Culture device with delayed shooting function and culture method

Abstract

Provided are a culture device with a delayed imaging function and a culture method that can automatically record only pores in a culture vessel in which cells are present. Provided is a culture device with a delayed imaging function, the culture device being provided with cells for each well of a culture vessel having at least one well, culturing the cells, and performing delayed imaging, the culture device being provided with: an imaging means (40) for performing imaging at a prescribed delayed imaging cycle for each well of the culture vessel; a detection means (41) for detecting the presence or absence of cells for each well of the culture vessel; and an image recording unit (42) that records an image of a hole detected as having a cell by the detection unit (41), from among the images captured by the imaging unit (40) for each hole.

Description

Technical Field

[0001] The present invention relates to a culture device with a time-lapse shooting function and a culture method for culturing cells such as embryos and performing time-lapse shooting. Background Art

[0002] A culture device with a time-lapse photography function for culturing cells such as embryos and performing time-lapse photography is known (for example, see 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 a patient are arranged in multiple wells divided in a culture container and culture is started, and time-lapse photography is performed at a certain period until they become embryos that can be transplanted (3 to 7 days), and the captured image data is stored in a recording medium.

[0003] Then, the images of the growth process of multiple embryos captured in time-lapse are morphologically evaluated to determine whether the embryos are good, and to determine whether the embryos are transplanted, cryopreserved, or excluded. The evaluation of embryo images is generally performed using image display software that assists in embryo evaluation. The image display software displays the images of the embryos in a time series to confirm the images and make a judgment.

[0004] Prior art literature

[0005] Patent Literature

[0006] Patent Document 1: Japanese Patent Application No. 2009-539387

[0007] Patent Document 2: Japanese Patent Application No. 2017-529844

[0008] Patent Document 3: Japanese Patent Application Publication No. 2016-123366 Summary of the invention

[0009] However, in order to cryopreserve or transplant a part of embryos in the early stage of culture, culture is sometimes terminated or temporarily removed from the device for microscopic observation or culture medium replacement. In time-lapse photography, a hole for placing embryos is specified in advance in the culture container when the culture dish is placed at the beginning of culture, and the specified hole is periodically photographed. Therefore, even if the embryo is taken out, the image of the hole without the embryo is photographed and recorded in the recording medium. Therefore, the amount of recording medium used increases, which may cause insufficient recording medium.

[0010] Furthermore, when performing time series display using image display software and displaying moving images by frame-by-frame playback, unnecessary images without embryos are also displayed, making the display difficult to see, which leads to a problem of reduced efficiency of evaluation work.

[0011] 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 containing cells in a culture container.

[0012] The culture device with time-lapse photography function of the present invention arranges cells in each hole of a culture container having at least one hole and cultures them, and performs time-lapse photography. The culture device with time-lapse photography function includes: a photography unit, which photographs each hole of the culture container with a specified time-lapse photography period; a detection unit, which detects the presence or absence of cells in each hole of the culture container; and an image recording unit, which records images of the holes detected as having cells by the detection unit in the images taken by the photography unit for each hole.

[0013] According to the culture device with time-lapse photography function of the present invention, each well of the culture container is photographed at a predetermined time-lapse period to detect the presence of cells, and only images of the wells detected to have cells are recorded in the image recording unit.

[0014] It is desirable that the detection unit detects the presence of cells by analyzing the image captured by the imaging unit for each well. Thus, when the presence of cells is detected as a result of analyzing the image captured for each well, only the image of the well detected to have cells is recorded in the image recording unit.

[0015] It is desirable that the detection unit detects the presence of cells at each time-lapse imaging cycle. Thus, the presence of cells is detected every time imaging is performed at a predetermined time-lapse cycle, and only images of wells detected to contain cells are recorded in the image recording unit.

[0016] It is desirable that the culture device with a time-lapse photography function of the present invention has a notification unit for notifying when the detection result of the detection unit is different from the previous detection result. Thus, when the detection result of the detection unit is different from the previous detection result, a notification based on the notification unit is performed, so that the user can confirm the detection result.

[0017] It is desirable that the culture device with a time-lapse photography function of the present invention has an experience recording unit for recording the presence or absence of the experience of the cells in all the wells of the culture container in each time-lapse photography cycle. Thus, by recording the presence or absence of the experience of the cells in all the wells of the culture container in each time-lapse photography cycle by the experience recording unit, the presence or absence of the experience of the cells in all the wells of the culture container in each time-lapse photography cycle can be flexibly used in the image arrangement and history management in the timeline display.

[0018] The culture method of the present invention configures cells and cultures them in each hole of a culture container having at least one hole, and performs time-lapse photography. The culture method is characterized in that it includes: photographing each hole of the culture container with a specified time-lapse photography period; detecting the presence of cells in each hole of the culture container; and recording images of the holes detected as containing cells in the images photographed for each hole.

[0019] According to the culture method of the present invention, each well of the culture container is photographed at a predetermined time delay period to detect the presence of cells, and only images of the wells detected to have cells are recorded.

[0020] According to the present invention, it is possible to automatically record images of only the holes where cells exist for each hole of the culture container, and it is not necessary to pre-register the holes with cells that need to be photographed when the culture dish is placed, thereby reducing the work. In addition, the usage of the recording medium for recording images can be reduced. Furthermore, the images of unnecessary holes where cells do not exist are not recorded, so the freedom of the shooting cycle and schedule setting increases, and cultivation based on higher-precision image observation can be performed. Furthermore, when the recorded images are displayed, there are no images of unnecessary holes where cells do not exist, so it is possible to perform efficient evaluation. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 It is a schematic structural diagram of a culture device with a time-lapse photography function in an embodiment of the present invention.

[0022] Figure 2 yes Figure 1 Magnified image of the culture vessel.

[0023] Figure 3 yes Figure 1 Functional block diagram of a culture device with time-lapse photography function.

[0024] Figure 4 This is a diagram showing an example of a step of detecting the presence or absence of cells.

[0025] Figure 5 is a flow chart showing the time-lapse imaging process performed by the culture apparatus.

[0026] Figure 6 1 and 2 are diagrams showing examples of real-time image display of all wells, wherein (A) is a diagram showing a conventional display example, and (B) is a diagram showing a display example of the present invention.

[0027] Figure 7 1 and 2 are diagrams showing time line display examples of a plurality of wells, wherein (A) is a diagram showing a conventional display example, and (B) is a diagram showing a display example of the present invention.

[0028] Figure 8A diagram showing a comparative example of displaying a moving image by playing back a plurality of time-lapse shots frame by frame.

[0029] Fig. 9 It is a figure which shows an example of abnormality notification display. DETAILED DESCRIPTION

[0030] Figure 1 is a schematic structural diagram of a culture device with a time-lapse photography function in an embodiment of the present invention, Figure 2 yes Figure 1 Magnified image of the culture vessel. Figure 3 yes Figure 1 Functional block diagram of a culture device with time-lapse photography function.

[0031] like Figure 1 As shown, the culture device with time-lapse photography function (hereinafter, sometimes referred to as "culture device") 1 in the embodiment of the present invention is a culture device as follows: in the chamber part 2, there are at least one hole 11 (refer to Figure 2 ) are arranged in each well 11 of a culture container 10 and cultured, and time-lapse photography is performed by the photography unit 3. Time-lapse photography refers to a photography method of taking a plurality of still images at a certain time interval (time-lapse period), or combining the plurality of still images taken to form a dynamic image.

[0032] In addition, the culture device 1 has a culture control unit 4 that controls the chamber unit 2, a shooting control unit 5 that controls the shooting unit 3, a recording unit 6 that stores images shot by the shooting unit 3, an input / display unit 7 that inputs instructions for the operation of the device or displays images, and an overall control unit 8 that controls the entire device.

[0033] The chamber 2 is a chamber part for maintaining the temperature, CO2 / O2 gas concentration and other environments required for cell culture. One or more culture containers 10 can be stored in the chamber 2. A heater, CO2 gas flow path, and O2 gas flow path (not shown) are connected to the chamber 2. The culture control unit 4 controls the heater and CO2 / O2 gas flow rate to keep the temperature and CO2 / O2 gas concentration constant. Figure 1 In the figure, two culture containers 10 are shown in the chamber part 2, but it can also be composed of a single chamber or a plurality of chambers divided for each of a plurality of culture containers.

[0034] The imaging unit 3 is composed of a camera 30, an illumination unit 31, an objective lens 32, a movable unit 33, and the like. In the imaging unit 3, the illumination unit 31 irradiates light to the cells in the culture container 10, and the objective lens 32 receives the transmitted light and diffracted light, and the image magnified by the objective lens 32 is input to the camera 30. The movable unit 33 adjusts the arrangement direction of the camera 30 and the illumination unit 31 relative to the hole of the culture container 10 ( Figure 2 The movable part 33 is movable in the X, Y directions (in the X, Y directions) as a whole. In addition, the movable part 33 photographs the cells in the plurality of culture containers 10 by making the objective lens 32 movable relative to the Z axis (optical axis). In addition, it is also possible to have a structure in which a plurality of cameras 30 and lighting 31 are arranged for each culture container 10 and the chamber part 2. In addition, as another mode, it is also possible to have the following structure: the camera 30 and lighting 31 are fixed, the chamber part 2 is integrated with a movable part (not shown) provided separately, and the culture container 10 is movable in the X, Y directions and the Z direction. Furthermore, the camera 30 and lighting 31 may be fixed, and both the chamber part 2 and the culture container 10 may be movable.

[0035] The shooting control unit 5 controls the optical settings, position control, time-lapse shooting conditions, and image storage of the shooting unit 3 during image shooting. Figure 3 As shown, the shooting control unit 5 includes: a shooting unit 40, which shoots each hole 11 of the culture container 10 with a specified delayed shooting cycle; a detection unit 41, which detects the presence or absence of cells in each hole 11 of the culture container 10; and an image recording unit 42, which records the image of the hole 11 detected as having cells by the detection unit 41 among the images shot by the shooting unit 40 for each hole 11.

[0036] The photographing unit 40 photographs each well 11 of the culture container 10 through the photographing unit 3 at a predetermined delay period. The delay period can be arbitrarily set between several minutes and several hours. The photographing unit 3 is, for example, a microscope camera composed of an objective lens with a magnification of about 10 times, a CCD or CMOS image sensor, etc.

[0037] The detection unit 41 detects the presence or absence of cells by analyzing the image captured for each well by the imaging unit 40. The presence or absence of cells is detected in units of wells. Figure 4 An example of a step for detecting the presence or absence of cells is shown. Figure 4 In the example shown, the detection unit 41 detects the hole from the image of the hole captured by the imaging unit 40 as an image of only the embryo as a cell, and detects the presence of the embryo from the image of only the embryo. The detection of the presence of the embryo from the image of only the embryo can be performed by a method using image processing, artificial intelligence (machine learning, deep learning), etc.

[0038] For example, there is a method of extracting an object by identifying an image and detecting the presence of an embryo based on the object. The embryo image has the following features, and the presence of an embryo can be detected by focusing on any one of them or combining a plurality of them from the object.

[0039] Round shape

[0040] Size: 100~200μm

[0041] Transparent objects with rings in their outlines (areas of roughly uniform brightness in the image)

[0042] Has structure (texture) inside

[0043] Alternatively, instead of using detection based on object extraction, a method for detection using various characteristics of the image, such as light and dark characteristics of the entire image, extraction of feature points, or model comparison, may be used through deep learning or the like.

[0044] The image recording unit 42 records only the images of the wells detected as having cells by the detection unit 41 among the images captured by the imaging unit 40 for each well in the recording unit 6. The recording unit 6 is, for example, a hard disk drive (HDD), a disk such as a floppy disk (FD), an optical disk such as a compact disk (CD), a DVD, an optical magneto-optical disk such as an MO, a solid state drive (SSD), a memory card, a flash memory such as a USB memory, a magnetic tape, or other recording media. In addition, as the recording unit 6, a recording medium such as a network attached storage (NAS) or a cloud storage may also be used.

[0045] In addition, the timing for detecting the presence of cells by the detection unit 41 may be the start of time-lapse photography, the middle of time-lapse photography, or both, but it is desirable to detect the presence of cells at each time-lapse cycle. Thus, the presence of cells is detected every time photography is performed at a predetermined time-lapse cycle, and only the images of the wells detected as having cells are recorded in the image recording unit 42, so that only the images of the minimum number of wells are recorded in the image recording unit 42.

[0046] 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 the user to the culture control unit 4 and the imaging control unit 5, and displays images captured by the camera 30 and images read from the recording unit 6, etc.

[0047] The overall control unit 8 controls the modules in the device and the overall device. In addition, the overall control unit 8 has an interface with the outside of the device. In addition, the overall control unit 8 can also be equipped with software for observing and evaluating embryos. Thus, the overall control unit 8 configures the image to the input / display unit 7 in an easy-to-understand manner for analysis and display, which can assist the user's cultivation evaluation business.

[0048] Next, a time-lapse imaging method based on the culture device 1 having the above-described structure will be described. Figure 5 : is a flowchart showing the time-lapse imaging process performed by the culture apparatus 1 .

[0049] (S100) When the culture container (culture dish) 10 is placed in the culture device 1 (culture dish is placed), the process starts.

[0050] ( S101 ) Automatic detection (auto focusing) of the XY coordinates and the Z-axis focal center of all the wells 11 is performed using marks or the like provided in the culture container 10 .

[0051] ( S102 ) The imaging unit 40 moves the imaging section 3 to the first hole 11 .

[0052] ( S103 ) The imaging unit 40 images the hole 11 through the imaging section 3 . The detection unit 41 detects the hole 11 from the image captured by the imaging unit 40 .

[0053] ( S104 ) The detection unit 41 automatically detects the presence or absence of the embryo in the hole 11 .

[0054] (S105) The detection unit 41 determines the presence or absence of an embryo. If there is no embryo in the well 11, the process proceeds to step (S108).

[0055] (S106) When there is an embryo in the hole 11, the imaging unit 40 captures a plurality of slice images of the hole 11 shifted forward and backward from the Z-axis focal position. However, when slice images are not required, only the focal center may be captured.

[0056] ( S107 ) The image recording means 42 stores the image of the hole 11 in the recording unit 6 .

[0057] (S108) It is determined whether the imaging has been completed for all the holes 11. If the imaging has not been completed, the process proceeds to step (S103) in order to image the next hole 11.

[0058] (S109) When imaging of all holes 11 is completed, the process ends.

[0059] (S110) In the time-lapse photography after the second time-lapse period, the process starts from step (S102).

[0060] In this example, the method of first detecting the hole 11 and then detecting the cells is adopted, but the following method can also be adopted: without detecting the hole 11 itself, the regularity of the hole arrangement is used to directly search for cells and detect the presence of cells.

[0061] In addition, in step (S101), a method of detecting the X, Y, and Z coordinates of all the wells 11 is adopted, but the following method may also be adopted: a plurality of segmented images consisting of a plurality of wells 11 are taken in the entire culture container 10 and they are stitched together, or a high-resolution camera is used to take a large-scale image of all the wells 11 in one image, and thereafter the image is segmented and the image of each well is extracted to detect the presence or absence of cells.

[0062] In addition, if Figure 3 As shown, the culture device 1 in this embodiment can be configured as follows, wherein the shooting control unit 5 comprises: an experience recording unit 43, which records the experience or non-experience of cells in all holes 11 of the culture container 10 in each time-lapse shooting cycle; and a notification unit 44, which notifies when the detection result of the detection unit 41 is different from the previous detection result.

[0063] The history recording unit 43 records the presence or absence of the history of the cells in all the wells 11 of the culture container 10 in the recording unit 6 for each time-lapse imaging cycle based on the detection result of the detection unit 41. Thus, the presence or absence of the history of the cells in all the wells 11 of the culture container 10 is recorded by the history recording unit 43 for each time-lapse imaging cycle, so that the image arrangement and history management in the timeline display can be flexibly utilized based on the presence or absence of the history of the cells in all the wells 11 of the culture container 10 for each time-lapse imaging cycle.

[0064] Figure 6 2 are diagrams showing examples of real-time image display of all holes, (A) is a diagram showing a conventional display example, and (B) is a diagram showing an example of display of the present invention. Figure 6 As shown in (A), in the past, images of all the wells 11 of the culture container 10 were recorded, and images of all the wells 11 were displayed regardless of whether there were embryos in each well 11. On the other hand, in the present invention, only images of the wells 11 with embryos among all the wells 11 of the culture container 10 are recorded in the recording unit 6, and the history of the presence or absence of embryos in all the wells 11 is also recorded by the history recording unit 43. Therefore, based on the history of the presence or absence of the embryos, Figure 6 (B) shows only the image of the well 11 with the embryo, and does not show the well 11 without the embryo. Therefore, when the embryo is compared, observed or evaluated by observation software or evaluation software for the image recorded in the recording unit 6, it is easy to see clearly at a glance, and it is easy to grasp the state and process, and the cultivation observation and evaluation can be performed efficiently.

[0065] Figure 7 1 and 2 are diagrams showing time line display examples of a plurality of wells, wherein (A) is a diagram showing a conventional display example, and (B) is a diagram showing a display example of the present invention. Figure 7An image showing a plurality of holes arranged horizontally and holes arranged vertically for each delay period is shown. In cell culture, embryos are sometimes extracted from the hole 11 for cryopreservation or transplantation during the culture. In addition, embryos are sometimes taken out of the culture container (culture dish) 10 for external microscopic observation or culture medium replacement. In this case, in the past, as Figure 7 As shown in (A), all the holes 11 with embryos extracted are also displayed. On the other hand, in the present invention, based on the history of the embryos recorded by the history recording unit 43, as shown in FIG. Figure 7 As shown in (B), the wells 11 with embryos extracted for cryopreservation or transplantation during culture are not displayed. In addition, when the wells 11 are taken out to the outside together with the culture container 10 for external microscopic observation or culture medium replacement, they are not displayed. Then, when the culture container 10 is re-arranged, the wells 11 with embryos are displayed again.

[0066] Figure 8 is a diagram showing a comparative example of displaying a dynamic image by playing back a plurality of time-lapse shots frame by frame. Figure 8 As shown, in the past, owing to read the time-lapse image data of the embryo of regulation from the storage device and display continuously, the image 81 of only hole without cell is mixed.Therefore, the image of not having embryo appears momentarily when dynamic image is displayed, becomes discontinuous and the dynamic image display that is difficult to observe.But, in the culture device 1 with the time-lapse shooting function in the present embodiment, the image 81 of only hole without embryo is excluded, therefore the dynamic image that is easy to observe without discontinuity can be obtained.That is, in the culture device 1 with the time-lapse shooting function in the present embodiment, by continuously displaying the time-lapse image, the growth state of cell can be visualized continuously, and the process of change, growth can be grasped easily and understandably.

[0067] The notification unit 44 notifies when the detection result of the detection unit 41 is different from the previous detection result. The notification unit 44 may be, for example, a notification buzzer sounding, a notification lamp lighting, or abnormality notification display on a display. Fig. 9 4 shows an example of abnormality notification display. In the case where the detection result of the detection unit 41 is different from the previous detection result, for example, Fig. 9As shown, the notification unit 44 displays an abnormal notification on the display through a dialog box, thereby prompting the user of the culture device 1 to pay attention and confirm whether shooting is required. At this time, the shooting action of the corresponding hole 11 can also be set to a standby state, and the operator can set it to "OK" when the cryopreservation or transplantation is completed and the presence of the embryo can be confirmed, and set it to "CANCEL" when there may be an error in the judgment, indicating that another shooting is performed. In addition, as another embodiment, the detection result of the presence or absence of cells can also be notified to the user, and information notification can be provided on whether it is necessary to confirm shooting, abnormal setting of the culture dish, etc.

[0068] As described above, in the culture device 1 of the present embodiment, only the image of the well 11 in which cells are present can be automatically recorded for each well 11 of the culture container 10, and the usage of the recording unit 6 for recording images can be reduced. In addition, since the images of unnecessary wells 11 in which cells are not present are not recorded, the degree of freedom in setting the cycle and schedule of photographing increases, and culture based on higher-precision image observation can be performed. Furthermore, when the recorded images are displayed, there are no images of unnecessary wells in which cells are not present, so that efficient evaluation can be performed.

[0069] Industrial Applicability

[0070] The present invention is useful as a culture device and a culture method with a time-lapse photography function for culturing cells such as embryos and performing time-lapse photography.

[0071] Description of Reference Numerals

[0072] 1Cultivation device with time-lapse photography function

[0073] 2 Chamber

[0074] 3. Filming Department

[0075] 4. Cultivation Control Department

[0076] 5. Shooting control unit

[0077] 6 Records Department

[0078] 7 Input / display unit

[0079] 8 Overall Control Department

[0080] 10. Culture container

[0081] 11 holes

[0082] 30 Camera

[0083] 31 lighting

[0084] 32 Objective lens

[0085] 33 Movable parts

[0086] 40 Shooting Units

[0087] 41 Detection Unit

[0088] 42 Image recording unit

[0089] 43 Experience Record Unit

[0090] 44 Notification Unit

Claims

1. A culture device with a time-lapse photography function, which arranges cells in each well of a culture container having at least one well and cultures them, and performs time-lapse photography, the culture device with a time-lapse photography function comprising: A photographing unit, which photographs each hole of the culture container at a prescribed delayed photographing cycle; A detection unit for detecting the presence or absence of cells in each well of the culture container; as well as An image recording unit records images of the wells detected as containing cells by the detection unit, among the images captured by the imaging unit for each well.

2. The culture device with time-lapse photography function according to claim 1, wherein: The detection unit detects the presence or absence of the cells by analyzing the image captured by the imaging unit for each well.

3. The culture device with time-lapse photography function according to claim 1 or 2, wherein: The detection unit detects the presence or absence of cells in each time-lapse shooting cycle.

4. The culture device with a time-lapse photography function according to claim 1 or 2, further comprising a notification unit for notifying when the detection result of the detection unit is different from the previous detection result. 5 . The culture device with a time-lapse photography function according to claim 1 , further comprising a history recording unit for recording the presence or absence of cells in all wells of the culture container in each time-lapse photography cycle.

6. A culture method, which comprises disposing cells in each well of a culture container having at least one well and culturing the cells, and performing time-lapse photography, the culture method comprising: Taking pictures of each hole of the culture container at a prescribed time-lapse shooting cycle; Detecting the presence or absence of cells in each well of the culture container; as well as Among the images captured for each of the wells, images of wells in which cells were detected were recorded.

Images