JP2019115346A - Method of determining cell maturity, observation device, program, control device, and method of producing cells - Google Patents

Abstract

To improve determination accuracy of cell line maturity.SOLUTION: The method of determining cell maturity of the present invention comprises: determining the relationship between the maturity of cells contained in a standard colony and the area of the standard colony, based on the relationship between the area of the standard colony containing cells of the same type as the subject colony and the area per cell contained in the standard colony; calculating the area of a subject colony; and determining the maturity of cells contained in the subject colony, based on the calculated area of the subject colony and the relationship between the maturity of the cells contained in the standard colony and the area of the standard colony.SELECTED DRAWING: Figure 1

Description

  The present invention relates to a method of determining the degree of cell maturation, an observation device, a program, a control device, and a method of producing a cell.

  In general, techniques for evaluating cell culture conditions have become fundamental technologies in a wide range of fields including advanced medical fields such as regenerative medicine and drug screening. For example, in the field of regenerative medicine, there exist processes for proliferating and differentiating cells in vitro. And in said process, in order to control the success or failure of differentiation of a cell, and canceration of a cell, and the presence or absence of infection, it is essential to evaluate the culture condition of a cell appropriately. As an example, a method for evaluating cancer cells using a transcription factor as a marker is disclosed (see Patent Document 1).

  Stem cells such as ES (Embryonic Stem, embryonic stem) cells or iPS (induced Pluripotent Stem, induced pluripotent stem) cells are, in theory, almost infinite while maintaining pluripotency to differentiate into almost all tissues. Because of their ability to be proliferated, attention has been focused on their application to drug development and regenerative medicine.

U.S. Pat. No. 7,060,445

  When applying such stem cells to drug discovery research and regenerative medicine, the condition is good (the size of the colony is a moderate size, and the density of cells present in the colony is a moderate density) In order to do so, it is required to accurately determine the degree of maturation of the cell line in culture. However, conventionally, the degree of maturation of the cell line has been determined by visual observation by a researcher, so there is a problem that the determination accuracy of the degree of maturity of the cell line can not be improved.

  Therefore, the present invention has been made in view of the above problems, and a method, an observation device, a program, a control device, and a method of determining the degree of cell maturation which can improve the determination accuracy of the degree of cell line maturation. An object is to provide a method for producing cells.

  [1] In order to solve the above problems, one aspect of the present invention is the area of a standard colony containing cells of the same type as a target colony, and the area per cell contained in the standard colony. Determining the relationship between the degree of maturation of the cells contained in the standard colony and the area of the standard colony based on the relationship between the standard colony and the step of calculating the area of the target colony; Determining the degree of maturation of the cells contained in the target colony based on the relationship between the area of the target colony and the degree of maturity of the cells contained in the standard colony and the area of the standard colony And determining the degree of maturation of the cell.

  [2] In addition, in order to solve the above problems, one aspect of the present invention relates to a temperature-controlled room for culturing cells of a first cell line contained in a target colony, and the same cell line as the first cell line Information obtained by measuring the first information indicating the relationship between the area of a colony and the area per cell contained in the colony, and the degree of maturation of the cells contained in the colony, A storage unit for storing second information indicating a relationship with an area, an observation unit for observing the target colony, an area calculation unit for calculating an area of the observed target colony, and an area of the target colony to be calculated And a maturity determining unit that determines the maturity of the cells included in the target colony based on the second information and the second information.

  [3] In addition, in order to solve the above problems, one aspect of the present invention provides a computer with an area calculation procedure for calculating the area of a target colony containing cells of a first cell line in culture in a temperature-controlled room, Information obtained based on first information indicating the relationship between the area of a colony of the same cell line as the first cell line and the area per cell contained in the colony, which is included in the colony The target colony is obtained based on an acquisition procedure for acquiring, from the storage unit, second information indicating a relationship between the degree of maturation of the target cell and the area of the colony, and the area of the target colony calculated and the second information. And a program for causing a process of determining the degree of maturity of the cells contained therein to be performed.

  [4] In addition, in order to solve the above problems, one aspect of the present invention is a control unit that calculates the area of a target colony including cells of a first cell line being cultured in a temperature-controlled room, Information obtained based on first information indicating the relationship between the area of a colony of a cell line and the same cell line and the area per cell contained in the colony, and maturation of the cells contained in the colony And a storage unit for storing second information indicating a relationship with the area of the colony, and the control unit acquires the second information from the storage unit, and calculates the area of the target colony calculated. It is a control apparatus which determines the maturity degree of the cell contained in the said object colony based on the acquired said 2nd information.

  [5] In addition, in order to solve the above problems, one aspect of the present invention calculates a culture procedure for culturing cells of a first cell line contained in a target colony in a temperature-controlled room, and calculating the area of the target colony. This information is obtained based on first information indicating a relationship between an area calculation procedure, an area of a colony of the same cell line as the first cell line, and an area per cell contained in the colony. An acquisition procedure for acquiring, from the storage unit, second information indicating the relationship between the degree of maturation of the cells contained in the colony and the area of the colony, and based on the area of the target colony to be calculated and the second information And determining the degree of maturation of the cells contained in the target colony.

  According to the present invention, it is possible to improve the determination accuracy of the degree of maturation of a cell line.

It is a block diagram showing an example of composition of an observation device by an embodiment of the present invention. It is a block diagram which shows an example of a structure of the control apparatus with which the observation apparatus of this embodiment is provided. It is a front view of the observation device of this embodiment. It is a top view of the observation device of this embodiment. It is a graph which shows an example of the time change of the colony area for every cell strain of this embodiment. It is a graph which shows an example of the relationship between the colony area for every cell strain of this embodiment, and the number of cells. It is a graph which shows an example of the relationship between the colony area of the cell of this embodiment, and the density of the cell contained in this colony. It is a schematic diagram which shows an example of the whole observation image which the imaging device of this embodiment imaged. It is a graph which shows an example of the relationship between the time change of the colony area of this embodiment, and a 1st calibration curve. It is a flowchart which shows an example of operation | movement of the calibration curve registration by the observation apparatus of this embodiment. It is a flowchart which shows an example of the cell number estimation operation | movement by the observation apparatus of this embodiment.

[Embodiment]
Hereinafter, embodiments of the present invention will be described with reference to the drawings. First, an overview of the configuration of an incubator (observation apparatus) according to an embodiment of the present invention will be described with reference to FIGS. 1 to 4. FIG. 1 is a block diagram showing an example of the configuration of the incubator 11 according to an embodiment of the present invention. FIG. 2 is a block diagram showing an example of the configuration of the control device 41 provided in the incubator 11 of the present embodiment. Moreover, FIG. 3, FIG. 4 is the front view and top view of the incubator 11 of this embodiment.

  The incubator 11 is a device for culturing cells and imaging the cultured cells with a microscope camera to observe the state of the cells. The incubator 11 has an upper casing 12 and a lower casing 13. In the assembled state of the incubator 11, the upper casing 12 is mounted on the lower casing 13. The internal space between the upper casing 12 and the lower casing 13 is vertically divided by the base plate 14.

  First, an outline of the configuration of the upper casing 12 will be described. Inside the upper casing 12, a temperature-controlled room 15 for culturing cells is formed. The temperature-controlled room 15 has a temperature control unit 15a and a humidity control unit 15b, and the temperature-controlled room 15 is maintained in an environment suitable for cell culture (for example, an atmosphere with a temperature of 37 ° C. and a humidity of 90%) In addition, illustration of the temperature control apparatus 15a in FIG. 3, FIG. 4 and the humidity control apparatus 15b is abbreviate | omitted).

  A large door 16, a middle door 17, and a small door 18 are disposed on the front of the temperature-controlled room 15. The large door 16 covers the front of the upper casing 12 and the lower casing 13. The middle door 17 covers the front surface of the upper casing 12 and isolates the environment between the temperature-controlled room 15 and the outside when the large door 16 is opened. The small door 18 is a door for carrying in and out the culture container 19 for culturing cells, and is attached to the middle door 17. By carrying the culture container 19 in and out of the small door 18, it is possible to suppress the environmental change of the temperature controlled room 15. The large door 16, the middle door 17 and the small door 18 are kept airtight by the packings P1, P2 and P3, respectively.

  Further, in the temperature-controlled room 15, a stocker 21, an observation unit 22, a container transfer device 23, and a transfer stand 24 are disposed. Here, the transfer stand 24 is disposed in front of the small door 18, and carries the culture container 19 out of the small door 18.

  The stocker 21 is disposed on the left side of the temperature-controlled room 15 as viewed from the front of the upper casing 12 (the lower side in FIG. 4). The stocker 21 has a plurality of shelves, and each shelf of the stocker 21 can store a plurality of culture vessels 19. In each culture container 19, cells to be cultured are accommodated together with a culture medium. The stocker 21 is not essential.

  The observation unit 22 is disposed on the right side of the temperature-controlled room 15 as viewed from the front of the upper casing 12. The observation unit 22 can perform time-lapse observation of cells in the culture vessel 19.

  Here, the observation unit 22 is fitted in the opening of the base plate 14 of the upper casing 12 and disposed. The observation unit 22 has a sample stage 31, a stand arm 32 for disposing an illumination light source protruding above the sample stage 31, and a main body 33 having an observation optical system and an imaging device 34 built therein. The sample stage 31 and the stand arm 32 are disposed in the constant temperature chamber 15, while the main body portion 33 is accommodated in the lower casing 13.

  The sample stage 31 is made of a translucent material, and the culture vessel 19 can be placed thereon. The sample stage 31 is configured to be movable in the horizontal direction, and can adjust the position of the culture vessel 19 placed on the upper surface. Further, the stand arm 32 incorporates an LED light source 39. Then, the imaging device 34 can obtain a microscope image of the cells by imaging the cells of the culture vessel 19 transmitted and illuminated from the upper side of the sample stage 31 by the stand arm 32 through the micro-optical system.

  The container transfer device 23 is disposed at the center of the temperature-controlled room 15 as viewed from the front of the upper casing 12. The container transfer device 23 transfers the culture container 19 between the stocker 21, the sample table 31 of the observation unit 22 and the transfer table 24. As described above, when the stocker 21 is not provided, the container transport device 23 is also unnecessary.

  As shown in FIG. 4, the container transfer device 23 includes a vertical robot 38 having an articulated arm, a rotation stage 35, a mini stage 36, and an arm unit 37. The rotation stage 35 is attached to the tip of the vertical robot 38 rotatably 180 ° in the horizontal direction via a rotation shaft 35 a. Therefore, the rotation stage 35 can cause the arm unit 37 to face the stocker 21, the sample stage 31, and the transport stage 24.

  In addition, the mini stage 36 is slidably attached to the rotation stage 35 in the horizontal direction. An arm 37 for holding the culture vessel 19 is attached to the mini stage 36.

  Next, an outline of the configuration of the lower casing 13 will be described. Inside the lower casing 13, a main body portion 33 of the observation unit 22 and a control device 41 of the incubator 11 are accommodated.

  The control device 41 is connected to the temperature adjustment device 15a, the humidity adjustment device 15b, the observation unit 22, and the container conveyance device 23, respectively. The control device 41 generally controls each part of the incubator 11 in accordance with a predetermined program.

  As an example, the control device 41 controls the temperature adjustment device 15a and the humidity adjustment device 15b to maintain the inside of the temperature-controlled room 15 at a predetermined environmental condition. Further, the control device 41 controls the observation unit 22 and the container conveyance device 23 based on a predetermined observation schedule to automatically execute the observation sequence of the culture container 19. Further, the control device 41 executes a culture state evaluation process of evaluating the culture state of the cells based on the image acquired in the observation sequence.

[Standard curve showing the time change of cell colony area (first standard curve)]
Here, with reference to FIG. 5, the time-dependent change of the colony area of a cell is demonstrated.
FIG. 5 is a graph showing an example of temporal change in colony area for each cell line. As cells proliferate over time, the area of the colony containing these cells (hereinafter, also simply referred to as colony area) increases over time. The time change of the colony area is different for each cell line. As an example, the time-dependent change of the colony area of cell line A is shown by the standard curve L1A of the same figure. In addition, the time change of the colony area of the cell line B is shown by the standard curve L1B in the same figure. The calibration curve L1A and the calibration curve L1B are collectively referred to as a first calibration curve L1. Once the cell line is identified, the change in colony area of the cell line with the passage of time can be estimated based on the predetermined relationship shown in FIG. 5, that is, the first calibration curve L1.

  Here, by observing the time change of the colony area, it can be determined whether or not the colony proliferates normally. That is, when the time variation of the colony area of a certain colony is larger than the time variation of a standard colony area (for example, when the colony area is abnormally increased), the cells contained in this colony are included. May be differentiated. That is, a colony in which the colony area is abnormally increased can be regarded as an abnormal colony by regarding the cells as being differentiated. In addition, when the time variation of the colony area of a certain colony is smaller than the time variation of the standard colony area (for example, when the colony area does not change with time), the cells contained in the colony are killed. There is a possibility. That is, a colony whose colony area does not change with time can be determined as an abnormal colony by regarding the cells as dead. The incubator 11 of the present embodiment determines whether the colony is normally grown or not by registering (memory) the first calibration curve L1 in advance for each cell line.

[Standard curve showing the relationship between the colony area of cells and the number of cells contained in this colony (second standard curve)]
Here, the relationship between the colony area of cells and the number of cells will be described with reference to FIG. The unit of the horizontal axis in FIG. 6 is μm 2 (micro square meter), and the unit of the vertical axis is individual.
FIG. 6 is a graph showing an example of the relationship between the colony area of one colony per cell line (hereinafter, colony area) and the number of cells in one colony (hereinafter, the number of cells in a colony). In addition, the colony used as object at the time of producing a standard curve selects the colony which suits a standard curve by the user in appearance. The selection point is preferably selected, for example, from individuals in which colonies do not adhere to each other and individual colonies exist independently. Also, the size of the roller is visually judged, and a wide range from small to large colonies is selected. These may be implemented by visual judgment by the user, or it is also possible to store selection conditions in advance and automatically discriminate by image analysis. There is a predetermined relationship between the colony area of cells and the number of cells contained in this colony. Also, this predetermined relationship is different for each cell line. As an example, the relationship between the colony area of cell line A and the number of cells contained in this colony is shown by the standard curve L2A in the same figure. Further, the relationship between the colony area of the cell line B and the number of cells contained in this colony is shown by the calibration curve L2B in the same figure. The calibration curve L2A and the calibration curve L2B are collectively referred to as a second calibration curve L2. If a cell line is identified, the number of cells contained in this colony can be estimated from the colony area of this cell line based on the second calibration curve L2 shown in the same figure. The incubator 11 of the present embodiment estimates (calculates) the number of cells contained in the colony from the image of the colony by registering (memory) the second calibration curve L2 in advance for each cell line. Specifically, for a cell line, the relationship between the number of cells and the area of the colony is measured based on the phase contrast image and the fluorescence image which is an image of the cell line subjected to fluorescence staining. In addition, the relationship between the measured cell number and the colony area is stored in advance as a second calibration curve L2.
An image used to count the number of cells is, for example, an image obtained by imaging cells in a colony under fluorescent staining. Then, the number of cells of the obtained image is measured for cells showing a predetermined luminance value. At this time, it is possible to perform a smoothing process on the data of the luminance value obtained from the image, and based on this data, specify as a cell to be counted cells having a predetermined luminance value. By controlling the processing level when this smoothing process is performed, it is possible to adjust the sensitivity for identifying cells.

[Standard curve showing the relationship between the colony area of cells and the density of cells contained in this colony (third standard curve)]
Here, with reference to FIG. 7, the relationship between the colony area of cells and the density of cells contained in the colony will be described.
FIG. 7 is a graph showing an example of the relationship between the colony area of cells and the density of cells contained in the colony. The density of cells contained in the colony increases with the degree of cell maturation over time. Also, the density of cells contained in the colony decreases less with the passage of time when reaching a certain degree of maturation. This means that although the number of cells in the colony increases as the area of the colony increases, the change in area per individual cell in the colony is small.
Therefore, the degree of cell maturation can be determined by observing the density of cells contained in the colony.

  Specifically, the density of cells contained in this colony is represented by the relationship between the colony area and the area per cell contained in this colony. As time goes on, cells grow and colony area increases. When the degree of maturity of cells contained in this colony is increased, the inside of the colony is in a state of being packed. That is, when the cells contained in the colony mature, the density of the cells inside the colony increases. Furthermore, when cells grow further with the inside of the colony being packed, the density of the cells reaches the upper limit, the increase in density is suppressed, and the colony area increases. Therefore, the degree of maturation of the cells contained in the colony can be determined by observing the temporal change in density of the cells inside the colony.

  The density of cells contained in this colony varies from cell line to cell line. As an example, the relationship between the colony area of cell line A and the area per cell contained in this colony is shown by the calibration curve L3A in the same figure. The relationship between the colony area of the cell line B and the area per cell contained in this colony is shown by the standard curve L3B in the same figure. The calibration curve L3A and the calibration curve L3B are collectively referred to as a third calibration curve L3. Once the cell line is identified, the degree of maturation of this cell can be determined based on the third calibration curve L3 shown in the same figure. Specifically, for the cell line A, when the area per cell contained in the colony reaches the threshold ThA of the calibration curve L3A shown in the figure, it is determined that the cell line A has matured . Further, with regard to cell line B, when the area per cell contained in the colony reaches the threshold ThB of the calibration curve L3B shown in the same figure, it is determined that this cell line B has matured.

  That is, the incubator 11 of the present embodiment determines the degree of cell maturity from the image of the colony by registering (memory) the third calibration curve L3 in advance for each cell line. Specifically, when creating a standard curve, for a cell line, based on a phase contrast image and a fluorescence image that is an image of the cell line stained fluorescently, the colony area and the density of the cells contained in this colony Measure the relationship between Further, the relationship between the measured colony area and the density of the cells contained in the colony is stored in advance as a third calibration curve L3.

  Returning to FIG. 1, the configuration of the control device 41 will be described. The control device 41 includes a control unit 42, a storage unit 43, and an input unit 44.

  The storage unit 43 is configured of a hard disk, a non-volatile storage medium such as a flash memory, and a volatile storage medium such as a DRAM or an SRAM. The storage unit 43 stores management data on each culture container 19 stored in the stocker 21, data of the entire observation image captured by the imaging device, and data of the microscope image. Furthermore, in the storage unit 43, a program executed by the control unit 42 is stored. In addition, in the storage unit 43, various calculation results by the control unit 42 are temporarily stored.

  In the above management data, (a) index data indicating each culture vessel 19, (b) storage position of the culture vessel 19 in the stocker 21, (c) type and shape of the culture vessel 19 (well plate, (D) type of cells cultured in culture vessel 19 (information identifying cell line) (e) observation schedule of culture vessel 19 (f) imaging conditions at time-lapse observation (objective (objective) The magnification of the lens, the observation point in the container, etc. are included. In addition, in the case of a culture container 19 capable of simultaneously culturing cells in a plurality of small containers such as a well plate, management data is generated for each small container.

  In this example, different types of cell lines are observed as cell lines to be observed. In this case, information for identifying a cell line is required, but if there is only one cell line to be observed and there is no need to identify a cell line, cell line identification information is not essential. Of course, one or more cell lines to be observed may be input.

In addition, in the storage unit 43, calibration curve information indicating the relationship between the area of a colony and the number of cells included in the colony is stored in association with one another.
In addition, when observing different types of cell lines, it is preferable to store cell line information for identifying cell lines of cells in the storage unit 43, and store the cell line information in association with the respective information. Further, it is preferable to store feature amount information indicating a feature amount of the area of the colony, and store the feature amount information in association with each information.

  The input unit 44 includes an input device such as a keyboard and a mouse. Various information such as cell line information is input to the input unit 44 by the operation of the user.

  Next, the configuration of the control unit 42 will be described with reference to FIG. The control unit 42 includes an image reading unit 4203, an area calculation unit 4211, a write control unit 4221, a cell number calculation unit 4222, and a density calculation unit 4223.

  Note that in the figure, it is assumed that the quality of the colony is determined based on the first calibration curve L1, and a configuration including the quality determination unit 4224 is shown. Further, in the figure, assuming that the degree of cell maturity is determined based on the third calibration curve L3, a configuration including the degree of maturity determination unit 4225 is shown. In the incubator 11 of the present embodiment, the quality determination unit 4224 and the maturity determination unit 4225 are not essential.

  The control unit 42 is, for example, a processor that executes various arithmetic processing of the control device 41. The control unit 42 operates as an image reading unit 4203, an area calculation unit 4211, a quality determination unit 4224, a maturity determination unit 4225, a writing control unit 4221, and a cell number calculation unit 4222 by execution of a program. Each may function.

  The write control unit 4221 controls writing of information output by each unit of the control device 41 to the storage unit 43.

  The image reading unit 4203 reads the image data of the entire observation image or the microscope image captured by the imaging device 34, and supplies the read image data to each unit of the control device 41. Further, the image reading unit 4203 reads the image data of the entire observation image or the microscope image stored in the storage unit 43, and supplies the read image data to each unit of the control device 41.

  When the area calculation unit 4211 stores the first calibration curve L1 in the storage unit 43 and determines the quality of the colony based on the image of the colony captured by the imaging device 34 and the first calibration curve L1. In two cases, calculate the area of the colony. Here, first, the case where the first calibration curve L1 is stored in the storage unit 43 will be described, and then, the case where the quality of the colony is determined based on the first calibration curve L1 will be described.

  The area calculating unit 4211 calculates a colony area, and the first calibration curve L1 in which the calculated colony area is associated with the number of cells included in the colony is used as the calibration curve information via the writing control unit 4221. It is stored in the storage unit 43. That is, the area calculation unit 4211 stores the first calibration curve L1 in the storage unit 43. When there are a plurality of cell lines, cell line information (cell line ID) is stored in the storage unit 43 via the input unit 44, and calibration curve information is stored for each cell line.

When there are multiple cell lines, it is necessary to input information (cell line ID) indicating the cell line type. In addition, a user who knows the type of the cell line to be observed may input the information indicating the type of the cell line, or the type of the cell to be observed, cells such as brightness, etc. are identified, and matching technology It is also possible to automatically create and input information indicating the type of cell line by using a technology that automatically determines the type of cell line by using the method.
In this embodiment, a case will be described in which information indicating a cell line (cell line ID) is input from the user via the input unit 44.

  In addition, the area calculation unit 4211 calculates the area of the colony based on the image in which the colony of the cell is imaged. A specific example of an image for which the area calculation unit 4211 calculates the area of the colony will be described with reference to FIG.

  FIG. 8 is a schematic view showing an example of the entire observation image captured by the imaging device 34 of the present embodiment. Among the whole observation images shown in the figure, the whole observation image PIC01 is an image (frame 1) of a colony detection result one day after the culture start. The whole observation image PIC02 to the whole observation image PIC05 (frames 1 to 5) are images of colony detection results after 5 days from the start of culture.

  The entire observation image PIC01 includes an image of the first colony CO1 and an image of the second colony CO2 one day after the start of the culture. Further, the entire observation image PIC02 includes an image of the first colony CO1 and an image of the second colony CO2 two days after the start of the culture. As shown in the figure, the first colony CO1 two days after the start of the culture is proliferated compared to the first colony CO1 one day after the start of the culture, and its area is increased. In addition, as shown in the figure, the second colony CO2 two days after the start of the culture is proliferated compared to the second colony CO2 one day after the start of the culture, and the area is increased. .

  The whole observation image PIC03 includes an image of the first colony CO1 three days after the start of the culture, an image of the second colony CO2, and an image of the third colony CO3. As shown in the figure, the first colony CO1 three days after the start of the culture is proliferated compared to the first colony CO1 two days after the start of the culture, and its area is increased. Also, as shown in the figure, the second colony CO2 after 3 days from the start of culture is proliferating compared to the second colony CO2 after 2 days from the start of culture, and the area is increased. .

  Here, with respect to the temporal change of the area of the colony, the whole observation image PIC01 to the whole observation image PIC03 will be described side by side in time series and compared. In this case, the area of the first colony CO1 monotonously increases in the entire observation image PIC01 to the entire observation image PIC03. On the other hand, in the area of the second colony CO2, the increase in the entire observation image PIC01 to the entire observation image PIC02 is significantly different from the increase in the entire observation image PIC02 to the entire observation image PIC03. That is, the area of the second colony CO2 has a large increase in the whole observation image PIC02 to the whole observation image PIC03 as compared to the increase in the whole observation image PIC01 to the whole observation image PIC02. That is, the area of the second colony CO 2 rapidly increases three days after the start of the culture. This indicates that the second colony CO2 might have differentiated and abnormally grown three days after the start of culture.

Here, with reference to FIG. 9, the relationship between the time change of a colony area and a 1st calibration curve is demonstrated.
FIG. 9 is a graph showing an example of the relationship between the time change of the colony area and the first calibration curve according to the present embodiment. In the figure, the horizontal axis indicates time, and the vertical axis indicates colony area. FIG. 9A shows the area of the colony included in the entire observation image PIC01 obtained by acquiring the image of the colony. That is, FIG. 9A shows the area of the first colony CO1 and the area of the second colony CO2 one day after the start of the culture. As shown in the figure, the area CO11 of the first colony CO1 and the area CO21 of the second colony CO2 are both plotted on the first calibration curve L1. That is, one day after the start of the culture, the area CO11 of the first colony CO1 and the area CO21 of the second colony CO2 both show normal values.

  Moreover, the area of the colony contained in whole observation image PIC02-whole observation image PIC05 which were acquired by acquiring the image of a colony in FIG.9 (B)-(E) is each shown. Among these, FIG. 9 (B) shows the area CO12 of the first colony CO1 and the area CO22 of the second colony CO2 two days after the start of the culture. As shown in the figure, the area CO12 of the first colony CO1 and the area CO22 of the second colony CO2 are both plotted on the first calibration curve L1. That is, two days after the start of the culture, the area CO12 of the first colony CO1 and the area CO22 of the second colony CO2 both show normal values.

  FIG. 9C shows the area CO13 of the first colony CO1 and the area CO23 of the second colony CO2 three days after the start of the culture. As shown in the figure, the area CO13 of these first colonies CO1 is plotted on a first calibration curve L1. On the other hand, the area CO23 of the second colony CO2 is not plotted on the first calibration curve L1. That is, three days after the start of the culture, the area CO12 of the first colony CO1 shows a normal value, and the area CO23 of the second colony CO2 shows an abnormal value. This indicates that the second colony CO2 might have differentiated and abnormally grown three days after the start of culture. That is, based on whether the colony area is present on the first calibration curve L1, it can be determined whether the area of the colony is a normal value or an abnormal value. In this example, the second colony CO 2 is larger than the normal colony area, and there is a possibility that the second colony CO 2 differentiated and abnormally grew three days after the start of the culture. A countermeasure is taken to stop (for example, remove from the culture medium) the subsequent culture of colony CO2.

  In the present embodiment, the quality determination unit 4224 determines the quality of the colony. That is, the quality determination unit 4224 determines the quality of the colony based on the change over time of the area of the colony calculated by the area calculation unit 4211. Specifically, the quality determination unit 4224 determines the quality of the colony based on the colony area calculated by the area calculation unit 4211 and the first calibration curve L1 stored in the storage unit 43.

  Returning to FIG. 8, after 3 days from the start of the culture, a third colony CO3 which has not been present is generated. This is because iPS cells, unlike normal cells, have uneven adhesion timing. Specifically, the first colony CO1 and the second colony CO2 adhere and grow one day after the start of the culture. On the other hand, the third colony CO3 does not adhere until 2 days after the start of the culture, and adheres and starts to proliferate after 3 days after the start of the culture. As described above, the imaging device 34 generates a whole observation image by performing imaging a plurality of times with the lapse of time from the start of culture.

  The whole observation image PIC04 (frame 4) contains an image of the first colony CO1 four days after the start of the culture, an image of the second colony CO2, and an image of the third colony CO3. . As shown in the figure, the first colony CO1 four days after the start of the culture is proliferated compared to the first colony CO1 three days after the start of the culture, and the area thereof is increased. Also, as shown in the same figure, the second colony CO2 after 4 days from the start of culture is proliferating compared to the second colony CO2 after 2 days from the start of culture, and the area is increased. .

  That is, the imaging device 34 captures a time-lapse image from the start of culture. As a result, the incubator 11 of the present embodiment can accurately observe the state of colonies even for cells such as iPS cells whose adhesion timing is uneven.

Returning to FIG. 2, the area calculation unit 4211 acquires the entire observation image captured by the imaging device 34 from the image reading unit 4203. Further, the area calculation unit 4211 generates a colony detection result image (whole observation image PIC01 to whole observation image PIC05) shown in FIG. 8 based on the acquired whole observation image.
In addition, the area calculation unit 4211 calculates the area of the colony of cells based on the generated image of the result of colony detection. Specifically, the area calculation unit 4211 masks the colony portion using an object detection algorithm with a known learning function, and sets the masked portion (a region surrounded by a curve in the colony detection result image in FIG. 8). The area where colonies are present is calculated, and the colony area is calculated from the masked area. The method of calculating the colony area is not limited to this.

  When the information (cell line ID) indicating the cell line is input from the user via the input unit 44, the cell number calculation unit 4222 uses the cell line ID as a search key and stores the calibration curve information stored in the storage unit 43. A search is performed, and based on the search result, a second calibration curve L2 (relationship of colony area to cell number) with which the cell line ID is matched is obtained. Further, the cell number calculation unit 4222 calculates the number of cells based on the area of the colony calculated by the area calculation unit 4211, using the acquired second calibration curve L2. That is, the cell number calculation unit 4222 calculates the number of cells included in the target colony whose area is calculated by the area calculation unit 4211 based on the image captured by the imaging device 34.

  The density calculation unit 4223 calculates the density of cells included in the colony based on the area of the colony calculated by the area calculation unit 4211 and the number of cells included in the colony calculated by the cell count calculation unit 4222. Specifically, the density calculation unit 4223 calculates the area per cell contained in the colony based on the colony area calculated by the area calculation unit 4211 and the number of cells calculated by the cell number calculation unit 4222. Do.

  The maturity determining unit 4225 determines the degree of maturity of the cells included in the colony based on the density of the cells calculated by the density calculating unit 4223. Specifically, for the cell line A, when the area per cell contained in the colony reaches the threshold value ThA of the calibration curve L3A shown in the same figure, the maturity determining unit 4225 determines the cell line A. Determine that A is mature. In the cell line B, when the area per cell contained in the colony reaches the threshold ThB of the calibration curve L3B shown in the figure, the maturity determining unit 4225 determines that the cell line B matures. It is determined that

[Operation of incubator (observation device)]
Next, an example of the operation of the incubator 11 will be described. The incubator 11 calculates the number of cells by estimating the number of cells contained in the colony from the area of the colony of the cells based on the registered standard curve. Here, the standard curve is information indicating the relationship between the area of a colony of cells and the number of cells contained in the colony. Here, an operation of registering a calibration curve will be described first, and then, an operation of calculating the number of cells based on the registered calibration curve will be described.

[Operation of calibration curve registration]
First, the operation of the calibration curve registration will be described with reference to FIG.
FIG. 10 is a flowchart showing an example of an operation of calibration curve registration by the incubator 11 (observation apparatus) of the present embodiment. The incubator 11 stores standard curves (first standard curve L1, second standard curve L2, and third standard curve L3) for each cell line.

  As an example, the incubator 11 detects an image of a colony in the image data based on the above-described image data, and calculates the area of the image of the colony. The incubator 11 registers, for each cell line, the first calibration curve L1 for a cell line by correlating the calculated colony area with the elapsed time from the start of culture.

  Further, the incubator 11 relates the second calibration curve L2 of a cell line to a calibration line by correlating the calculated colony area with the number of cells in the colony counted by the fluorescence staining observation image for each cell line. Register as information.

  Moreover, the incubator 11 registers the third calibration curve L3 for a cell line as calibration curve information by correlating the calculated colony area with the area per cell in this colony for each cell line. Do.

  Here, before the start of the calibration curve registration operation, the control unit 42 receives an instruction of the calibration curve registration operation input by the user via the input unit 44 in advance. The instruction of this standard curve registration operation includes information (cell line ID) indicating a target cell line for which a standard curve is to be registered (if there is one cell line to be observed, the standard curve is registered) It is not essential to input information (cell line ID) indicating the cell line of interest, and the storage unit 43 is set to start observation after one day and five days after the start of culture. The observation start time is stored in advance as an observation schedule for management data, and the method in which the user counts the number of cells in the colony is fluorescent staining of the cells and the counting method in which cells are counted by fluorescence observation, etc. In the case of a method in which cells in the container 19 are destroyed and observed, the number of culture containers 19 corresponding to the number of observation is prepared, for example, one day, two days, and three days after the start of culture. Observe In the case, at least three culture vessels 19 are stored in the stocker 21 of the temperature-controlled room 15. The same cell lines are respectively cultured in these culture vessels 19. These culture vessels 19 are one day Observe the cell lines from 1 day to 5 days after the start of culture by observing one cell at a time. Preparation of calibration curve performed by this incubator 11, ie calculation of colony area and specific registration of calibration curve In the present embodiment, although the following is performed by the control unit 42 included in the incubator 11, the control unit externally provided to the incubator 11 may execute the following operation.

  Step S101: The control unit 42 compares the observation schedule of the management data of the storage unit 43 with the current date and time, and determines whether the observation start time of the culture container 19 has arrived. When the observation start time has come (YES side), the control unit 42 shifts the process to step S102. On the other hand, if it is not the observation time (NO side), the control unit 42 stands by until the time of the next observation schedule.

  Step S102: The control unit 42 instructs the container transfer device 23 to transfer the culture container 19 corresponding to the observation schedule. Then, the container transfer device 23 carries out the instructed culture container 19 from the stocker 21 and places the container on the sample stage 31 of the observation unit 22. When the culture vessel 19 is placed on the sample stand 31, a bird's-eye view camera (not shown) built in the stand arm 32 captures an entire observation image of the culture vessel 19. Thereby, the image of the culture container 19 containing the image of a colony is imaged. As described above, the stacker 21 is not essential, and when the stacker 21 is not provided, the steps relating to the transport of the container are unnecessary.

Step S103: The image reading unit 4203 of the control unit 42 stores the entire observation image captured in step S102. The area calculation unit 4211 of the control unit 42 detects an image of a colony from the stored entire observation image, and calculates the area of each colony cell in the culture container 19 or the total area of each colony as a colony area.
When creating a standard curve, acquire the image of the culture vessel containing the colony image, count the number of cells in each colony from the fluorescence observation image (or add the number of cells in each colony), and calculate the colony area from the phase contrast image You may Alternatively, the total cell number of each colony may be counted by a hemocytometer, and the colony area may be calculated from the phase contrast image.

  Step S104: The control unit 42 instructs the container transfer device 23 to transfer the culture container 19 to the small door 18 after the end of the observation schedule. Then, the container conveyance device 23 conveys the instructed culture container 19 from the sample stage 31 of the observation unit 22 to the position of the small door 18.

  Step S105: The user opens the small door 18 and takes out the culture vessel 19. In addition, the user counts the number of cells of the culture container 19 taken out by a known method. For example, the user counts the number of cells by fluorescent staining. Also, the user inputs the counted number of cells to the input unit 44. Also, here, the user calculates the density of the cells in the colony, and inputs the calculated density to the input unit 44.

  Step S106: The write control unit 4221 of the control unit 42, the number of cells in the colony and the density of the cells in the colony input to the input unit 44, the colony area calculated in step S103, and information indicating the cell line (Cell line ID) is associated and stored in the storage unit 43. Thereby, the colony area, the cell number, and the cell line ID are associated in the storage unit 43 and stored as calibration curve information (in the case of observation of one cell line, the association with the cell line ID is not essential) ). Thereafter, the control unit 42 ends the observation sequence and returns the process to step S101.

  The calibration curve information is stored in the storage unit 43 by repeating steps S101 to S106 in this manner. Further, by repeating steps S101 to S106 for a plurality of cell lines, calibration curve information for each of the plurality of cell lines is stored in the storage unit 43. Specifically, by repeating steps S101 to S106 for each of cell line A and cell line B, calibration curve information of cell line A and calibration curve information of cell line B are stored in storage unit 43, respectively. .

[Operation of cell number estimation and determination of cell quality]
Next, an example of the cell number estimation operation of the incubator 11 will be described with reference to FIG.
FIG. 11 is a flowchart showing an example of the cell number estimating operation by the incubator 11 (observation apparatus) of the present embodiment. In this example, it is assumed that data (referred to as comparison data) regarding the number of cells with respect to the change in colony area of a specific cell line to be observed is acquired and stored in advance. From this data, the judgment of the quality of the cells to be observed is in an unknown state.

  The incubator 11 observes the culture vessel 19 carried into the temperature-controlled room 15 in a time-lapse manner in accordance with the registered observation schedule. In the culture vessel 19, a plurality of specific cell lines are cultured. For example, in the culture container 19A, the cell line A is cultured in the culture container 19A. In the culture container 19B, the cell line B is cultured in the culture container 19B. The incubator 11 sequentially conveys the culture container 19A and the culture container 19B to the vertical robot 38 observation unit 22 in accordance with the observation schedule, and the entire image of the culture container 19 (whole observation image) and a part of the culture container 19 And an enlarged microscope image. Further, the calibration curve information of the cell line A and the calibration curve information of the cell line B are stored in advance in the storage unit 43 according to the registration procedure of the calibration curve information described above. The operation of cell number estimation in time-lapse observation of the incubator 11 will be described below.

  Step S201: The control unit 42 compares the observation schedule of the management data of the storage unit 43 with the current date and time, and determines whether the observation start time of the culture container 19 has arrived. If the observation start time has come (YES side), the control unit 42 shifts the process to step S202. On the other hand, when it is not the observation time of the culture container 19 (NO side), the control unit 42 stands by until the time of the next observation schedule.

Step S202: The control unit 42 instructs the container transfer device 23 to transfer the culture container 19 corresponding to the observation schedule. Then, the container transfer device 23 carries out the instructed culture container 19 from the stocker 21 and places the container on the sample stage 31 of the observation unit 22. When the culture vessel 19 is placed on the sample stand 31, a bird's-eye view camera (not shown) built in the stand arm 32 captures an entire observation image of the culture vessel 19.
In the present embodiment, the observation apparatus shown in FIGS. 1, 3 and 4 is an apparatus provided with a temperature-controlled room for culturing cells to be observed. Therefore, the cells to be imaged in step S202 are those cultured in a thermostatic chamber of the observation apparatus. Thus, this step can also start from the step of culturing the cells. Further, as in the present embodiment, the observation apparatus may not be equipped with a constant temperature room, and the constant temperature room for culturing cells may be an apparatus separate from the observation apparatus.

Step S203: The cell number calculation unit 4222 which calculates the number of cells in the colony acquires information (cell line ID) indicating a cell line from the control data stored in the storage unit 43.
Step S204: The cell number calculation unit 4222 searches the calibration curve information stored in the storage unit 43 using the obtained cell line ID as a search key, and acquires calibration curve information in which the cell strain IDs coincide.

  Step S205: The image reading unit 4203 acquires the image captured in step 202. This image contains an image of the colony.

  Step S206: The area calculation unit 4211 calculates the area of the colony included in the image based on the image acquired in step S205.

Step S207: The cell number calculation unit 4222 estimates the cell number based on the area of the colony calculated in step S206 and the second calibration curve L2 of the calibration curve information acquired in step S204 (calculation (calculation) ).
The area calculation unit 4211 calculates the area of the recognized colony in the acquired image.
Note that this step may be performed when the culture container is transported to the observation unit (S202) and when the observation becomes possible. In this case, after the colony area is calculated, information indicating the cell line is obtained.

  Step S208: The density calculation unit 4223 calculates the density of cells based on the area of the colony calculated in step S206 and the number of cells calculated in step S207.

Step S209: When the quality determination unit 4224 is provided, the quality determination unit 4224 uses the colony area calculated from the acquired image and the first calibration curve L1 of the calibration curve information acquired in step S204. Based on the quality of the colony is determined. When the quality determination unit 4224 is not provided, the user determines the quality of the colony based on the calculated colony area and the first calibration curve L1 of the calibration curve information acquired in step S204. judge.
When the maturity determining unit 4225 is provided, the maturity determining unit 4225 uses the acquired image to calculate the area of the colony calculated in step S206, the density of the cells calculated in step S208, and the step S204. The degree of cell maturity is determined based on the third calibration curve L3 among the calibration curve information acquired in 4. When the maturation level determination unit 4225 is not provided, the area of the colony calculated in step S206, the density of the cells calculated in step S208, and the third of the calibration curve information acquired in step S204. The user determines the degree of cell maturation based on the standard curve L3 of

Step S210: The control unit 42 instructs the container transfer device 23 to transfer the culture container 19 after the end of the observation schedule. Then, the container conveyance device 23 conveys the instructed culture container 19 from the sample stage 31 of the observation unit 22 to a predetermined storage position of the stocker 21. Thereafter, the control unit 42 ends the observation sequence and returns the process to S201.
In addition, after it is determined whether or not the cells are in the good state as a result of the determination in step S209, the cells determined to be in the good state are taken out from the observation device, for example, drug discovery research and regenerative medicine Can be provided as cells for use in

  By repeating steps S201 to S210 for each cell line in this manner, the estimation result of the number of cells in each observation schedule is stored in the storage unit 43. Furthermore, it becomes possible to determine the quality of the cell based on the estimation result of the cell number. Furthermore, only selected good cells can be provided to research institutes and the like.

  As described above, the incubator 11 (observation apparatus) of the present embodiment determines the number of cells contained in the colony based on the calibration curve information and the area of the colony based on the non-invasively obtained image. A cell number calculation unit 4222 to calculate is provided. Thus, the incubator 11 can calculate the number of cells by a non-invasive method, and can improve the accuracy of the calculated number of cells. In addition, cells determined from the use of images obtained by noninvasive observation (for example, phase contrast observation) for determination of maturity and good or bad are continuously performed without hindrance to drug discovery research and regenerative medicine which are subsequent steps. It becomes possible to use.

  In addition, the control device 41 handles a plurality of microscope images obtained by imaging a plurality of points of the same culture container 19 (for example, 5 points observation or the whole culture container 19) in the same observation time zone as an image for one time lapse observation. You may

  In addition, although the area calculation part 421 demonstrated the example which detects the image of a colony based on a whole observation image, it is not restricted to this. The area calculation unit 4211 may detect an image of a colony by performing image processing on the phase contrast microscope image.

  Further, a program for executing each process of the incubator 11 (observation apparatus) in the embodiment of the present invention is recorded in a computer readable recording medium, and the computer system reads the program recorded in the recording medium. The above-described various processes may be performed by execution.

  Note that the “computer system” referred to here may include an OS and hardware such as peripheral devices. The "computer system" also includes a homepage providing environment (or display environment) if the WWW system is used. In addition, “computer readable recording medium” refers to flexible disks, magneto-optical disks, ROMs, writable nonvolatile memories such as flash memories, portable media such as CD-ROMs, hard disks incorporated in computer systems, etc. Storage devices.

  Furthermore, the “computer-readable recording medium” is a volatile memory (for example, DRAM (Dynamic Memory) inside a computer system that becomes a server or a client when a program is transmitted via a network such as the Internet or a communication line such as a telephone line). As Random Access Memory), it is assumed that the program which holds the program for a fixed time is included. The program may be transmitted from a computer system in which the program is stored in a storage device or the like to another computer system via a transmission medium or by transmission waves in the transmission medium. Here, the “transmission medium” for transmitting the program is a medium having a function of transmitting information, such as a network (communication network) such as the Internet or a communication line (communication line) such as a telephone line. Further, the program may be for realizing a part of the functions described above. Furthermore, it may be a so-called difference file (difference program) that can realize the above-described functions in combination with a program already recorded in the computer system.

  The embodiment of the present invention has been described in detail with reference to the drawings, but the specific configuration is not limited to this embodiment, and includes design and the like within the scope of the present invention.

  11: observation device, 41: control device, 4211: area calculation unit, 4222: cell number calculation unit, 4223: density calculation unit, 4224: quality determination unit, 4225: maturity determination unit, 43: storage unit

Claims (15)

Maturation of the cells contained in the standard colony based on the relationship between the area of a standard colony containing cells of the same type as the target colony and the area per cell contained in the standard colony Determining the relationship between the degree of contact and the area of the standard colony;
Calculating the area of the target colony;
Based on the calculated area of the target colony and the relationship between the degree of maturity of the cells contained in the standard colony and the area of the standard colony, the degree of maturation of the cells contained in the target colony is determined And determining the degree of cell maturation.   The method according to claim 1, wherein the step of calculating the area of the target colony includes calculating the area of the target colony based on a phase difference image obtained by photographing the target colony.   The step of determining the degree of maturation may be performed by determining the degree of maturity of the cells included in the target colony using time change of the area per cell included in the target colony based on the area of the target colony. The method according to claim 1 or claim 2, comprising.   The method according to claim 3, comprising the steps of: estimating the number of cells contained in the target colony based on the calculated area of the target colony and calculating the area per cell contained in the target colony .   The step of determining the degree of maturation is included in the target colony by comparing the time variation of the area per cell included in the target colony with the degree of maturity of cells included in the standard colony. 5. A method according to claim 3 or claim 4 comprising determining the degree of cell maturation.   The step of determining the degree of maturation includes determining the degree of maturity of cells included in the target colony based on an area per cell included in the target colony and a threshold value. Item 6. The method according to any one of Items 5. A constant temperature chamber for culturing cells of a first cell line contained in a target colony;
Information obtained by measuring first information indicating the relationship between the area of a colony of the same cell line as the first cell line and the area per cell contained in the colony, which is included in the colony A storage unit for storing second information indicating a relationship between the degree of maturation of the cell to be cultured and the area of the colony;
An observation unit for observing the target colony;
An area calculation unit that calculates an area of the observed target colony;
A maturity determining unit that determines the maturity of cells included in the target colony based on the area of the target colony calculated and the second information. The storage unit stores the second information for each cell line information that recognizes a cell line of cells,
The maturation degree determination unit determines the maturation degree of the cells included in the target colony based on the second information corresponding to the cell line information input and the area of the target colony. Observation device. The observation according to claim 7 or 8, wherein the maturity determining unit determines that the cells contained in the target colony have matured in a state in which the change in area per cell contained in the target colony is reduced. apparatus. In the temperature-controlled room, cells of a second cell line different from the first cell line contained in a second target colony are cultured,
The storage unit is information obtained based on third information indicating a relationship between the area of a colony of the same cell line as the second cell line and the area per cell contained in the colony. The observation device according to any one of claims 7 to 9, storing fourth information indicating a relationship between the degree of maturation of cells included in the colony and the area of the colony.   The first information includes calibration curve information indicating a relationship between an area of a colony of the same cell line as the first cell line and an area per cell contained in the colony. The observation device according to any one of 10.   The observation device according to any one of claims 7 to 11, further comprising: a quality determination unit that determines the quality of the target colony based on a temporal change in the area of the target colony. On the computer
An area calculation procedure for calculating an area of a target colony including cells of a first cell line being cultured in a temperature-controlled room,
Information obtained based on first information indicating the relationship between the area of a colony of the same cell line as the first cell line and the area per cell contained in the colony, which is included in the colony An acquisition procedure for acquiring, from the storage unit, second information indicating a relationship between the degree of maturation of the cell to be cultured and the area of the colony;
A maturity determining step of determining the maturity of the cells contained in the target colony based on the area of the target colony calculated and the second information;
A program to run a program. A control unit that calculates an area of a target colony including cells of a first cell line being cultured in a temperature-controlled room,
Information obtained based on first information indicating the relationship between the area of a colony of the same cell line as the first cell line and the area per cell contained in the colony, which is included in the colony A storage unit for storing second information indicating a relationship between the degree of maturation of the cell to be cultured and the area of the colony;
The control unit acquires the second information from the storage unit, and determines the degree of maturation of cells included in the target colony based on the calculated area of the target colony and the acquired second information. apparatus. A culture procedure for culturing cells of a first cell line contained in a target colony in a temperature-controlled room,
An area calculation procedure for calculating an area of the target colony;
Information obtained based on first information indicating the relationship between the area of a colony of the same cell line as the first cell line and the area per cell contained in the colony, which is included in the colony An acquisition procedure for acquiring, from the storage unit, second information indicating a relationship between the degree of maturation of the cell to be cultured and the area of the colony;
A maturity determining step of determining the maturity of the cells contained in the target colony based on the area of the target colony calculated and the second information;
A method of producing a cell having

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