JP2012095627A - Cell culture apparatus and program - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide means for detecting dynamic changes of a cultured cell in a short period of time while performing a wide-ranging and exhaustive observation.SOLUTION: An imaging part of a cell culture apparatus takes an observation image of the cultured cell in a constant temperature chamber. An image analysis part obtains form information of the cultured cell from a first image taken by the imaging part at a first time interval, and determines whether the form of the cultured cell meets given observation conditions based on the form information. An observation control part controls the imaging part to take a picture of the cultured cell at a second time interval that is shorter than the first time interval when the form of the cultured cell meets the given observation conditions.

Description

  The present invention relates to a cell culture device and a program.

  Some conventional cell culture devices have a function of performing time-lapse observation for each sample and capturing an observation image of cells in the sample at each time. In addition, a configuration for comparing and displaying temporal changes in a plurality of samples in observing cultured cells has also been proposed (see, for example, Patent Document 1). According to such a cell culture device, the user can grasp how the cultured cells change with time.

  In addition, for example, when observing differentiation of stem cells, it is difficult to specify in advance the generation time and generation location of stem cell differentiation. Therefore, when detecting and evaluating the differentiation of stem cells, generally, a wide and comprehensive observation of a plurality of samples is continuously performed over a long-term culture process.

JP 2009-162708 A

  Some differentiated cells exhibit characteristic dynamic changes in a very short cycle, such as cardiomyocytes that undergo spontaneous contraction. When evaluating differentiation induction into cells as described above, it is necessary to determine cell movement by observing images with small temporal resolution in time series.

  However, when performing a wide and comprehensive time-lapse observation for a plurality of samples, it is impossible to detect a dynamic change of cultured cells in a short cycle from a still image obtained by the time-lapse observation. Further, in the method in which the user observes the cultured cells and evaluates the differentiated state of the cells, a great burden is imposed on the user over a long-term culture process.

  In view of the above circumstances, a means for detecting a dynamic change of a cultured cell in a short cycle is provided while performing a comprehensive and comprehensive observation.

  The cell culture device according to one aspect includes a temperature-controlled room whose room is maintained at a predetermined environmental condition, an imaging unit, an image analysis unit, and an observation control unit. An imaging part images the observation image of the cultured cell in a constant temperature room. The image analysis unit acquires the morphological information of the cultured cells from the first image captured by the imaging unit at the first time interval, and determines whether the cultivated cell morphology satisfies a predetermined observation condition based on the morphological information. judge. The observation control unit causes the imaging unit to image the cultured cells at a second time interval shorter than the first time interval when the form of the cultured cells satisfies a predetermined observation condition.

  The image analysis unit according to one aspect may detect the contraction movement of the cultured cell by comparing a plurality of second images captured by the imaging unit at the second time interval. Further, the image analysis unit may change a predetermined observation condition depending on whether or not the contraction motion is detected. Further, the image analysis unit may specify the position of the cultured cell from the first image and correct a predetermined observation condition from the second image when the contraction motion is detected.

  Note that any program that causes the cell culture device to execute image analysis processing and observation control processing, and a storage medium that stores the above-described program are all effective as specific embodiments of the present invention.

  In the cell culture device according to one aspect, it is possible to detect a dynamic change of a cultured cell in a short cycle while performing a wide and comprehensive observation.

1 is a schematic diagram showing a configuration example of a cell culture device according to an embodiment. The flowchart which shows the operation example of the cell culture apparatus in one Embodiment Flowchart continued from FIG. Schematic diagram regarding detection processing of contraction movement of cultured cells in one embodiment

<Configuration example of cell culture device>
FIG. 1 is a schematic diagram illustrating a configuration example of a cell culture device according to an embodiment. The cell culture device is, for example, a device that cultures cells to be observed and time-lapse observations of the cells to be observed. In one embodiment, an observation example in the case of inducing differentiation of cardiomyocytes from induced pluripotent stem cells (iPS cells) will be described.

  The cell culture apparatus includes a temperature-controlled room 11, an environment control unit 12, a stage 13, a container transport unit 14, an imaging unit 15, a control unit 16, a storage unit 17, an input unit 18, a monitor 19, and a communication unit 20. Here, the environment control unit 12, the stage 13, the container transport unit 14, the imaging unit 15, the storage unit 17, the input unit 18, the monitor 19, and the communication unit 20 are each connected to the control unit 16.

  The temperature-controlled room 11 is a room for culturing cells. For example, in the temperature-controlled room 11, a plurality of culture containers (petri dishes, well plates, etc.) that hold the cells to be observed together with the culture medium are held. A container transport unit 14 and a stage 13 are disposed in the temperature-controlled room 11.

The environment control unit 12 controls environmental condition parameters (temperature, humidity, CO ₂ concentration, etc.) in the temperature-controlled room 11. By the operation of the environment control unit 12, the inside of the temperature-controlled room 11 is maintained at predetermined environmental conditions (for example, temperature 37 ° C., humidity 90%, CO ₂ concentration 5%) suitable for cell culture.

  A culture vessel is placed on the stage 13 during observation. The stage 13 has a drive mechanism that is driven in accordance with an instruction from an observation control unit 16a described later, and finely adjusts the position of the culture vessel during observation.

  The container transport unit 14 transports the culture container in the temperature-controlled room 11 in accordance with an instruction from an observation control unit 16a described later. For example, the container transport unit 14 transports the culture container at the storage position in the temperature-controlled room 11 onto the stage 13 during observation. And the container conveyance part 14 returns the culture container on the stage 13 to said accommodation position after completion | finish of observation. The container transport unit 14 carries the culture container into and out of the temperature-controlled room 11.

  The imaging unit 15 is an electronic camera module that captures an observation image of a cell to be observed under the environment of the temperature-controlled room 11. The optical system of the imaging unit 15 is an optical system of a transmission microscope (for example, a phase contrast microscope). And the imaging part 15 images the image of the cell in the culture container mounted on the stage 13 according to the instruction | indication of the below-mentioned observation control part 16a. The image output of the imaging unit 15 is connected to the storage unit 17.

  The imaging unit 15 in one embodiment has a still image and moving image shooting function. Note that the imaging unit 15 may be configured to capture still images and moving images with the same imaging device. Alternatively, the imaging unit 15 may be configured to branch the optical path with a half mirror or the like and to capture the still image and the moving image with different imaging elements.

  The control unit 16 is a processor that comprehensively controls the cell culture apparatus. The control unit 16 functions as an observation control unit 16a and an image analysis unit 16b by executing the program.

  The observation control unit 16a controls the stage 13, the container transport unit 14, and the imaging unit 15 in accordance with a preset observation schedule, and executes a time lapse observation process of the cells. In addition, the observation control unit 16a has a time interval shorter than the time interval in the time lapse observation when the shape of the cultured cell satisfies the moving image acquisition condition (observation condition as to whether or not to perform the observation with the moving image) during the time lapse observation. Then, the imaging unit 15 is caused to capture a moving image of the cultured cells. In addition, the image analysis unit 16 b analyzes the still image and the moving image acquired by the imaging unit 15.

  The storage unit 17 is configured by a storage medium such as a hard disk or a nonvolatile semiconductor memory, for example. The storage unit 17 stores a program executed by the control unit 16, information on the observation schedule of the culture vessel, information on the observation point of the culture vessel, teacher information for specifying a target cultured cell, and video shooting. More detailed observation condition information and the like are stored. The storage unit 17 can also store information on various images input from the imaging unit 15.

  The input unit 18 includes, for example, an input device such as a button or a touch panel. The input unit 18 supplies a signal corresponding to a user operation to the control unit 16. The monitor 19 executes various displays under the control of the control unit 16.

  The communication unit 20 performs data transmission / reception with a terminal device 22 outside the cell culture device via a wireless or wired communication line 21. In addition, said terminal device 22 is comprised, for example with a personal computer.

<Operation example of cell culture device>
Next, an example of the operation of the cell culture device according to one embodiment will be described with reference to the flowcharts of FIGS. 2 and 3 is executed by the control unit 16 in response to an instruction to start the culture process by the user.

  Step # 101: The container transport unit 14 carries the culture container containing the cells to be observed (iPS cells) and the medium into the temperature-controlled room 11 under the control of the observation control unit 16a. In the case where cells are cultured in parallel in a plurality of culture containers in a single culture process, the plurality of culture containers are carried into the temperature-controlled room 11 by the process of # 101. At this stage, the environment control unit 12 starts control to keep the environmental conditions in the temperature-controlled room 11 constant.

  Step # 102: The control unit 16 sets the observation point of the culture vessel at the time-lapse observation according to the user input from the input unit 18 or the terminal device 22. The control unit 16 in # 102 may set an arbitrary position (for example, 5 points) of the culture vessel designated by the user as an observation point. Alternatively, the control unit 16 may set the observation points in a tile shape so as to cover the entire range of the culture vessel. Note that the information on the observation point is stored in the storage unit 17 under the control of the control unit 16.

  Step # 103: The control unit 16 executes a teaching process for extracting a target cell from the observation image. For example, the control unit 16 records teacher information prepared in advance by the user in the storage unit 17.

  Here, the teacher information may be a template image of a target cell or a cell colony made of the cell, or may be a feature vector extracted from the template image. In the present embodiment, the teacher information includes the threshold value of the size of the cell colony and the contraction / expansion ratio with respect to the time change of each cell. In # 103 of one embodiment, one or a plurality of teacher information may be prepared for the same type of cultured cells, and it is not necessary to prepare the teacher information individually for all the cultured cells in the culture container.

  Step # 104: The control unit 16 sets an observation schedule according to a user input from the input unit 18 or the terminal device 22. The observation schedule information is stored in the storage unit 17 under the control of the control unit 16.

  For example, items that can be set in the observation schedule include `` the whole period of the culture process '', `` start time of time lapse observation '', `` time interval for performing time lapse observation '', `` analysis interval of still images acquired by time lapse observation '', The “moving image shooting interval” and “moving image shooting time” are included. When cells are simultaneously cultured in a plurality of culture containers, the control unit 16 sets an observation schedule for each culture container. In addition, the “moving image capturing time” set in # 104 is set to be longer than one cycle of cardiac muscle cell pulsation.

  Step # 105: The control unit 16 sets an initial value of the moving image acquisition condition. Here, the moving image acquisition condition is a determination condition applied to the determination of the presence or absence of moving image shooting, and is set by paying attention to the form of a cell colony in which cultured cells acquired by time-lapse observation are gathered.

  In the example of the embodiment, the control unit 16 sets the lower limit value of the size of the cell colony as the moving image acquisition condition. And since the observation control part 16a in one Embodiment has the high possibility that it is a colony which consists of a target cell when the magnitude | size of the colony of a cell exceeds a lower limit, the moving image of the cell is made into the imaging part 15. Let them shoot. In general, in the process of differentiating iPS cells into other cells, there is a high possibility that cell colonization occurs before differentiation. For this reason, the cell culture device according to an embodiment uses the size of a cell colony as a moving image acquisition condition in order to narrow down analysis targets in moving images.

  Step # 106: The control unit 16 sets on / off of the moving image analysis process in the culturing step in response to a user input from the input unit 18 or the terminal device 22.

  Step # 107: The control unit 16 determines whether or not the moving image analysis process (# 106) in the culture process is set to ON. If the above requirement is satisfied (YES side), the process proceeds to # 108. On the other hand, if the above requirement is not satisfied (NO side), the process proceeds to # 110.

  Step # 108: The control unit 16 reads out the detection conditions for detecting repetitive contraction motion (cardiomyocyte pulsation) of the cultured cells in response to the input made in step # 103, and the control unit 16 performs the subsequent processing. Set as a detection condition. For example, the control unit 16 sets a threshold indicating the cell area variation rate in a predetermined time width as the detection condition. And the image analysis part 16b in one Embodiment determines with the repetitive contraction motion having arisen in the cultured cell, when the area variation rate of the determination object cell exceeds a threshold value in the acquired moving image.

  The control unit 16 in # 108 also sets a value of “moving image analysis interval” as an additional setting item of the observation schedule.

  Step # 109: The control unit 16 sets on / off of the automatic adjustment processing of the moving image acquisition condition in the culturing step in response to a user input from the input unit 18 or the terminal device 22. Note that when the automatic adjustment processing of the moving image acquisition condition is on, the image analysis unit 16b determines whether or not to perform observation with a moving image according to the analysis result of the moving image (the size of the colony for moving image shooting). Adjust the lower limit value). This adjustment method will be described later in detail.

  Step # 110: The observation control unit 16a compares the observation schedule (# 104) with the current date and time to determine whether or not the time lapse observation time for the culture container has come. If the above requirement is satisfied (YES side), the process proceeds to # 111. On the other hand, if the above requirement is not satisfied (NO side), the process proceeds to # 126.

  Step # 111: The observation control unit 16a performs time-lapse observation of the culture vessel. For example, the observation control unit 16a executes the following process.

  First, the observation control unit 16a instructs the container transport unit 14 to transport the culture container. Then, the container transport unit 14 places the instructed culture container on the stage 13. Thereafter, the observation control unit 16a instructs the imaging unit 15 to capture a still image (observation image) of the cultured cell (see FIG. 4). At this time, the imaging unit 15 sequentially captures observation images for each observation point of the culture vessel set in advance. Note that observation image data at each observation point acquired by time-lapse observation is stored in the storage unit 17 under the control of the control unit 16.

  Step # 112: The image analysis unit 16b analyzes the observation image (# 111). The analysis process in # 112 is executed every time the “analysis interval of still images acquired by time-lapse observation” set in # 104 elapses.

  Specifically, the image analysis unit 16b in # 112 extracts the cultured cell image and other images separately from the observed image (see FIG. 4). And the image analysis part 16b acquires the magnitude | size (morphological information) of the colony containing the extracted cultured cell. For example, when a cell is imaged with a phase-contrast microscope, a halo appears around a region where the phase difference changes greatly, such as a cell membrane. Therefore, the image analysis unit 16b only has to extract a halo corresponding to the cell membrane by a known edge extraction method and estimate a group of closed spaces (cells) surrounded by edges by the contour tracking process as a colony.

  Step # 113: The image analysis unit 16b determines whether or not an observation condition (moving image acquisition condition) indicating whether or not to perform observation with a moving image is satisfied. Specifically, the image analysis unit 16b determines that the moving image acquisition condition is satisfied when the size of the colony acquired in # 112 exceeds the threshold value of the observation condition based on the moving image. If the above requirement is satisfied (YES side), the process proceeds to # 114. On the other hand, if the above requirement is not satisfied (NO side), the process proceeds to # 124.

  Step # 114: The observation control unit 16a newly registers the current observation position that satisfies the moving image acquisition condition as a moving image observation point.

  Step # 115: The observation control unit 16a instructs the imaging unit 15 to capture a moving image of the cultured cell at the moving image observation point. The moving image photographed in # 115 is used to detect repetitive contraction motion of the cultured cells. Therefore, the frame rate of the moving image # 115 is set to be smaller than the shooting interval for time lapse observation. More specifically, the frame rate of the moving image # 115 is set so that the temporal resolution is smaller than the pulsation of the cardiomyocytes.

  Step # 116: The control unit 16 determines whether or not the moving image analysis process (# 106) in the culture process is set to ON. If the above requirement is satisfied (YES side), the process proceeds to # 117. On the other hand, if the above requirement is not satisfied (NO side), the process proceeds to # 124.

  Step # 117: The image analysis unit 16b analyzes the moving image (# 115). The analysis process in # 117 is executed every time the “moving image analysis interval” set in # 108 elapses.

  Specifically, the image analysis unit 16b in # 117 calculates the area of the cultured cell in each frame of the moving image. Then, the image analysis unit 16b determines that the repetitive contraction motion is generated in the cultured cell when the area variation rate of the determination target cell exceeds the threshold value (# 108) in the acquired moving image (FIG. 4).

  Step # 118: The image analysis unit 16b determines whether or not the contraction movement of the cultured cell has been detected in the analysis process at # 117. If the above requirement is satisfied (YES side), the process proceeds to # 119. On the other hand, if the above requirement is not satisfied (NO side), the process proceeds to # 121.

  Step # 119: The control unit 16 outputs a notification indicating the detection of the contraction motion of the cultured cells to the monitor 19 or the terminal device 22. As a result, the user can confirm that differentiation into cardiomyocytes has occurred.

  Step # 120: The image analysis unit 16b performs an additional teaching process using the frame of the moving image in which the contraction movement of the cultured cell is detected. For example, the image analysis unit 16 b generates teacher information from a plurality of frames of the moving image, and adds the teacher information to the storage unit 17. Thereby, in the still image analysis processing (# 112) performed after the next time, the image analysis unit 16b uses the teacher information generated from the moving image of the cultured cell in which the contraction motion is detected, so that the image analysis unit 16b uses the cultured image from the observed image. To extract. Note that after the process of # 120 is completed, the process proceeds to # 124.

  Here, when the contraction motion of the cultured cell is detected, the additional teaching process is performed for the following reason. Since cardiomyocytes undergo repetitive contraction, the cell shape changes from time to time. In other words, depending on the acquisition timing of the observation image in time-lapse observation, there is a possibility that cells cannot be extracted from the initial teacher information even though there are cardiomyocytes performing contraction motion in the image. Therefore, in one embodiment, the accuracy of cell extraction in the still image analysis processing (# 112) is improved by adding teacher information from the moving image in which the contraction motion of the cultured cell is detected.

  Step # 121: The controller 16 determines whether or not the moving image acquisition condition automatic adjustment process (# 109) is set to ON. If the above requirement is satisfied (YES side), the process proceeds to # 122. On the other hand, if the above requirement is not satisfied (NO side), the process proceeds to # 124.

  Step # 122: The observation control unit 16a deletes the registration of the moving image observation point where the contraction movement of the cultured cell could not be detected.

  Step # 123: The image analysis unit 16b executes an automatic adjustment process for the moving image acquisition condition. Specifically, the image analysis unit 16b in # 123 decreases the threshold value of the moving image acquisition condition by a preset adjustment amount. As a result, in the determination of the moving image acquisition condition (# 113) performed after the next time, the image analysis unit 16b determines that a smaller colony also satisfies the moving image acquisition condition. Therefore, since shooting and analysis of a moving image is executed for a larger number of cultured cell colonies, the image analysis unit 16b can more easily detect differentiation into cardiomyocytes.

  Step # 124: The observation control unit 16a determines whether or not time-lapse observation has been completed at all observation points. If the above requirement is satisfied (YES side), the process proceeds to # 125. On the other hand, when the above requirement is not satisfied (NO side), the observation control unit 16a drives the stage 13 to adjust the field of view of the imaging unit 15 to the next observation point. Thereafter, the observation control unit 16a returns to the process of # 111 and repeats the above operation.

  Step # 125: The observation control unit 16a instructs the container transport unit 14 to transport the culture container. And the container conveyance part 14 returns the culture container on the stage 13 to the accommodation position in the temperature-controlled room 11.

  Step # 126: The observation control unit 16a refers to the observation schedule (# 104) and determines whether or not the culturing process has been completed. If the above requirement is satisfied (YES side), the control unit 16 ends the series of processes. On the other hand, when the above requirement is not satisfied (NO side), the observation control unit 16a performs the process of step # 127.

  Step # 127: The control unit 16 determines whether or not the automatic adjustment process (# 109) of the moving image acquisition condition is set to ON. If the above requirement is satisfied (YES side), the process proceeds to # 128. On the other hand, when the above requirement is not satisfied (NO side), the process returns to the process of # 110 and the above operation is repeated.

  Step # 128: The image analysis unit 16b obtains a ratio of the number of points where the contraction motion is detected in step # 118 to the number of moving image observation points that satisfy the moving image acquisition condition in step # 113. When the ratio is not greater than a certain value, differentiation may occur even in a cell colony having a size equal to or smaller than a threshold value. Therefore, the image analysis unit 16b reduces the threshold value of the cell colony size so that a smaller colony can be detected. As a result, the number of differentiated cell colonies may increase, and at the same time, undifferentiated cell colonies can be detected. Note that the number of cell colonies that differentiate into the target cells is relatively decreased with respect to the total number of detected cells. Therefore, when the ratio between the target cell colony and the total cell colony detected is equal to or less than a predetermined value, the image analysis unit 16b does not perform downward correction of the threshold with the target as the focusing target. Then, the control part 16 returns to the process of # 110 and repeats the said operation | movement.

  Above, description of the flowchart of FIG. 2, FIG. 3 is complete | finished.

  In the cell culture device in one embodiment, the image analysis unit 16b analyzes an observation image (# 111) acquired by time-lapse observation. Then, when the form of the cultured cell satisfies the moving image acquisition condition, the observation control unit 16a causes the imaging unit 15 to capture the moving image of the cultured cell at a time interval shorter than the time lapse observation time interval (# 115). Thereby, in one Embodiment, since the moving image of the cultured cell with high possibility that the spontaneous contraction motion has arisen can be acquired, the user can detect the dynamic change of the cultured cell in a short period. In addition, since the cell culture device according to one embodiment performs the above-described processing at a plurality of observation points, a wide range and comprehensive observation of cultured cells can be performed.

  In addition, the image analysis unit 16b according to the embodiment detects the contraction motion of the cultured cell based on the area variation rate of the cultured cell in the moving image (# 117). Thereby, in the cell culture apparatus of one Embodiment, since the pulsation of a cardiac muscle cell can be automatically detected in a culture process, the burden of the user in a culture process can be remarkably reduced.

<Supplementary items of the embodiment>
In the cell culture device of the above embodiment, an example has been described in which the functions of the image analysis unit 16b and the observation control unit 16a are realized by software using a program. It doesn't matter.

  In the example of the above embodiment, the example in which the control unit 16 of the cell culture device functions as the image analysis unit 16b and the observation control unit 16a has been described. However, the terminal device 22 functions as the image analysis unit 16b and the observation control unit 16a. You may do.

  In addition, the program memorize | stored in the memory | storage part 17 in the said embodiment may be a firmware program updated by version upgrade. That is, the function of the cell culture device of the present invention may be provided afterwards by updating the firmware of the existing cell culture device.

  From the above detailed description, features and advantages of the embodiments will become apparent. It is intended that the scope of the claims extend to the features and advantages of the embodiments as described above without departing from the spirit and scope of the right. Further, any person having ordinary knowledge in the technical field should be able to easily come up with any improvements and modifications, and there is no intention to limit the scope of the embodiments having the invention to those described above. It is also possible to use appropriate improvements and equivalents within the scope disclosed in.

DESCRIPTION OF SYMBOLS 11 ... Constant temperature chamber, 12 ... Environmental control part, 13 ... Stage, 14 ... Container conveyance part, 15 ... Imaging part, 16 ... Control part, 16a ... Observation control part, 16b ... Image analysis part, 17 ... Memory | storage part, 18 ... Input unit, 19 ... monitor, 20 ... communication unit, 21 ... communication line, 22 ... terminal device

Claims (5)

A temperature-controlled room where the room is maintained at predetermined environmental conditions;
An imaging unit for imaging an observation image of cultured cells in the temperature-controlled room;
The imaging unit acquires morphological information of the cultured cell from a first image captured at a first time interval, and determines whether the cultivated cell configuration satisfies a predetermined observation condition based on the morphological information. An image analysis unit to
An observation control unit that causes the imaging unit to image the cultured cells at a second time interval shorter than the first time interval when the form of the cultured cells satisfies the predetermined observation condition;
A cell culture apparatus comprising: The cell culture device according to claim 1,
The image analysis unit is a cell culture device that detects a contraction movement of the cultured cell by comparing a plurality of second images captured by the imaging unit at the second time interval. The cell culture device according to claim 2,
The cell culturing apparatus, wherein the image analysis unit changes the predetermined observation condition according to whether or not the contraction motion is detected. In the cell culture device according to claim 2 or 3,
The cell culture device, wherein the image analysis unit specifies a position of the cultured cell from the first image, and corrects the predetermined observation condition from the second image when the contraction motion is detected. A program for operating a cell culture device comprising a temperature-controlled room where the room is maintained at a predetermined environmental condition and an imaging unit that captures an observation image of the cultured cells in the temperature-controlled room,
The imaging unit acquires morphological information of the cultured cell from a first image captured at a first time interval, and determines whether the cultivated cell configuration satisfies a predetermined observation condition based on the morphological information. Image analysis processing to
When the form of the cultured cell satisfies the predetermined observation condition, an observation control process that causes the imaging unit to image the cultured cell at a second time interval shorter than the first time interval;
A program that causes a computer to execute.

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