Culture device having time-lapse imaging function, and culture method

Abstract

To provide a culture device having a time-lapse imaging function and a culture method, whereby it becomes possible to automatically record only a well in which a cell is present in a culture container.SOLUTION: Provided is a culture device having a time-lapse imaging function, in which a cell is placed in each well in a culture container having one or more wells and is cultured while time-lapse imaging is performed, the culture device being provided with: imaging means 40 that performs imaging at a predetermined time-lapse imaging period for each well in the culture container; detection means 41 that detects the presence of a cell in each well in the culture container; and image recording means 42 that records an image of a well that is determined as a well in which a cell is present by the detection means 41 among images captured for the individual wells by the imaging means 40.SELECTED DRAWING: Figure 3

Description

[Technical field]

[0001] The present invention relates to a culture device with a time lapse photography function that cultures cells such as embryos and performs time lapse photography, and a culture method. [Background technology]

[0002] There is known a culture device with a time lapse photography function that cultures cells such as embryos and also performs time lapse photography (see, for example, Patent Documents 1 to 3). For example, in a culture device with a time lapse photography function, multiple fertilized eggs (embryos) collected and processed from one patient are placed in multiple divided wells in one culture vessel to start culturing, and time lapse photography is performed at regular intervals until the embryos become transplantable (3 to 7 days), and the captured image data is stored on a recording medium.

[0003] The time-lapse images of the development of multiple embryos are then morphologically evaluated to determine whether the embryos are good or bad, and then the embryos are transferred, cryopreserved, or discarded. The evaluation of embryo images is generally performed using image display software that assists in embryo evaluation, and the images of the embryos over time are displayed in a chronological order on the image display software, allowing the user to confirm and make a judgment. [Prior art documents] [Patent documents]

[0004] [Patent Document 1] Special Publication No. 2009-539387 [Patent Document 2] Special Publication No. 2017-529844 [Patent Document 3] JP 2016-123366 A Summary of the Invention [Problem to be solved by the invention]

[0005] However, in the early stage of the culture, some embryos may be frozen and transferred, and the culture may be terminated, or may be temporarily removed from the device for microscopic observation or medium replacement. In time-lapse photography, wells containing embryos are designated in advance in the culture vessel at the time of dish-in to start the culture, and periodic photography is performed on the designated wells. Therefore, even when the embryos are removed, images of wells without embryos are taken and recorded on the recording medium. This increases the amount of recording medium used, and there is a possibility of a recording medium shortage.

[0006] Furthermore, when images are displayed in chronological order using image display software or when video is displayed frame by frame, unnecessary images that do not contain embryos are also displayed, resulting in a difficult-to-view display and reducing the efficiency of the evaluation work.

[0007] Therefore, an object of the present invention is to provide a culture device and a culture method with a time lapse photography function that can automatically record only the wells in a culture vessel in which cells exist. [Means for solving the problem]

[0008] The culture device with time lapse photography function of the present invention is a culture device with time lapse photography function that places cells in each well of a culture vessel having at least one well, cultivates them, and performs time lapse photography, and includes an imaging means that images each well of the culture vessel at a predetermined time lapse photography period, a detection means that detects the presence or absence of cells in each well of the culture vessel, and an image recording means that records images of wells that are detected as having cells by the detection means from the images captured for each well by the imaging means.

[0009] According to the culture device with time lapse photography function of the present invention, images are taken of each well of a culture vessel at a predetermined time lapse period, the presence or absence of cells is detected, and only images of wells detected as having cells are recorded in an image recording means.

[0010] The detection means preferably detects the presence or absence of cells by analyzing the image captured by the imaging means for each well, whereby, when the presence of cells is detected as a result of analyzing the image captured for each well, only the image of the well in which the presence of cells is detected is recorded in the image recording means.

[0011] The detection means is preferably one that detects the presence or absence of cells at each time lapse photography period, whereby the presence or absence of cells is detected each time photography is performed at a predetermined time lapse period, and only images of wells where the presence of cells is detected are recorded in the image recording means.

[0012] The culture device with time lapse photography function of the present invention preferably has a notification means for notifying when the detection result by the detection means is different from the previous detection result. In this way, when the detection result by the detection means is different from the previous detection result, the notification means notifies the user, so that the user can check the detection result.

[0013] The culture device with time lapse photography function of the present invention preferably has a history recording means for recording the history of the presence or absence of cells in all wells of the culture vessel for each time lapse photography cycle. As a result, the history of the presence or absence of cells in all wells of the culture vessel is recorded by the history recording means for each time lapse photography cycle, and this history of the presence or absence of cells in all wells of the culture vessel for each time lapse photography cycle can be used for image arrangement in a timeline display and history management.

[0014] The culture method of the present invention is a culture method in which cells are placed in each well of a culture vessel having at least one well, cultured, and time-lapse photography is performed, and is characterized by including the steps of photographing each well of the culture vessel at a predetermined time-lapse photography period, detecting the presence or absence of cells in each well of the culture vessel, and recording images of wells that are detected to contain cells from the images photographed for each well.

[0015] According to the culture method of the present invention, images are taken of each well of a culture vessel at a predetermined time lapse period, the presence or absence of cells is detected, and only images of wells in which the presence of cells is detected are recorded. Effect of the Invention

[0016] According to the present invention, it is possible to automatically record only images of wells in which cells are present for each well of a culture vessel, and it is not necessary to register in advance wells in which cells are present and which need to be photographed when the dish is inserted, which reduces the amount of work. It is also possible to reduce the amount of recording media used to record images. Furthermore, since images of unnecessary wells in which no cells are present are not recorded, the degree of freedom in setting the photographing cycle and schedule is increased, and culture by image observation with higher accuracy is possible. Furthermore, since there are no images of unnecessary wells in which no cells are present when the recorded images are displayed, it is possible to perform efficient evaluation. [Brief description of the drawings]

[0017] [Figure 1] FIG. 1 is a schematic configuration diagram of a culture device with a time lapse photography function according to an embodiment of the present invention. [Diagram 2] FIG. 2 is an enlarged view of the culture vessel of FIG. 1. [Diagram 3] FIG. 2 is a functional block diagram of the culture device with a time lapse photography function of FIG. 1. [Figure 4] FIG. 1 is a diagram showing an example of a procedure for detecting the presence or absence of cells. [Diagram 5] FIG. 13 is a flow diagram showing a time lapse photography process using the culture device. [Figure 6] FIG. 1 shows examples of live image display of all wells, where (A) shows a conventional display example, and (B) shows a display example of the present invention. [Figure 7] FIG. 1 shows examples of timeline display of multiple wells, where (A) shows a conventional display example, and (B) shows a display example of the present invention. [Figure 8] FIG. 13 is a diagram showing a comparative example in which a moving image is displayed by frame-by-frame advancement of a plurality of time-lapse images. [Figure 9]FIG. 13 is a diagram showing an example of an abnormality notification display. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0018] FIG. 1 is a schematic diagram of a culture device with a time lapse photography function in an embodiment of the present invention, FIG. 2 is an enlarged view of a culture vessel in FIG. 1, and FIG. 3 is a functional block diagram of the culture device with a time lapse photography function in FIG.

[0019] As shown in Fig. 1, a culture device with a time lapse photography function (hereinafter, sometimes simply referred to as a "culture device") 1 in an embodiment of the present invention is a culture device that arranges cells in each well 11 of a culture vessel 10 having at least one well 11 (see Fig. 2) in a chamber section 2, cultures the cells, and performs time lapse photography using a photography section 3. Time lapse photography is a photography method in which a plurality of still images are taken at regular time intervals (time lapse period) and these taken still images are stitched together to create a video.

[0020] The culture device 1 also has a culture control unit 4 that controls the chamber section 2, an imaging control unit 5 that controls the imaging unit 3, etc., a recording unit 6 that stores images captured by the imaging unit 3, an input / display unit 7 that inputs and commands the operation of the device and displays images, etc., and an overall control unit 8 that controls the entire device.

[0021] The chamber section 2 maintains the environment, such as the temperature and CO2 / O2 gas concentration, required for cell culture. One or more culture vessels 10 can be stored in the chamber section 2. A heater, a CO2 gas flow path, and an O2 gas flow path (not shown) are connected to the chamber section 2. The culture control unit 4 controls the heater and the CO2 / O2 gas flow rate so that the temperature and the CO2 / O2 gas concentration are constant. Although two culture vessels 10 are shown in FIG. 1, the chamber section 2 may be configured with multiple chambers divided into one or multiple culture vessels.

[0022] The photographing unit 3 is composed of a camera 30, an illumination 31, an objective lens 32, a movable unit 33, and the like. In the photographing unit 3, the illumination 31 shines light on the cells in the culture vessel 10, and the objective lens 32 receives the transmitted light and diffracted light, and the image enlarged by the objective lens 32 is input to the camera 30. The movable unit 33 moves the camera 30 and the illumination 31 together in the arrangement direction of the wells of the culture vessel 10 (X and Y directions in FIG. 2). The movable unit 33 also photographs the cells in the multiple culture vessels 10 by moving the objective lens 32 in the Z-axis (optical axis) direction. Note that a configuration in which multiple cameras 30 and illuminations 31 are arranged for each culture vessel 10 and chamber unit 2 can also be used. In another embodiment, the camera 30 and illumination 31 can be fixed, and the chamber unit 2 can be integrated with a separately provided movable unit (not shown), and the culture vessel 10 can be moved in the X, Y, or Z directions.

[0023] The imaging control unit 5 controls the optical settings of the imaging unit 3 during image capture, position control, time lapse imaging conditions, image storage, etc. As shown in Fig. 3, the imaging control unit 5 has an imaging means 40 that captures images for each well 11 of the culture vessel 10 at a predetermined time lapse imaging period, a detection means 41 that detects the presence or absence of cells for each well 11 of the culture vessel 10, and an image recording means 42 that records images of wells 11 detected by the detection means 41 as having cells among the images captured for each well 11 by the imaging means 40.

[0024] The photographing means 40 photographs each well 11 of the culture vessel 10 with the photographing unit 3 at a predetermined time lapse period set in advance. The time lapse period can be set arbitrarily between several minutes and several hours. The photographing unit 3 is a microscope camera composed of an objective lens with a magnification of about 10 times, a CCD or CMOS image sensor, etc.

[0025] The detection means 41 detects the presence or absence of cells by analyzing the images captured by the imaging means 40 for each well. Detection of the presence or absence of cells is performed on a well-by-well basis. FIG. 4 shows an example of a procedure for detecting the presence or absence of cells. In the example shown in FIG. 4, the detection means 41 detects wells from the images of the wells captured by the imaging means 40, produces an image of only embryos as cells, and detects the presence or absence of embryos from this image of only embryos. Detection of the presence or absence of embryos from the image of only embryos can be performed by a method using image processing, artificial intelligence (machine learning or deep learning), etc.

[0026] For example, there is a method of extracting objects by labeling an image and detecting the presence or absence of an embryo from the object. Embryo images have the following characteristics, and the presence or absence of an embryo can be detected by focusing on one of these characteristics or a combination of multiple characteristics from the object. -Circular -Size is 100~200μm - There is a transparent ring at the edge (almost uniform brightness area in the image) -Has an internal structure (texture)

[0027] Alternatively, instead of detection by object extraction, a method may be adopted in which detection is performed by deep learning using various image characteristics, such as light and dark characteristics or feature point extraction or model comparison in the entire image.

[0028] The image recording means 42 records only images of wells detected as having cells by the detection means 41 among the images captured for each well by the imaging means 40 in the recording unit 6. The recording unit 6 is, for example, a recording medium such as a magnetic disk such as a hard disk drive (HDD) or a floppy disk (FD), an optical disk such as a compact disk (CD) or a DVD, a magneto-optical disk such as an MO, a solid state drive (SSD), a flash memory such as a memory card or a USB memory, or a magnetic tape. It is also possible to use a recording medium such as a network attached storage (NAS) or a cloud storage as the recording unit 6.

[0029] The timing of detection of the presence or absence of cells by the detection means 41 can be at the start of time lapse photography, during time lapse photography, or both, but it is preferable to detect the presence or absence of cells every time lapse cycle. This allows the presence or absence of cells to be detected every time photography is performed at a predetermined time lapse cycle, and only images of wells where cells are detected to be present are recorded in the image recording means 42, so that only images of a minimum number of wells are recorded in the image recording means 42.

[0030] The input / display unit 7 is composed of a display device with a touch panel, etc. The input / display unit 7 provides input instructions from a user to the culture control unit 4 and the imaging control unit 5, and also displays images captured by the camera 30 and images read out from the recording unit 6.

[0031] The overall control unit 8 controls the various blocks within the device and the entire device. The overall control unit 8 also has an interface with the outside of the device. Note that software for observing and evaluating embryos may be provided in the overall control unit 8. This allows the overall control unit 8 to assist the user in the culture evaluation work by clearly arranging images on the input / display unit 7 and analyzing and displaying them.

[0032] Next, a description will be given of a time lapse photography method using the above-configured culture apparatus 1. Fig. 5 is a flow chart showing the time lapse photography process using the culture apparatus 1.

[0033] (S100) When a culture container (dish) 10 is placed in the culture device 1 (dish-in), processing is started. (S101) The XY coordinates and Z-axis focal center of all wells 11 are automatically detected (autofocused) using marks or the like provided in the culture vessel 10. (S102) The imaging means 40 moves the imaging part 3 to the first well 11. (S103) The photographing means 40 photographs the well 11 using the photographing section 3. The detection means 41 detects the well 11 from the image photographed by the photographing means 40. (S104) The detection means 41 automatically detects the presence or absence of an embryo in the well 11. (S105) The detection means 41 determines whether or not an embryo is present. If no embryo is present in the well 11, the process proceeds to step (S108). (S106) If an embryo is present in the well 11, the imaging means 40 moves back and forth from the Z-axis focal position to capture multiple slice images of the well 11. However, if slice images are not required, it is sufficient to only capture images at the center of the focal point. (S107) The image recording means 42 stores the image of the well 11 in the recording unit 6. (S108) It is determined whether or not imaging has been completed for all wells 11. If imaging has not been completed, the process proceeds to step (S103) to image the next well 11. (S109) When imaging of all wells 11 is completed, the process ends. (S110) In time lapse photography from the second cycle onwards of the time lapse period, processing is carried out from step (S102).

[0034] In this example, well 11 is detected first, and then cells are detected. However, it is also possible to employ a method in which, without detecting well 11 itself, cells are directly searched for by utilizing the regularity of the well arrangement, and the presence or absence of cells is detected.

[0035] In addition, although the method in step (S101) involves detecting the X, Y, and Z coordinates of all wells 11, it is also possible to employ a method in which multiple divided images of multiple wells 11 are taken across the entire culture vessel 10 and then tiled, or a high-resolution camera is used to take a wide-area image of all wells 11 in a single image, and then the image is divided to extract the individual well images to detect the presence or absence of cells.

[0036] In addition, as shown in FIG. 3, the culture device 1 in this embodiment can be configured so that the photography control unit 5 has a history recording means 43 that records the history of the presence or absence of cells in all wells 11 of the culture container 10 for each time lapse photography period, and a notification means 44 that notifies when the detection result by the detection means 41 differs from the previous detection result.

[0037] The history recording means 43 records the history of the presence or absence of cells in all wells 11 of the culture vessel 10 in each time lapse photography cycle in the recording unit 6 based on the detection result by the detection means 41. As a result, since the history of the presence or absence of cells in all wells 11 of the culture vessel 10 is recorded by the history recording means 43 in each time lapse photography cycle, it becomes possible to utilize the history of the presence or absence of cells in all wells 11 of the culture vessel 10 in each time lapse photography cycle for image arrangement in a timeline display and history management.

[0038] FIG. 6 shows an example of live image display of all wells, (A) shows a conventional display example, and (B) shows a display example of the present invention. As shown in FIG. 6(A), conventionally, images of all wells 11 of the culture vessel 10 are recorded, and images of all wells 11 are displayed regardless of the presence or absence of an embryo in each well 11. On the other hand, in the present invention, only images of wells 11 with embryos among all wells 11 of the culture vessel 10 are recorded in the recording unit 6, and the history of the presence or absence of an embryo in all wells 11 is also recorded by the history recording means 43. Therefore, based on this history of the presence or absence of an embryo, it is possible to display only images of wells 11 with embryos and not display wells 11 without embryos, as shown in FIG. 6(B). Therefore, when the images recorded in the recording unit 6 are used for comparative observation or evaluation of embryos using observation software or evaluation software, it is easy to see at a glance, and the state and progress are easy to grasp, making it possible to efficiently perform culture observation and evaluation.

[0039] FIG. 7 is a diagram showing an example of a timeline display of multiple wells, where (A) is a diagram showing a conventional display example, and (B) is a diagram showing a display example of the present invention. In FIG. 7, multiple wells are arranged horizontally, and images of the wells for each time lapse period are arranged vertically. In cell culture, embryos may be extracted from wells 11 during the culture for cryopreservation or transplantation. In addition, the culture vessel (dish) 10 may be taken out for external microscopic observation or culture medium exchange. In this case, in the past, as shown in FIG. 7(A), wells 11 from which extracted embryos were present are all displayed. On the other hand, in the present invention, based on the embryo presence / absence history recorded by the history recording means 43, wells 11 from which embryos were extracted for cryopreservation or transplantation during the culture are not displayed as shown in FIG. 7(B) based on the embryo presence / absence history recorded by the history recording means 43. In addition, when the culture vessel 10 is taken out for external microscopic observation or culture medium exchange, it is not displayed. Then, when the culture vessel 10 is rearranged, wells 11 in which embryos are present are displayed again.

[0040] FIG. 8 is a diagram showing a comparative example in which a video is displayed by frame-by-frame advancement of multiple time-lapse images. As shown in FIG. 8, in the past, time-lapse image data of a specific embryo was read from a storage device and displayed continuously, so images 81 of only wells without cells were mixed in. Therefore, when displaying a video, an image without an embryo appears for a moment, resulting in a discontinuous and difficult-to-view video display. However, in the culture device 1 with time-lapse photography function in this embodiment, the image 81 of only wells without an embryo is eliminated, so that a video image that is easy to view without discontinuity can be obtained. That is, in the culture device 1 with time-lapse photography function in this embodiment, the growth state of cells can be visualized continuously by continuously displaying time-lapse images, and the process of change and growth can be easily understood.

[0041] The notification means 44 notifies the user when the detection result by the detection means 41 is different from the previous detection result. The notification means 44 may, for example, sound a notification buzzer, turn on a notification lamp, or display an abnormality notification on a display. FIG. 9 shows an example of an abnormality notification display. When the detection result by the detection means 41 is different from the previous detection result, the notification means 44 may, for example, display an abnormality notification on the display as a dialogue as shown in FIG. 9 to alert the user of the culture device 1 and ask the user to confirm whether or not imaging is required. At this time, the imaging operation of the corresponding well 11 is put into a standby state, and the operator can select "OK" if the presence or absence of an embryo has been confirmed by cryopreservation or transplantation, or select "CANCEL" if there is a possibility of an error in the judgment, and instruct the user to take another image. In another embodiment, the detection result of the presence or absence of cells may be notified to the user, and the user may be notified of the necessity of imaging or abnormality in the dish installation.

[0042] As described above, in the culture device 1 of this embodiment, it is possible to automatically record only images of wells 11 in which cells exist for each well 11 of the culture container 10, and the amount of usage of the recording unit 6 for recording images can be reduced. In addition, since images of unnecessary wells 11 in which no cells exist are not recorded, the degree of freedom in setting the shooting cycle and schedule is increased, and culture by image observation with higher accuracy is possible. Furthermore, since there are no images of unnecessary wells in which no cells exist when the recorded images are displayed, efficient evaluation can be performed. [Industrial Applicability]

[0043] INDUSTRIAL APPLICABILITY The present invention is useful for a culture device with a time lapse photography function that cultures cells such as embryos while performing time lapse photography, and a culture method thereof. [Explanation of symbols]

[0044] 1. Culture device with time-lapse photography function 2. Chamber section 3. Photography Department 4. Culture control unit 5. Shooting control section 6 Recording section 7 Input / display section 8 Overall control section 10 Culture vessel 11 wells 30 Camera 31 Lighting 32 Objective Lens 33 Moving parts 40 Filming Method 41 Detection methods 42 Image recording means 43 Career Recording Means 44 Means of Notification

Claims

1. A culture device with a time lapse photography function that arranges cells in each well of a culture vessel having at least one well, cultures the cells in each well, and performs time lapse photography, an imaging means for imaging each well of all wells of the culture vessel at a predetermined time lapse imaging period; a detection means for detecting the presence or absence of cells in each of all wells of the culture vessel for each of the time lapse photography periods; an image recording means for recording an image of a well detected by the detecting means as having a cell among the images taken for each well by the photographing means; A culture device with time-lapse photography function.

2. 2. The culture device with time lapse photography function according to claim 1, wherein the detection means detects the presence or absence of the cells by analyzing the images taken for each well by the photography means.

3. A culture device with a time-lapse photography function as described in claim 1 or 2, wherein the photography means photographs multiple slice images of a well that is detected by the detection means as containing cells.

4. The culture device with time lapse photography function according to claim 1 or 2, further comprising a notification means for notifying when the detection result by said detection means is different from the previous detection result.

5. 3. The culture device with time lapse photography function according to claim 1, further comprising a history recording means for recording the history of the presence or absence of cells in all wells of said culture vessel for each time lapse photography period.

6. A culture method in which cells are placed in each well of a culture vessel having at least one well and cultured while performing time-lapse photography, taking photographs of all wells of the culture vessel at a predetermined time lapse photography period for each well; detecting the presence or absence of cells for each of all wells of the culture vessel for each time lapse photography period; Recording images of wells detected as having cells among the images taken for each well. A culture method comprising:

Images