at each of the first, second, and third imaging time points, calculates statistical index values ​​of the primary information of the cells captured at the plurality of observation points at each of the first, second, and third imaging time points, calculates primary time-course change index values ​​of the statistical index values ​​at the first and second imaging time points and primary time-course change index values ​​of the statistical index values ​​at the second and third imaging time points, and calculates secondary time-course change index values ​​from the primary time-course change index values ​​at the first and second imaging time points and the primary time-course change index values ​​at the second and third imaging time points, The processing device is a passaging timing determination system that determines that the passaging timing has been reached at the photographing time point corresponding to the time point at which the secondary time-course change index value has reached a predetermined threshold value.

29. A passage timing determination system for determining the passage timing of cells expanding or growing in a medium in a culture vessel, comprising: an imaging means for capturing images of cells at a plurality of observation points, which are selected from the entire area of ​​the culture medium in the culture vessel, and acquiring the images as cell images; a processing device that acquires primary information about any one of the number of cells captured in the cell image, the density of the number of cells, the area occupied by the cells, the area of ​​each cell, and the relative distance between the cells; the imaging means photographs cells at the plurality of observation points at a first photographing time point, a second photographing time point after the first photographing time point, and a third photographing time point after the second photographing time point, and obtains cell images of the plurality of observation points at each of the first photographing time point, the second photographing time point, and the third photographing time point, and obtains primary information about any of the number of cells, the density of the number of cells, the area occupied by cells, the area of ​​a single cell, and the relative distance between cells photographed in the cell images of the plurality of observation points at each of the first photographing time point, the second photographing time point, and the third photographing time point, and calculate a primary time-course change index value of the primary information of the photographed cells at the plurality of observation locations and a primary time-course change index value of the primary information of the photographed cells at the plurality of observation locations at the second photographing time point and the third photographing time point, calculate a secondary time-course change index value from the primary time-course change index value of the plurality of observation locations at the first photographing time point and the second photographing time point and the primary time-course change index value of the plurality of observation locations at the second photographing time point and the third photographing time point, and calculate statistical index values ​​of the secondary time-course change index value at each of the first photographing time point, the second photographing time point and the third photographing time point, The processing device is a passaging timing determination system that determines that the passaging timing has been reached at the photographing time point corresponding to the time point at which the statistical index value has reached a predetermined threshold value.

30. A passage timing determination system for determining the passage timing of cells expanding or growing in a medium in a culture vessel, comprising: an imaging means for capturing images of cells at a plurality of observation points, which are selected from the entire area of ​​the culture medium in the culture vessel, and acquiring the images as cell images; a processing device that acquires primary information about any one of the number of cells captured in the cell image, the density of the number of cells, the area occupied by the cells, the area of ​​each cell, and the relative distance between the cells; the imaging means photographs cells at the plurality of observation points at a first imaging time point, a second imaging time point after the first imaging time point, and a third imaging time point after the second imaging time point, and acquires cell images of the plurality of observation points at the first imaging time point, the second imaging time point, and the third imaging time point, respectively; the processing device acquires primary information about any one of the number of cells, the density of the number of cells, the area occupied by a cell, the area of ​​an individual cell, and the relative distance between cells, captured in the cell images of the plurality of observation locations at each of the first, second, and third imaging time points; calculates primary time-course change index values ​​of the primary information of the cells captured at the plurality of observation locations at each of the first, second, and third imaging time points and primary time-course change index values ​​of the primary information of the cells captured at the plurality of observation locations at each of the second and third imaging time points; calculates statistical index values ​​of the primary time-course change index values ​​of the plurality of observation locations at each of the first, second, and third imaging time points; and calculates secondary time-course change index values ​​from the statistical index values ​​at the first and second imaging time points and the statistical index values ​​at the second and third imaging time points; The processing device is a passaging timing determination system that determines that the passaging timing has been reached at the photographing time point corresponding to the time point at which the secondary time-course change index value has reached a predetermined threshold value.

31. The passage timing determination system according to claim 26, A system for determining passaging timing, wherein the time-course change index value is a differential change amount, a rate of change amount, a rate of change per unit time, a rate of change, a change time, a specific growth rate, or a predetermined doubling time of the statistical index value.

32. The passage timing determination system according to claim 27, A system for determining passaging timing, wherein the time-course change index value is a differential change amount, a rate of change amount, a rate of change per unit time, a rate of change, a change time, a specific growth rate, or a predetermined doubling time of the primary information.

33. The passage timing determination system according to claim 28, A system for determining passaging timing, wherein the primary time-course change index value is a differential change amount, a rate of change amount, a rate of change per unit time, a rate of change, a change time, a specific growth rate, or a predetermined doubling time of the statistical index value.

34. The passage timing determination system according to claim 29, A system for determining passaging timing, wherein the primary time-course change index value is a differential change amount, a rate of change amount, a rate of change per unit time, a rate of change, a change time, a specific growth rate, or a predetermined doubling time of the primary information.

35. The passage timing determination system according to claim 30, A system for determining passaging timing, wherein the primary time-course change index value is a differential change amount, a rate of change amount, a rate of change per unit time, a rate of change, a change time, a specific growth rate, or a predetermined doubling time of the primary information.

36. The passaging timing determination system according to any one of claims 26 to 30, A system for determining passaging timing, wherein the statistical index value is an average value.

37. The passaging timing determination system according to any one of claims 26 to 30, A system for determining passaging timing, wherein the statistical index value is a sum.

38. The passaging timing determination system according to any one of claims 26 to 30, The passaging timing determination system, wherein the statistical index value is a variance or deviation.

39. The passage timing determination system according to any one of claims 26 to 30, A system for determining the timing of passage, wherein the predetermined threshold value is determined by a preliminary test in advance as a statistical index value at which cell spreading or proliferation no longer appears.

40. The passage timing determination system according to claim 38, A system for determining passage timing, wherein the predetermined threshold is determined in advance by a preliminary test as an inflection point in the time change of the variance or the deviation.

41. The passage timing determination system according to claim 31, The rate of change is calculated as a value at any one photographing time point, A system for determining the timing of passage, wherein the predetermined threshold is determined by a preliminary test in advance, as the time point at which the index value of change over time no longer shows an increasing trend.

42. The passage timing determination system according to claim 31, The rate of change is calculated as a ratio to the value at the time of immediately preceding imaging, A system for determining passaging timing, wherein the predetermined threshold is determined as an inflection point in the time change of the time-course index value in a preliminary test that is carried out in advance.

43. The passage timing determination system according to claim 33, The rate of change is calculated as a value at any one photographing time point, A system for determining the timing of passage, wherein the predetermined threshold is determined by a preliminary test in advance, as the time point at which an increasing trend in the secondary time-course change index value is no longer observed.

44. The passage timing determination system according to claim 33, The rate of change is calculated as a ratio to the value at the time of immediately preceding imaging, A system for determining passaging timing, wherein the predetermined threshold is determined in advance by a preliminary test as an inflection point in the time change of the secondary time-course index value in the test.

45. The passage timing determination system according to claim 32 or 34, The rate of change is calculated as a value at any one photographing time point, A system for determining the timing of passage, wherein the predetermined threshold is determined by a preliminary test conducted in advance, as the time point at which an increasing trend in the statistical index value is no longer observed.

46. The passage timing determination system according to claim 32 or 34, The rate of change is calculated as a ratio to the value at the time of immediately preceding imaging, A system for determining passaging timing, wherein the predetermined threshold is determined in advance by a preliminary test as an inflection point in the change over time of the statistical index value in the test.

47. The passage timing determination system according to claim 35, The rate of change is calculated as a value at any one photographing time point, A system for determining the timing of passage, wherein the predetermined threshold is determined by a preliminary test in advance, as the time point at which an increasing trend in the secondary time-course change index value is no longer observed.

48. The passage timing determination system according to claim 35, The rate of change is calculated as a ratio to the value at the time of immediately preceding imaging, A system for determining passaging timing, wherein the predetermined threshold is determined in advance by a preliminary test as an inflection point in the time change of the secondary time-course index value in the test.

49. The passage timing determination system according to any one of claims 28 to 30, A system for determining passage timing, wherein the time interval between the first photographing time point and the second photographing time point and the time interval between the second photographing time point and the third photographing time point are the same time interval.

50. The passaging timing determination system according to any one of claims 26 to 30, A system for determining the timing of passage, wherein the predetermined threshold is determined by machine learning in advance through a preliminary test.

51. The passaging timing determination system according to any one of claims 26 to 30, The processing device is arranged away from the imaging means and is capable of communicating with the imaging means.

52. 1. A cell culture system for cells spreading or growing in a medium in a culture vessel, comprising: The passage timing determination system according to claim 26 ; A cell culture system having at least one of a culture medium unit that replaces the culture medium in the culture vessel, a culture unit that stores the culture vessel at a predetermined temperature, and a transfer unit that transfers the culture vessel between the passage timing determination system and the culture unit.

53. 1. A cell culture system for cells spreading or growing in a medium in a culture vessel, comprising: The passage timing determination system according to claim 27 ; A cell culture system having at least one of a culture medium unit that replaces the culture medium in the culture vessel, a culture unit that stores the culture vessel at a predetermined temperature, and a transfer unit that transfers the culture vessel between the passage timing determination system and the culture unit.

54. 1. A cell culture system for cells spreading or growing in a medium in a culture vessel, comprising: The passage timing determination system according to claim 28; A cell culture system having at least one of a culture medium unit that replaces the culture medium in the culture vessel, a culture unit that stores the culture vessel at a predetermined temperature, and a transfer unit that transfers the culture vessel between the passage timing determination system and the culture unit.

55. 1. A cell culture system for cells spreading or growing in a medium in a culture vessel, comprising: The passage timing determination system according to claim 29 ; A cell culture system having at least one of a culture medium unit that replaces the culture medium in the culture vessel, a culture unit that stores the culture vessel at a predetermined temperature, and a transfer unit that transfers the culture vessel between the passage timing determination system and the culture unit.

56. 1. A cell culture system for cells spreading or growing in a medium in a culture vessel, comprising: The passage timing determination system according to claim 30; A cell culture system having at least one of a culture medium unit that replaces the culture medium in the culture vessel, a culture unit that stores the culture vessel at a predetermined temperature, and a transfer unit that transfers the culture vessel between the passage timing determination system and the culture unit.

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