Automated culturing apparatus and automated culturing method

Abstract

To achieve high-efficiency culturing.SOLUTION: According to one embodiment, an automated culturing apparatus includes a plurality of incubators, a liquid control unit, an imaging unit, and a condition control unit. The plurality of incubators is configured to store culture containers in which a cell or a biological tissue is cultured. The liquid control unit is configured to control delivery and discharge of a cell suspension and a medium for the culture containers stored in each of the incubators. The imaging unit is configured to capture an image of the cell or the biological tissue in the culture containers in the incubators. The condition control unit is configured to independently control a culturing condition of the cell or the biological tissue for each of the incubators.SELECTED DRAWING: Figure 1

Description

[Technical field]

[0001] The embodiments disclosed in the present specification and drawings relate to an automated culture device and an automated culture method. [Background technology]

[0002] With the advancement of regenerative medicine, cell culture such as induced pluripotent stem cells (iPS cells) has been attracting attention. In cell culture, reproducibility often varies depending on pipetting technique, and variability between operators is a problem. For this reason, the use of automated culture devices that enable cell culture without human intervention is being considered, but because it is not possible to set different culture conditions for each culture vessel, it is difficult to set optimal culture conditions according to the growth rate of each individual cell. [Prior art documents] [Patent documents]

[0003] [Patent Document 1] JP 2020-5553 A Summary of the Invention [Problem to be solved by the invention]

[0004] One of the problems to be solved by the embodiments disclosed in this specification and the drawings is to realize highly efficient culture. However, the problems to be solved by the embodiments disclosed in this specification and the drawings are not limited to the above problem. Problems corresponding to the effects of each configuration shown in the embodiments described later can also be positioned as other problems. [Means for solving the problem]

[0005] The automated culture device according to this embodiment includes a plurality of incubators, a liquid control unit, an imaging unit, and a condition control unit. The plurality of incubators house culture vessels for culturing cells or biological tissues. The liquid control unit controls the supply and discharge of cell suspension and culture medium to and from the culture vessels housed in each of the plurality of incubators. The imaging unit images the cells or the biological tissues in the culture vessels in the plurality of incubators. The condition control unit controls the culture conditions of the cells or the biological tissues independently for each incubator. [Brief description of the drawings]

[0006] [Figure 1] FIG. 1 is a block diagram showing the automatic culture device according to this embodiment. [Diagram 2] FIG. 2 is a diagram showing a first configuration example of the imaging section according to the present embodiment. [Diagram 3] FIG. 3 is a diagram showing a second configuration example of the imaging section according to the present embodiment. [Figure 4] FIG. 4 is a flowchart showing the culture process of the automatic culture device according to this embodiment. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0007] Hereinafter, an automatic culture device and an automatic culture method according to the present embodiment will be described with reference to the drawings. In the following embodiments, parts with the same reference numerals perform similar operations, and duplicated descriptions will be omitted as appropriate.

[0008] A conceptual diagram of the automatic culture device according to this embodiment will be described with reference to the block diagram of FIG. The automatic culture device 1 according to this embodiment includes a processing circuit 10, a plurality of incubators 11, a plurality of liquid supply / discharge units 12, a plurality of culture condition setting units 13, an imaging unit 14, and a memory 15. The incubators 11, the liquid supply / discharge units 12, and the culture condition setting units 13 are in one-to-one correspondence with each other. That is, in the example of FIG. 1, the liquid supply / discharge units 12 and the culture condition setting units 13 are connected to four incubators 11, respectively. However, this is not limiting, and it is sufficient that there are a plurality of incubators 11, and it is sufficient that a plurality of incubators 11 are arranged according to the specifications of the automatic culture device 1.

[0009] The processing circuit 10 is a processor, and includes a liquid control function 101 , a judgment function 102 , and a condition control function 103 . The liquid control function 101 controls, via the liquid supply / discharge unit 12, the supply / discharge, amount of liquid supply, intervals of liquid supply, etc. of cell suspension or culture medium to / from culture containers for culturing cells or biological tissues housed in each of the multiple incubators 11.

[0010] In the following, an example of culturing cells will be described, but the culture process can be similarly performed for biological tissue. Examples of cells include various cultured cell lines such as Chinese hamster ovary-derived CHO cells, mouse connective tissue L929 cells, mouse skeletal muscle myoblasts (C2C12 cells), human fetal lung-derived normal diploid fibroblasts (TIG-3 cells), human fetal kidney-derived cells (HEK293 cells), human alveolar basal epithelial adenocarcinoma-derived A549 cells, and human cervical carcinoma-derived HeLa cells, as well as epithelial cells and endothelial cells that constitute various tissues and organs in the body, skeletal muscle cells that exhibit contractility, smooth muscle cells, cardiac muscle cells, neuron cells that constitute the nervous system, glial cells, and fibroblasts, hepatic parenchymal cells, non-parenchymal liver cells, and adipocytes that are involved in the metabolism of the body, and cells with differentiation potential such as iPS cells, embryonic stem (ES) cells, embryonic germ (EG) cells, embryonic carcinoma (EC) cells, mesenchymal stem cells, hepatic stem cells, pancreatic stem cells, skin stem cells, muscle stem cells, and germline stem cells, or precursor cells of various tissues, and further cells induced to differentiate therefrom. The culture vessel is assumed to be, for example, a plastic vessel called a dish, but any vessel that is chemically stable and capable of culturing desired cells may be used.

[0011] The determination function 102 determines the growth state of the cells. Specifically, the determination function 102 determines the growth state, for example, the number of cell colonies, the size of the colonies, and the degree of cell growth, such as confluence. Confluence is the proportion of cells covering the bottom surface of the culture vessel. The condition control function 103 performs control so that the culture conditions for the cells or the biological tissue are set independently for each incubator, for example, based on an image captured by the imaging unit 14 .

[0012] The incubator 11 is a thermostatic device that keeps the temperature, humidity, and gas composition such as carbon dioxide concentration and oxygen concentration constant. A culture vessel such as a dish can be stored in the incubator 11. Although it is assumed that a single culture vessel is stored in one incubator 11, multiple culture vessels may be stored. The incubator 11 has a structure that allows the image of the culture vessel inside to be captured by the imaging unit 14 described later. For example, a transparent acrylic panel may be provided in a part of the housing of the incubator 11 so that the culture vessel inside the incubator 11 can be captured. Alternatively, the incubator 11 may be provided with an opening / closing mechanism such as a window or a lid, and the incubator 11 may be structured so that the image can be captured by the imaging unit 14 with a part of the housing open.

[0013] The liquid supply / discharge unit 12 is connected to the incubator 11 via a tubing member such as a tube. The liquid supply / discharge unit 12 supplies a cell suspension or a culture medium through piping to a culture vessel in the incubator 11. In a culture medium replacement process, the liquid supply / discharge unit 12 sucks the culture medium from the culture vessel and discharges it to the outside via, for example, a pipetting unit (not shown) and a tube arranged in the incubator 11, and supplies new culture medium to the culture vessel. In a cell recovery process, the liquid supply / discharge unit 12 recovers cells from the culture vessel via the pipetting unit and a tube.

[0014] The culture condition setting unit 13 is connected to the incubator 11 via tubular members such as tubes, a heat plate, etc. According to control instructions from the condition control function 103, the culture condition setting unit 13 sets the temperature, humidity, and gas composition such as carbon dioxide or oxygen in the incubator 11.

[0015] The imaging unit 14 is, for example, a phase-contrast microscope, and captures images of cells in a culture vessel in each incubator 11. This allows the user to observe the cells in the culture vessel and check the cell growth state, such as the number of colonies, the size of the colonies, and the confluence. Note that the imaging unit 14 is not limited to a phase-contrast microscope, and may be an optical microscope such as a bright-field microscope or a fluorescent microscope, or may be an electron microscope or an optical camera.

[0016] The memory 15 is a non-volatile memory such as a hard disk drive (HDD) or a solid state drive (SSD), and stores data such as images of the inside of the culture vessel captured by the imaging unit 14 and the growth state of cells in the culture vessels stored in each incubator 11.

[0017] Although not shown, the automatic culture device 1 may further include a display for displaying captured images and data related to the imaging conditions, and an input / output interface for outputting the data to the outside and for acquiring input from the user.

[0018] Next, a first configuration example of the imaging unit 14 according to this embodiment is shown in FIG. FIG. 2 is a conceptual diagram showing the layout relationship between the imaging unit 14 and a plurality of incubators 11. As shown in FIG. The imaging unit 14 is disposed so as to be movable in one dimension along a first shaft 20 extending along the arrangement direction of the multiple incubators 11. For example, the imaging unit 14 may be connected to the first shaft 20 via a linear bush (not shown) or the like, and linearly guided along the extension direction of the first shaft 20. Note that the first shaft 20 is disposed above the placement surface of the incubators 11 so that the imaging unit 14 does not collide with the incubators 11. This allows the imaging unit 14 to capture images of the inside of the culture containers in each incubator 11 individually.

[0019] Next, a second configuration example of the imaging unit 14 according to the present embodiment is shown in FIG. 2 shows an example in which a plurality of incubators 11 are arranged in a line, while FIG. 3 shows an example of the configuration of the imaging unit 14 in which a plurality of incubators 11 are arranged two-dimensionally.

[0020] Here, eight incubators 11 are illustrated in a two-dimensional arrangement. The first shaft 20 is connected to two second shafts 22 via a moving section 21. The moving section 21 may be formed with a structure capable of linear guiding, such as a linear bush. When the incubator 11 is viewed from above, the second shaft 22 extends in a direction perpendicular to the extension direction of the first shaft 20 outside the arrangement area of ​​the incubator 11, and the moving section 21 is connected to the second shaft 22. In addition, the first shaft 20, the moving section 21, and the second shaft 22 are arranged above the arrangement surface of the incubator 11 so that the imaging section 14 does not collide with the incubator 11.

[0021] This allows the imaging unit 14 to be freely moved on a two-dimensional plane parallel to the surface on which the incubators 11 are arranged, so that even if the incubators 11 are arranged two-dimensionally, images of the culture vessels in each incubator 11 can be individually captured. Note that, without being limited to the second configuration example shown in Fig. 3, an arm that is movable in three dimensions may be placed above the incubator 11, and the imaging unit 14 may be connected to the tip of the arm, so that images of the culture vessels can be individually captured.

[0022] 2 and 3, the imaging unit 14 captures images from above each incubator 11, but the imaging unit 14 may be disposed below each incubator 11 and capture images of the culture containers in the incubators 11 from below. In this case, the incubator 11 may be formed in a structure that allows imaging from below the housing.

[0023] Next, the culture process of the automatic culture device 1 according to this embodiment will be described with reference to the flowchart of FIG.

[0024] In step SA1, the processing circuit 10 sets the initial values ​​of the culture conditions for each incubator 11 independently by the condition control function 103. Specifically, after one or more culture containers seeded with cells are stored in each incubator 11, the processing circuit 10 sets the culture conditions, specifically, temperature, humidity, and carbon dioxide concentration, via the culture condition setting unit 13 by the condition control function 103. For example, one incubator 11 is set to a temperature of 37°C, a humidity of 95%, and a carbon dioxide concentration of 5%, and another incubator 11 has its temperature changed and is set to a temperature of 36.5°C, a humidity of 95%, and a carbon dioxide concentration of 5%. In this way, different culture conditions may be set for each incubator 11. The initial values ​​of the culture conditions may be set differently for each incubator 11 based on empirically determined values ​​according to the type of cells to be cultured. It is also possible to set the same culture conditions in a plurality of incubators 11. For example, the same initial values ​​may be set for each of the plurality of incubators 11, two at a time.

[0025] In step SA2, the automatic culture device 1 starts culture based on the initial values ​​of the culture conditions that have been set. In step SA3, the imaging unit 14 captures images of the culture vessels in each incubator 11 at predetermined intervals to obtain images of each culture vessel. For example, with the configuration shown in Fig. 2 or 3, the imaging unit 14 captures images of the cells in the culture vessels while patrolling each incubator 11. The predetermined intervals may be, for example, once a day at a fixed time, or may be multiple time intervals throughout the day, such as 8:00, 12:00, and 16:00, or may be an interval at which cell growth can be appropriately observed, such as every two days.

[0026] In step SA4, the processing circuit 10 determines the growth state of the cells using the determination function 102 based on the image captured by the imaging unit 14. For example, the processing circuit 10 may calculate the number of cell colonies, the size of the colonies, and the confluence by analyzing the image using the determination function 102. In step SA5, the processing circuit 10 judges whether the cell growth state is according to the culture schedule by the judgment function 102. For example, if the cell growth state is immediately after the start of cell culture, and if the image shows that the cells have adhered to the culture vessel and divided, it is judged that the cell growth state is according to the schedule. If a certain number of days have passed since the start of culture, the growth prediction value for the date specified in the schedule is set as a threshold, and if at least one of the cell colony number, colony size, and confluence calculated by image analysis is equal to or greater than the threshold, it is judged that the cell growth state is according to the schedule. If the cell growth status is as scheduled, proceed to step SA6, and if the cell growth status is not as scheduled, proceed to step SA7.

[0027] In step SA6, the processing circuit 10 controls the liquid supply / discharge unit 12 by the liquid control function 101 to exchange the culture medium. The liquid supply / discharge unit 12 sucks out (collects) the culture medium in the culture vessel in the incubator 11, and sends new culture medium to the culture vessel. It is assumed that the culture medium exchange is performed without removing the culture vessel from the incubator 11. Specifically, the liquid supply / discharge unit 12 is connected to a pipette for collecting culture medium and a pipette for adding culture medium in the incubator 11, and each pipette is operated under the control of the liquid control function 101 to collect and add culture medium in the culture vessel. Furthermore, the processing circuit 10 may control the amount of culture medium fed based on the cell growth state using the liquid control function 101. For example, if the cells are growing quickly, a process may be performed in which the amount of medium fed is increased to increase the amount of medium. Furthermore, by repeating the processes from step SA3 to step SA6, the processing circuit 10 uses the liquid control function 101 to control the interval at which the culture medium is fed based on the cell growth state.

[0028] In step SA7, the processing circuit 10 controls the culture condition setting unit 13 by the condition control function 103, and changes the culture conditions (temperature, humidity, gas composition) for each incubator 11 that houses a culture vessel for cells whose growth is not progressing according to schedule. For example, the culture conditions set for the incubator 11 in which the cells are growing the fastest are set as the culture conditions for the other incubators 11. Specifically, the processing circuit 10 controls the culture condition setting unit 13 by the condition control function 103, and applies the temperature, humidity, and gas composition set for the incubator 11 that houses the culture vessel with the largest colony size, for example, to the other incubators 11. This is expected to provide management that accelerates the growth of cells housed in each incubator 11, although the degree of growth varies depending on the individual cells.

[0029] On the other hand, if the cells are growing faster than scheduled, the culture conditions set for the incubator 11 in which the cells are growing the slowest can be set as the culture conditions for the other incubators 11. As a result, although there are individual differences between cells, as in the case of accelerating growth, it is expected that the growth of the cells housed in each incubator 11 will be slowed down, and this can be applied to needs such as culturing high-quality cells in a small number of colonies.

[0030] In step SA8, the processing circuit 10 judges whether the culture has been completed or not by the judgment function 102 based on the image captured by the imaging unit 14. For example, when it can be judged based on the image that the cells have reached a so-called subconfluence, in which the occupancy rate of the area of ​​the medium is about 70 to 80%, it may be judged that the culture has been completed. If the culture is completed, the process proceeds to step SA9, and if the culture is not completed, the process returns to step SA3 and the same process is repeated. In step SA9, the culture container is removed from the incubator 11, and the cells are collected. This completes the culture process of the automatic culture device 1. In the culture process shown in Figure 4, an example is shown in which the culture medium is replaced when the cell growth state is as scheduled, but this is not limited to the above. The processing circuit 10 may perform culture medium replacement via the liquid supply and discharge unit 12 using the liquid control function 101 at any timing based on a user instruction, etc.

[0031] In this embodiment, it is assumed that there is one imaging unit 14, and the imaging unit 14 is moved to capture images of the culture vessels accommodated in each incubator 11, but the imaging unit 14 may be fixed and installed exclusively for each incubator 11. In other words, if there are eight incubators 11, eight imaging units 14 may be installed.

[0032] According to the present embodiment described above, the automatic culture device has a plurality of incubators that house culture vessels, and the image capturing unit moves to capture images of the culture vessels in each incubator. It is possible to control the incubators so that culture conditions can be set independently. This allows automatic culturing in multiple incubators, reducing variations in culturing caused by operators. Furthermore, the imaging unit can capture images of each incubator, allowing independent control while optimizing the culture conditions according to the growth rate of the cells. This allows for highly efficient culturing.

[0033] The term "processor" used in the above description means a circuit such as a central processing unit (CPU), a graphics processing unit (GPU), an application specific integrated circuit (ASIC), a programmable logic device (e.g., a simple programmable logic device (SPLD), a complex programmable logic device (CPLD), and a field programmable gate array (FPGA)). When the processor is a CPU, for example, the processor realizes a function by reading and executing a program stored in a memory circuit. On the other hand, when the processor is an ASIC, for example, instead of storing the program in a memory circuit, the function is directly incorporated as a logic circuit in the circuit of the processor. Note that each processor in this embodiment is not limited to being configured as a single circuit for each processor, but may be configured as one processor by combining multiple independent circuits to realize the function. Furthermore, multiple components in the figure may be integrated into one processor to realize the function. In other words, efficient and highly accurate inspection can be performed.

[0034] Although some embodiments have been described, these embodiments are presented as examples and are not intended to limit the scope of the invention. These embodiments can be implemented in various other forms, and various omissions, substitutions, modifications, and combinations of embodiments can be made without departing from the spirit of the invention. These embodiments and their modifications are included in the scope of the invention and its equivalents as described in the claims, as well as in the scope and spirit of the invention. [Explanation of symbols]

[0035] 10 Processing circuit 11. Incubator 12 Liquid supply and discharge section 13 Culture condition setting section 14 Imaging unit 15 Memory 20 1st Shaft 21 Mobile section 22 Second Shaft 101 Liquid Control Function 102 Judgment function 103 Conditional Control Function

Claims

1. A plurality of incubators each accommodating a culture vessel for culturing cells or biological tissues; a liquid control unit that controls the supply and discharge of cell suspension and culture medium to and from the culture vessels accommodated in each of the plurality of incubators; an imaging unit that images the cells or the biological tissues in the culture containers in the plurality of incubators; A condition control unit that controls the culture conditions of the cells or the biological tissue independently for each incubator; An automatic culture device comprising:

2. The automatic culture device according to claim 1 , wherein the imaging unit images the cells or the biological tissues in the culture containers in each incubator by moving in a one-dimensional direction along an arrangement direction of the plurality of incubators.

3. The automatic culture device according to claim 1 , wherein the imaging unit captures images of the cells or the biological tissues in the culture containers in each incubator by moving on a two-dimensional plane parallel to a surface on which the incubators are arranged.

4. A determination unit for determining a growth state of the cells or the biological tissue in the culture vessel, The automatic culture device according to claim 1 , wherein the condition control unit controls at least one of a temperature, a humidity, and a gas composition in each incubator based on the growth state.

5. A determination unit for determining a growth state of the cells or the biological tissue in the culture vessel, The automatic culture device according to claim 1 , wherein the liquid control unit controls at least one of an amount of the culture medium and a liquid feeding interval based on the growth state.

6. The automatic culture device according to claim 1 , wherein the condition control unit sets the culture conditions set in the incubator in which the cells or the biological tissue in the culture container are growing the fastest as the culture conditions in the other incubators.

7. The automatic culture device according to claim 1 , wherein the condition control unit sets the culture conditions set in the incubator in which the growth state of the cells or the biological tissue in the culture container is the slowest as the culture conditions in the other incubators.

8. The automatic culture device according to claim 4 , wherein the growth state is determined based on at least one of the number of colonies, the size of the colonies, and the confluence of the cells or the biological tissue.

9. The automated culture device according to claim 1 , wherein each of the multiple incubators accommodates a single culture vessel.

10. Controlling the supply and discharge of cell suspensions and culture media to and from culture containers for culturing cells or biological tissues housed in each of the multiple incubators; taking images of the cells or the biological tissues in the culture containers in the plurality of incubators; An automated culture method for independently controlling culture conditions for the cells or biological tissue for each incubator.

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