Cell culture device and cell culture method

Abstract

The purpose of the present invention is to prevent detachment of cells adhering to a carrier and to suppress a decrease in cell culture efficiency. A cell culture device (100) for culturing cells is provided with: a culture tank (10) for storing a culture solution (50); a partition unit (20) that partitions the culture tank into at least a first space (R1) and a second space (R2); and a stirring unit (30) that stirs the culture solution, the first space (R1) is a space in which a carrier (40) to which cells adhere is accommodated, the second space (R2) is a space in which the stirring unit (30) is provided, and the partition unit (20) is configured so that the culture solution can pass through and the carrier cannot enter the second space from the first space.

Description

Technical Field

[0001] This invention relates to cell culture apparatus and cell culture methods. Background Technology

[0002] In recent years, in order to achieve the United Nations Sustainable Development Goals (SDGs), truly sustainable food production systems are being developed in countries around the world, among which the development of "cellular foods" is expanding. Furthermore, as background technology in this field, for example, Patent Document 1 describes a liquid culture method for tissue cell cultures that uses indirect aeration via a tubular gas-permeable membrane through a permeator. As an apparatus for implementing this method, Patent Document 1 discloses a structure in which the tubular permeator forms a vertical central tube within a container for the cell culture apparatus. Moreover, a stirrer is fixed to the lower part of the permeator, and by rotating the stirrer, gas diffuses in the liquid.

[0003] Existing technical documents

[0004] Patent documents

[0005] Patent Document 1: Japanese Patent Publication No. 03-021149. Summary of the Invention

[0006] The problem the invention aims to solve

[0007] However, the upper part of the tubular permeator in the cell culture device described in Patent Document 1 is open, so cell cultures may enter the interior from the upper part of the permeator. If cell cultures bound to microcarriers enter the interior of the permeator, the cell cultures will be sheared by the agitator. As a result, cells may detach from the microcarriers or be destroyed, potentially leading to a decrease in cell culture efficiency.

[0008] This invention was made in view of such actual conditions, and its purpose is to prevent cells attached to the carrier from peeling off and to inhibit the reduction of cell culture efficiency.

[0009] Solution for solving the problem

[0010] To achieve the above objectives, a first aspect of the present invention is a cell culture apparatus for culturing cells, characterized in that it comprises: a culture tank for storing a culture medium; a partition unit for dividing the culture tank into at least a first space and a second space; and a stirring unit for stirring the culture medium, wherein the first space is a space for accommodating a carrier to which the cells are attached, the second space is a space in which the stirring unit is disposed, and the partition unit is configured such that the culture medium can pass through, and the carrier cannot intrude from the first space into the second space.

[0011] Furthermore, in order to achieve the above objectives, a second aspect of the present invention is a cell culture method, characterized in that it uses the cell culture apparatus described in the first aspect above. The cell culture method includes the steps of: introducing a carrier into the first space and supplying the culture medium to the culture tank; driving the stirring unit to stir the culture medium; removing a portion of the carrier from the first space; and introducing a new carrier into the first space and mixing it with the remaining carrier in the first space.

[0012] Invention Effects

[0013] According to the present invention, cell detachment from the carrier can be prevented, thus inhibiting the reduction of cell culture efficiency. Furthermore, other issues, structures, and effects beyond those described above will become clearer through the following description of embodiments. Attached Figure Description

[0014] Figure 1 This is a schematic diagram showing the overall structure of the cell culture apparatus and the longitudinal section of the culture tank according to the first embodiment of the present invention.

[0015] Figure 2 It is a schematic diagram showing a cross-section of the culture tank.

[0016] Figure 3 It is a three-dimensional view of the cylindrical component set inside the culture tank.

[0017] Figure 4 This is a flowchart illustrating the steps involved in cell culture.

[0018] Figure 5 This is a flowchart illustrating the steps of carrier recovery and replenishment.

[0019] Figure 6 This is a schematic diagram showing the overall structure of the cell culture apparatus and the longitudinal section of the culture tank according to the second embodiment of the present invention.

[0020] Figure 7 It is a three-dimensional view of the cylindrical component set inside the culture tank.

[0021] Figure 8A This is a diagram showing the state of the cylindrical component after its diameter has been expanded.

[0022] Figure 8B This is a diagram showing the state of the cylindrical component after its diameter has been reduced.

[0023] Figure 9 This is a flowchart illustrating the steps of recycling and replenishing the carrier according to the second embodiment.

[0024] Figure 10A This is a schematic diagram showing a longitudinal section of the cell culture apparatus of Modified Example 1.

[0025] Figure 10B It is shown schematically. Figure 10A The diagram shows a cross-sectional view of the culture tank.

[0026] Figure 11A This is a schematic cross-sectional view of the cell culture apparatus of Modified Example 2.

[0027] Figure 11B yes Figure 11A An enlarged view of part B shown.

[0028] Figure 12A This is a diagram showing the changes in the shape of the culture tank.

[0029] Figure 12B This is a diagram showing the changes in the shape of the culture tank.

[0030] Figure 12C This is a diagram showing the changes in the shape of the culture tank.

[0031] Figure 12D This is a diagram showing the changes in the shape of the culture tank.

[0032] Figure 13 This is a schematic diagram showing a longitudinal section of the cell culture apparatus of Modified Example 4.

[0033] Figure 14 This is a schematic diagram showing a longitudinal section of the cell culture apparatus of Modified Example 5.

[0034] Figure 15A This is a top view of the basket.

[0035] Figure 15B This is a longitudinal sectional view of the basket.

[0036] Figure 16A This is a top view of the basket in variation example 6.

[0037] Figure 16B yes Figure 16A A cross-sectional view of XVIB-XVIB. Detailed Implementation

[0038] The following describes embodiments of the present invention using examples of animal cell culture, with reference to the accompanying drawings.

[0039] (First Implementation)

[0040] Figure 1 This is a schematic diagram showing the overall structure of the cell culture apparatus 100 according to the first embodiment of the present invention and a longitudinal section of the culture tank 10. Figure 2 This is a schematic cross-sectional view of the culture tank 10. Figure 3This is a perspective view of the cylindrical component 20 installed inside the culture tank 10. Additionally, in Figure 2 In the middle, the following was omitted. Figure 1 The illustration shows the stirring unit 30.

[0041] like Figure 1 As shown, the cell culture apparatus 100 of this embodiment mainly includes a culture tank 10, a cylindrical component 20, and a stirring unit 30.

[0042] The culture tank 10 has a bottomed cylindrical container formed by a carcass portion 11 and a bottom 12, and a disc-shaped cover 13 covering the upper part of the carcass portion 11. The culture tank 10 stores a culture medium 50 inside. The culture tank 10 is made of, for example, stainless steel, but may also be made of other materials (glass, silicone, etc.). Furthermore, when the culture tank 10 is made of silicone, it is sometimes referred to as a culture bag.

[0043] The cover 13 is tightly fitted to the body portion 11 via a gasket (not shown). This seals the interior of the culture tank 10. Supply pipes 15 and 16 are provided on the cover 13. The carrier 40 (described later) and culture medium 50, supplied from the supply line, are introduced into the culture tank 10 via the supply pipes 15 and 16. In addition to the supply pipes 15 and 16, the cover 13 is also provided with various tubes, hoses, nozzles, etc. (not shown). These are used for various purposes such as connecting various sensors, supplying oxygen, or performing sampling.

[0044] A solenoid valve 60 is installed at the bottom 12 of the culture tank 10. Of course, there can be multiple solenoid valves 60. Furthermore, level switches 65 and 66 are installed in the body 11 of the culture tank 10 to detect the liquid level of the culture medium 50. Level switch 65 detects whether the liquid level of the culture medium 50 is at the appropriate level PL. When the liquid level of the culture medium 50 is higher than the appropriate level PL, level switch 65 outputs a high level signal. Here, the appropriate level PL refers to the position where the culture medium 50 approximately fills the culture tank 10.

[0045] On the other hand, the level switch 66 detects whether the liquid level of the culture medium 50 is at a specific level SL. If the liquid level of the culture medium 50 is lower than the specific level SL, the level switch 66 outputs a low level signal. Here, in this embodiment, the specific level SL is defined as approximately half the height of the appropriate level PL, but the specific level SL can be arbitrarily set to a position suitable for the recovery and replenishment of the carrier 40.

[0046] Solenoid valve 60 is connected to controller 70 via electrical wiring E1, and level switches 65 and 66 are connected to controller 70 via electrical wiring E3. Controller 70 controls the opening and closing of solenoid valve 60 based on inputs (low level signal / high level signal) from level switches 65 and 66. When solenoid valve 60 is open, any amount of carrier 40 and culture medium 50 in culture tank 10 is recovered (discharged) to the recovery line. When solenoid valve 60 is closed, the remaining carrier 40 and culture medium 50 are stored in culture tank 10. Subsequently, carrier 40 and culture medium 50 are further replenished, thereby allowing animal cells to attach to the replenished carrier 40.

[0047] The cylindrical component (separation unit) 20 is constructed from a cylindrical component, for example, made of stainless steel. Figure 2 As shown, the cylindrical component 20 is coaxially (central axis O) with the culture tank 10, dividing the internal space of the culture tank 10 into two concentric circles. Specifically, the culture tank 10 is divided into a second space R2 and a first space R1. The second space R2 is the internal space of the cylindrical component 20, and the first space R1 is an annular space formed between the culture tank 10 and the cylindrical component 20. The carrier 40 is housed in the first space R1. A stirring unit 30 is provided in the second space R2, but the carrier 40 is not present (see reference). Figure 1 ).

[0048] In addition, such as Figure 3 As shown, the cylindrical member 20 has multiple holes 21 arranged on its entire circumference. The diameter of the holes 21 is determined to be large enough to prevent the carrier 40 from passing through. For example, the diameter of the holes 21 is about 500 micrometers. Through the holes 21 of this size, the first space R1 and the second space R2 are connected by the multiple holes 21, and the culture medium 50 flows between the first space R1 and the second space R2 through the holes 21 of the cylindrical member 20. On the other hand, the carrier 40 is larger than the diameter of the holes 21, so the carrier 40 cannot enter the second space R2 from the first space R1. That is, the cylindrical member 20 is a structure that allows the culture medium 50 to pass through, but prevents the carrier 40 from passing through.

[0049] In addition, such as Figure 1 As shown, the cylindrical member 20 is constructed at approximately the same height as the body portion 11 of the culture tank 10, so that the carrier 40 cannot wrap around and intrude into the interior of the cylindrical member 20. In this way, the cylindrical member 20 completely isolates the culture tank 10 into two spaces R1 and R2, completely preventing the carrier 40 from intruding from the first space R1 and wrapping around into the second space R2.

[0050] Here, the cylindrical member 20 only needs to separate the two spaces R1 and R2 in such a way that the carrier 40 housed in the first space R1 does not intrude into the second space R2; it is not necessary to completely isolate the culture tank 10 into two spaces. It is assumed that even if there is a gap between the cylindrical member 20 and the cover 13, such a cylindrical member 20 is also a separating unit of the present invention, provided that the carrier 40 does not intrude into the second space R2 through the gap.

[0051] Alternatively, the cylindrical component 20 can be constructed from a metal mesh of, for example, 14 to 100 mesh (14 to 100 meshes per inch). Even with such a structure, it is possible to achieve [the desired effect]. Figure 3 The cylindrical component 20 shown has the same effect.

[0052] Next, the carrier 40 will be described. The carrier 40 is used to attach animal cells and is sheet-like, three-dimensional in shape, composed of a porous, gel-like structure. The material of the carrier 40 is not particularly limited; examples include materials made of resins such as PET and PP, and materials derived from plants or animals. In this embodiment, the carrier 40 is an edible carrier derived from plants or animals. The carrier 40 may contain, for example, proteins and polysaccharides. Here, "edible" means a composition that can be safely ingested by the body. Using this carrier 40 as a matrix, animal cells are attached and proliferated, thereby generating cellular food. Examples of animal cells attached to the carrier 40 include cells from muscle, skin, intestines, heart, brain, stomach, embryonic membranes, liver, kidneys, and lungs. Additionally, as... Figure 1 and Figure 2 As shown, the carrier 40 is uniformly and densely filled into the first space R1, thereby suppressing the flow within the first space R1.

[0053] Stirring unit 30 is used to stir culture medium 50, such as Figure 1 As shown, it includes an electric motor 31 as a drive source, a rotating shaft 32, and stirring blades 33. That is, in this embodiment, a propeller-type stirring unit 30 is used. By arranging the stirring blades 33 in multiple layers in the height direction, the culture medium 50 is more uniformly distributed from the center of the culture tank 10 to the first space R1 in the height direction. Furthermore, it can also be applied to large culture tanks 10. In this embodiment, the stirring blades 33 are arranged in multiple layers, but the stirring blades 33 can also be a single continuous stirring blade in the height direction, or a device that transports liquid to the outer periphery, such as a centrifugal pump, can be used.

[0054] The motor 31 is fixed to the cover 13. The output shaft of the motor 31 is connected to the rotating shaft 32 via a coupling (not shown). Multiple (e.g., three) stirring blades 33 are arranged in two layers on the rotating shaft 32. When the motor 31 rotates, the rotating shaft 32 rotates along with the motor 31, and the pair of stirring blades 33 rotate horizontally about the rotating shaft 32. This stirs the culture medium 50 within the second space R2. The culture medium 50 is then uniformly introduced into the first space R1 through multiple holes 21 provided in the cylindrical member 20. Furthermore, the motor 31 is connected to the controller 70 via electrical wiring E2, and its rotation is controlled according to instructions from the controller 70.

[0055] The controller 70 is configured as an arithmetic processing unit including a CPU, ROM and RAM as storage devices, and other peripheral circuits. The control program is stored in the ROM, and the CPU reads the control program and executes various processes. Various sensors (not shown) installed in the cell culture apparatus 100 are electrically connected to the controller 70. Based on input sensor data, the controller 70 controls, for example, the rotational speed of the motor 31, or, as described later, the opening and closing of the solenoid valve 60.

[0056] (Cell culture methods)

[0057] Next, the cell culture method using the cell culture apparatus 100 will be described. Figure 4 This is a flowchart illustrating the steps involved in cell culture. Figure 4 Each step shown is controlled by controller 70. First, as a preliminary stage, the operator sterilizes the cell culture apparatus 100 to remove microorganisms and prevent contamination. Next, according to instructions from controller 70, carrier 40 is added to the first space R1 from the supply line via supply pipes 15 and 16 (S1). Next, culture medium 50 is supplied to the second space R2 of the culture tank 10 until the liquid level of culture medium 50 reaches the appropriate level PL (S2). At this time, nutrients such as oxygen are also supplied to the culture tank 10 as needed. Then, according to a program pre-stored in controller 70, controller 70 periodically or continuously drives the motor 31 of the stirring unit 30 arbitrarily (S3). This stirs the culture medium 50, promoting the culture of animal cells. That is, the animal cells proliferate efficiently using carrier 40 as a substrate. When the animal cells have fully attached to carrier 40, a portion of carrier 40 is recovered, and new carrier 40 is added (S4). These steps S1 to S4 are repeated until the required amount of carrier 40 is recovered (S5).

[0058] Next, the steps for recycling and replenishing the carrier 40 shown in process S4 will be described in detail. Figure 5 This is a flowchart illustrating the steps involved in the recovery and replenishment of the carrier. For example... Figure 5As shown, according to the instruction from the controller 70, the solenoid valve 60 is opened (S41), allowing the carrier 40 and the culture medium 50 to be discharged together to the recovery line (S42). That is, a portion of the carrier 40 and the culture medium 50 are recovered from the first space R1 of the culture tank 10, and the liquid level of the culture medium 50 gradually decreases.

[0059] Next, based on the low liquid level signal input from the liquid level switch 66 (S43 / Yes), the controller 70 closes the solenoid valve 60 (S44). Then, according to the instruction from the controller 70, new carrier 40 is replenished from the supply line via supply pipes 15 and 16 to the first space R1, and culture medium 50 is replenished to the second space R2 (S45). Specifically, the same amount of new carrier 40 as the carrier 40 recovered in S42 is replenished to the first space R1. Thus, in S45, the old carrier 40 (carrier 40 with attached cells) and the new carrier 40 (carrier 40 without attached cells) are mixed in the first space R1 at a ratio of approximately 1:1.

[0060] Then, as the culture medium 50 is supplied, the liquid level of the culture medium 50 rises. After the liquid level of the culture medium 50 reaches the appropriate level PL, a high level signal from the level switch 65 is input to the controller 70. Based on the high level signal, the controller 70 stops supplying the culture medium 50 and ends the process (S46 / Yes). In this way, approximately half of the carrier 40 with attached animal cells is replaced with a new carrier 40.

[0061] According to the above-described embodiment, the following effects can be achieved.

[0062] The first space R1 and the second space R2 are separated by the cylindrical member 20, and the holes 21 of the cylindrical member 20 are structures through which the carrier 40 cannot pass. Therefore, the carrier 40 cannot invade the second space R2. As a result, collisions between the carrier 40 and the stirring blade 33 can be prevented, and the detachment of animal cells attached to the carrier 40 can be prevented. Thus, according to this embodiment, cell culture efficiency can be improved. Furthermore, according to this embodiment, it also contributes to achieving the United Nations Sustainable Development Goals (SDGs).

[0063] Furthermore, the culture medium 50 is stirred by the stirring blades 33 and evenly distributed across the carrier 40, thus enabling the animal cells to adhere uniformly to the carrier 40. Moreover, the multiple pores 21 buffer the eddies during stirring, thereby significantly reducing the pressure on the animal cells. This results in a high-quality cell-based food.

[0064] Furthermore, by including a step (S4) in which only about half of the carrier 40 is replaced, cell culture efficiency is improved compared to replacing all of the carrier 40. To explain in more detail, when all the carriers 40 attached with animal cells are harvested, the state of high cell proliferation is reset, and the cell proliferation must be restarted from the initial state of low cell quantity before starting the next production run.

[0065] Cells roughly double in number per day. Therefore, assuming an initial cell mass of 1 kg is cultured, it will grow to 100 kg over 7 days, which translates to approximately 14 kg / day of production. Conversely, if an initial cell mass of 50 kg is cultured, the cells roughly double in number per day, resulting in 100 kg of cells over 1 day of culture, which translates to approximately 50 kg / day of production.

[0066] In other words, by retaining about half of the old carrier 40 and mixing it with the new carrier 40, the remaining cells can be used instead of the cells that should have been added at the start of the next production run, allowing for cultivation to begin with a large initial cell quantity. This increases the daily production rate.

[0067] In addition, if you want to change the replacement ratio of the old carrier 40 to the new carrier 40, you only need to change the position of the liquid level switch 66.

[0068] Furthermore, the cell culture apparatus 100 of this embodiment has a simple structure consisting only of a culture tank 10 for storing culture medium 50, a cylindrical member 20 disposed in the culture tank 10, and a stirring unit 30 for stirring the culture medium 50. Therefore, it also has the advantage of being able to manufacture the equipment at low cost.

[0069] (Second Implementation)

[0070] Next, the cell culture apparatus of the second embodiment of the present invention will be described. Figure 6 This is a schematic diagram showing the overall structure of the cell culture apparatus 200 according to the second embodiment of the present invention and a longitudinal section of the culture tank 10. Figure 7 This is a perspective view of the cylindrical component 20-1 installed inside the culture tank 10. Figure 8A and Figure 8B This is a diagram showing the displacement state of the cylindrical component 20-1. Figure 8A This shows the expanded diameter state of cylindrical component 20-1. Figure 8B This shows the state of the cylindrical component 20-1 after its diameter reduction. Additionally, in... Figure 8A and Figure 8B The illustration of carrier 40 is omitted in the text.

[0071] The cell culture apparatus 200 of the second embodiment is characterized by having a structure in which the diameter of the cylindrical member 20-1 is expanded and contracted. For example... Figure 6 , 7 As shown, an expansion / contraction unit 25 is provided on the upper end side of the cylindrical member 20-1. The expansion / contraction unit 25 is connected to the controller 70 via electrical wiring E4. According to the instructions from the controller 70, the expansion / contraction unit 25 causes the cylindrical member 20-1 to expand or contract axially, thereby enlarging or reducing the outer diameter of the cylindrical member 20-1. In addition, the cylindrical member 20-1 can take any form as long as it is a structure that cooperates with the expansion / contraction unit 25 to expand or contract its diameter, such as a bracket type, a hinge type, a threaded type, or a pneumatic expansion type.

[0072] Moreover, in Figure 8A The cylindrical component 20-1 shown is in the expanded diameter state, and in... Figure 8B In the case of the cylindrical member 20-1 with its diameter reduced, the volume ratio of the first space R1 to the second space R2 changes. Specifically, compared to the case of the cylindrical member 20-1 with its diameter expanded, the volume ratio of the first space R1 increases in the case of the cylindrical member 20-1 with its diameter reduced. That is, when the cylindrical member 20-1 is reduced in diameter, the receiving space of the carrier 40 increases. In addition, in this embodiment, the cylindrical member 20-1 is expanded in normal conditions, as will be described later, and is reduced in diameter when the carrier 40 is being retrieved or replenished.

[0073] Next, the processing steps for recycling and replenishing the carrier 40 in the second embodiment will be described in detail. Figure 9 This is a flowchart illustrating the steps of carrier recovery and replenishment processing according to the second embodiment. In the second embodiment, characteristic processes are S44-1, S46-1, and S47-1. Therefore, these processes will be described in detail, and descriptions of processes identical to those in the first embodiment will be omitted.

[0074] like Figure 9 As shown, when solenoid valve 60 is closed (S44), controller 70 drives expansion unit 25, as... Figure 8B As shown, the cylindrical member 20-1 is reduced in diameter (S44-1). When the cylindrical member 20-1 is reduced in diameter, the volume of the first space R1 increases. Then, a new carrier 40 is added, and when the culture medium 50 rises to the appropriate level PL, the controller 70 maintains this state for a predetermined time. In this state, the volume of the first space R1 increases, so the carrier 40 can move relatively freely within the first space R1 compared to the state after the cylindrical member 20-1 is expanded in diameter. Therefore, compared to the first embodiment, the culture medium 50 fills the first space R1 more uniformly. Then, after a predetermined time has elapsed (S46-1 / Yes), the controller 70 drives the expansion / contraction unit 25 to expand the diameter of the cylindrical member 20-1 (S47-1).

[0075] As explained above, the second embodiment achieves the same effects as the first embodiment. Furthermore, in the second embodiment, the cylindrical member 20-1 has an expanded or reduced diameter structure, thus increasing the volume of the first space R1 when the carrier 40 is replenished, thereby enabling the culture medium 50 to be supplied uniformly to all carriers 40. Moreover, if the culture medium 50 is stirred by the stirring unit 30 when the first space R1 increases, the cell-attached carriers 40 and the newly replenished carriers 40 can be mixed more uniformly. As a result, the probability of cells cultured on the old carrier 40 as a substrate moving to and attaching to the new carrier 40 is increased, and the number of cells proliferating on the new carrier 40 as a substrate is increased, thus further improving cell culture efficiency.

[0076] Furthermore, this invention is not limited to the embodiments described above. Various modifications can be made without departing from the spirit of this invention, and all technical matters encompassed by the technical concept described in the claims are within the scope of this invention. The above embodiments show preferred examples, but those skilled in the art can implement various alternatives, modifications, variations, or improvements based on the content disclosed in this specification, and these are also included within the technical scope described in the appended claims.

[0077] For example, the above embodiments can be modified as follows.

[0078] (Variation Example 1)

[0079] Figure 10A This is a schematic diagram showing a longitudinal section of the cell culture apparatus 100-1 of Modified Example 1. Figure 10B It is shown schematically. Figure 10A The diagram shows a cross-sectional view of the culture tank 10. (See figure.) Figure 10A and Figure 10B As shown, the cell culture apparatus 100-1 of Modified Example 1 is a structure in which the culture tank 10 is divided into three spaces in a concentric circle using two cylindrical members 20 and 20-2. Furthermore, the cylindrical member 20-2 (the second cylindrical member) is identical to the cylindrical member 20 except for its diameter. The third space R3, formed on the outer periphery of the culture tank 10 (in other words, the third space R3 formed radially outside the first space R1), is the same as the second space R2, storing only the culture medium 50; the carrier 40 cannot penetrate (it is absent).

[0080] In this modified example 1, a circulation path C1 is provided to circulate the culture medium 50 in the third space R3 and the second space R2. A circulation pump 68 is provided in the circulation path C1. When the circulation pump 68 is driven, the culture medium 50 in the third space R3 is drawn in by the circulation pump 68 and discharged (returned) to the second space R2 via the circulation path C1. These circulation paths C and the circulation pump 68 correspond to the culture medium circulation unit of the present invention.

[0081] When the culture medium 50 is drawn into the third space R3 by the circulating pump 68, the hydraulic pressure in the third space R3 decreases slightly, thus increasing the hydraulic pressure in the second space R2. As a result, the culture medium 50 flows from the second space R2 to the third space R3 through the holes 21 of the cylindrical components 20 and 20-2, efficiently circulating the culture medium 50.

[0082] As a result, the supply of culture medium 50 to carrier 40 becomes more uniform, and the cell culture efficiency is further improved.

[0083] Furthermore, in this modified example 1, a structure is adopted in which culture medium 50 is drawn from the third space R3 and returned to the second space R2. However, a structure can also be adopted in which culture medium 50 is drawn from the second space R2 and returned to the third space R3. Even with this configuration, the culture medium 50 can be uniformly supplied to the carrier 40 in the first space R1 through the circulation of the culture medium 50.

[0084] (Variation Example 2)

[0085] Figure 11A This is a schematic cross-sectional view of the cell culture apparatus 100-2 of Modified Example 2. Figure 11B yes Figure 11A An enlarged view of part B is shown. (See attached image.) Figure 11A As shown, the cell culture apparatus 100-2 of Modified Example 2 is the same as that of Modified Example 1, and is a structure in which the culture tank 10 is divided into three spaces in a concentric circle by two cylindrical components 20, 20-2. Furthermore, the first space R1 is divided into four equal parts in the circumferential direction by a plurality of rectifier plates 45, namely the first dividing space R1-1, the second dividing space R1-2, the third dividing space R1-3, and the fourth dividing space R1-4.

[0086] Furthermore, the second space R2 and the third space R3 are connected via a flow path 46 formed between a pair of rectifier plates 45. In this embodiment, as... Figure 11A As shown, four rectifier plates 45 are arranged at 90-degree intervals in the circumferential direction, and flow paths 46 are formed between each pair of rectifier plates 45. Additionally, although not shown, multiple holes or slits are provided on the rectifier plates 45. Of course, the number and shape of the holes and slits are arbitrary.

[0087] Next, the effect of Modified Example 2 will be explained. When the culture medium 50 flows from the second space R2 to the first space R1 as described above, turbulence (not shown) may sometimes occur, especially when using a carrier 40 with an uneven shape. Turbulence is difficult to control, thus reducing the molecular uniformity of nutrients, oxygen, etc., within the culture tank 10, which may adversely affect the culture efficiency. Therefore, as shown in Modified Example 2, a flow path 46 connecting the second space R2 and the third space R3 is formed between a pair of flow straighteners 45, thereby ensuring that a portion as laminar flow F1 is always formed. In this way, by providing the flow path 46 for forming a laminar flow F1 that can control the flow of the culture medium 50, the generation of turbulence in the first space R1 is reduced, the molecular uniformity within the culture tank 10 is improved, and thus the culture efficiency is increased.

[0088] Furthermore, the number of flow paths 46, i.e. the number of rectifier plates 45, can be arbitrary. As long as the optimal number of flow paths 46 is set according to the size of the culture tank 10, the shape of the carrier 40, and other conditions, it is acceptable.

[0089] In addition, such as Figure 11B As shown, by setting an R-shape 47 at the inlet of the flow path 46, the inlet loss (resistance) of the flow path 46 can be reduced, so the flow of culture medium 50 from the second space R2 to the third space R3 becomes smooth, and a good culture environment can be maintained.

[0090] (Variation Example 3)

[0091] Figure 12A ~D shows the change in the shape of the culture tank 10. Figure 12A This is an example of a structure in which the internal space of the cylindrical culture tank 10 is divided into concentric circles, which is equivalent to the first embodiment. Figure 12B This is a structural example of dividing the internal space of the cylindrical culture tank 10-1 in the vertical direction. Figure 12C This is a structural example of dividing the internal space of a rectangular box-shaped culture tank 10-2 in the left-right direction. Figure 12D This is a structural example of dividing the internal space of a rectangular box-shaped culture tank 10-3 in the vertical direction. As shown in these structural examples, the culture tank of the present invention can be of various shapes, and the internal space can be divided in any direction. Furthermore, the number of divided spaces does not necessarily have to be two. It can also be divided into three or more (see [reference]). Figure 10A B).

[0092] Furthermore, it can be transformed into, such as Figure 12B , Figure 12C , Figure 12DThe structure shown uses the movable partitions 23-1, 23-2, and 23-3 to change the volume ratio of the first space R1 to the second space R2. In this case, simply connecting the partitions 23-1, 23-2, and 23-3 to, for example, a sliding mechanism, and driving the sliding mechanism via the controller 70 to change the volume ratio is sufficient. Alternatively, a structure that allows the partitions 23-1, 23-2, and 23-3 to move like a diaphragm to change the volume ratio can also be used.

[0093] (Variation Example 4)

[0094] Figure 13 This is a schematic diagram showing a longitudinal section of the cell culture apparatus 100-3 of Modified Example 4. Figure 13 As shown, the cell culture apparatus 100-3 of Modified Example 4 uses two cylindrical members 20 and 20-2 to divide the culture tank 10 into three spaces in a concentric circle, and further uses a circular plate member 80 to divide it into two spaces in the vertical direction. The cylindrical member 20-2 (the second cylindrical member) is identical to the cylindrical member 20 except for its diameter. Furthermore, the circular plate member 80 also has multiple holes or is formed into a mesh, through which the culture medium 50 can pass, but the carrier 40 cannot.

[0095] Therefore, in the third space R3 formed on the outer periphery of the culture tank 10, similar to the second space R2, only the culture medium 50 is stored, and the carrier 40 does not invade (is not present). Similarly, the fourth space R4 formed in the lower part of the culture tank 10 separated by the circular plate member 80 also only stores the culture medium 50, and the carrier 40 does not invade.

[0096] Furthermore, the cell culture apparatus 100-3 is provided with a circulation path C1 that circulates the culture medium 50 in the third space R3, the second space R2, and the fourth space R4. A circulation pump 68 is provided in the circulation path C1. When the circulation pump 68 is driven, the culture medium 50 in the third space R3 and the fourth space R4 is drawn in by the circulation pump 68 and discharged (returned) to the second space R2 via the circulation path C1. These circulation paths C and the circulation pump 68 correspond to the culture medium circulation unit of the present invention.

[0097] When the culture medium 50 is drawn into the third space R3 and the fourth space R4 by the circulation pump 68, the hydraulic pressure in the third space R3 and the fourth space R4 decreases slightly, thus the hydraulic pressure in the second space R2 becomes relatively higher. As a result, the culture medium 50 flows from the second space R2 through the holes 21 of the cylindrical components 20 and 20-2 to the third space R3 and the fourth space R4, efficiently circulating the culture medium 50. This further makes the supply of culture medium 50 to the carrier 40 more uniform, and further improves the cell culture efficiency.

[0098] Furthermore, in Modification 4, the partition member 81 is positioned to roughly divide the culture tank 10 in the vertical direction. This partition member 81, like the circular plate member 80, has multiple holes or is formed into a mesh, allowing the culture medium 50 to pass through, but preventing the carrier 40 from passing through. Therefore, a flow space T is formed directly below the partition member 81, allowing the culture medium 50 to flow. By allowing the culture medium 50 to pass through this flow space T, the culture efficiency of cells attached to the carrier 40 can be further improved.

[0099] Here, the partition member 81 can be installed on the inner circumferential surface of the cylindrical member 20-2 in a suitable manner. For example, if it is a structure that engages with a protrusion (not shown) provided on the cylindrical member 20-2, the partition member 81 can be easily installed and removed, thus simplifying the replacement of the carrier 40.

[0100] In addition, in this modified example 4, a structure is adopted in which culture medium 50 is drawn from the third space R3 and the fourth space R4 and returned to the second space R2. However, a structure in which culture medium 50 is drawn from the second space R2 and returned to the third space R3 and the fourth space R4 can also be adopted. Furthermore, the circular plate member 80 and the partition member 81 can be composed of a common member, or structures with different sizes of holes and meshes can be adopted.

[0101] (Variation Example 5)

[0102] Figure 14 This is a schematic diagram showing a longitudinal section of the cell culture apparatus 100-4 of Modified Example 5. (See diagram below.) Figure 14 As shown, in the cell culture apparatus 100-4 of Modified Example 5, four baskets 85-1, 85-2, 85-3, and 85-4, serving as partition units for separating the internal space, are coaxially stacked inside the culture tank 10. Furthermore, the bottommost basket 85-4 is supported by a support member 87, such that the four baskets 85-1, 85-2, 85-3, and 85-4 are positioned at a distance from the bottom 12 of the culture tank 10 equal to the height of the support member 87. In other words, a space (fourth space R4) is formed between the bottommost basket 85-4 and the bottom 12 of the culture tank 10.

[0103] Next, the structure of baskets 85-1, 85-2, 85-3, and 85-4 will be described in detail. Figure 15A This is a top view of the 85-1 basketball team. Figure 15B This is a longitudinal sectional view of basket 85-1. Furthermore, since the four baskets 85-1, 85-2, 85-3, and 85-4 have the same structure, basket 85-1 will be described here.

[0104] like Figure 15A and Figure 15BAs shown, basket 85-1 has an inner cylinder 85a-1, an outer cylinder 85b-1, and an annular bottom plate 85c-1 connecting the lower parts of the inner cylinder 85a-1 and the outer cylinder 85b-1. The inner cylinder 85a-1, outer cylinder 85b-1, and bottom plate 85c-1 all have multiple holes or are formed into a mesh, allowing the culture medium 50 to pass through, while the carrier 40 cannot. Through this structure, the carrier 40 is placed on the bottom plate 85c-1 and contained in the space between the inner cylinder 85a-1 and the outer cylinder 85b-1.

[0105] Furthermore, when cascading baskets 85-1, 85-2, 85-3, and 85-4, such as Figure 14 As shown, a second space R2 is formed inside the four inner cylinders 85a-1, 85a-2, 85a-3, and 85a-4; a first space R1 is formed between the four inner cylinders 85a-1, 85a-2, 85a-3, and 85a-4 and the four outer cylinders 85b-1, 85b-2, 85b-3, and 85b-4; a third space R3 is formed between the four outer cylinders 85b-1, 85b-2, 85b-3, and 85b-4 and the body portion 11 of the culture tank 10; and a fourth space R4 is formed between the bottom plate 85c-4 of the lowest basket 85-4 and the bottom 12 of the culture tank 10.

[0106] Furthermore, in Modified Example 5, four stirring blades 33 are provided, the same number as the number of layers in baskets 85-1, 85-2, 85-3, and 85-4. This allows the culture medium 50 to circulate efficiently within each basket via the stirring blades 33. Of course, the number of stirring blades 33 can also be less than the number of basket layers.

[0107] Furthermore, in this modified example 5, with the carrier 40 contained in baskets 85-1, 85-2, 85-3, and 85-4, a flow space T is formed in the upper part of each basket to allow the culture medium 50 to flow. Therefore, the culture medium 50 circulates in the flow space T of each basket as indicated by the blank arrows in the figure. This allows for efficient culture of cells attached to the carrier 40.

[0108] Furthermore, in variation 5, the basket does not necessarily have to be cylindrical. For example, it can be any shape, such as a rectangular cylindrical basket with a bottom.

[0109] (Variation Example 6)

[0110] Modification 6 is obtained by modifying the structure of the basket in the cell culture apparatus 100-4 of Modification 5. Figure 16A These are top views of baskets 85-1, 85-2, 85-3, and 85-4 in variant example 6. Figure 16B This is a longitudinal sectional view of the states of baskets 85-1, 85-2, 85-3, and 85-4 in the layered configuration variation example 6. Figure 16AXVIB-XVIB sectional view.

[0111] like Figure 16A and Figure 16B As shown, the top basket 85-1 has three access paths 86-1a, 86-1b, and 86-1c. In this example, the three access paths 86-1a, 86-1b, and 86-1c can be of the same shape or different shapes. In the following explanation, we will assume that the three access paths 86-1a, 86-1b, and 86-1c are of the same shape.

[0112] The passageway 86-1a is formed by a cylindrical body that passes through the base plate 85c-1 of the basket 85-1. The height (axial length) of the passageway 86-1a is approximately 1 / 2 to 2 / 3 of the height of the basket 85-1. The passageway 86-1a is shaped to a diameter that allows the carrier 40 to pass through.

[0113] The second-highest basket 85-2 has two passageways 86-2a and 86-2b with the same shape as passageway 86-1a. Passageway 86-2a is positioned at the same center as passageway 86-1a when viewed from above, and passageway 86-2b is positioned at the same center when viewed from above. The third-highest basket 85-3 has a passageway 86-3a with the same shape as passageway 86-1a. Passageway 86-3a is positioned at the same center as passageway 86-1a and passageway 86-2a when viewed from above. The lowest basket 85-4 has no passageway.

[0114] When these baskets are stacked in this way, as... Figure 16A As shown, because paths 86-1a, 86-2a, and 86-3a are consistent when viewed from above (configured on the Z1 axis), therefore... Figure 16B As shown, when the carrier 40 is placed into the passage path 86-1a along the Z1 axis, the carrier 40 passes through the passage path 86-1a, the passage path 86-2a, and the passage path 86-3a in sequence, and falls into the space between the inner cylinder 85a-4 and the outer cylinder 85b-4 of the bottom basket 85-4, where it is contained.

[0115] Similarly, because the view from above via path 86-1b is the same as that via path 86-2b (configured on the Z2 axis), therefore... Figure 16B As shown, when the carrier 40 is placed into the passage path 86-1b along the Z2 axis, the carrier 40 passes through the passage path 86-1b and the passage path 86-2b in sequence, and falls into the space between the inner cylinder 85a-3 and the outer cylinder 85b-3 of the basket 85-3 on the third layer, where it is contained.

[0116] Furthermore, when the carrier 40 is housed in the basket 85-2 on the second layer from the top, it is sufficient to allow the carrier 40 to fall along the Z3 axis to the passage path 86-1c.

[0117] According to this modified example 6, the carrier 40 can be easily fed into baskets 85-1, 85-2, 85-3, and 85-4, thus improving work efficiency compared to modified example 5.

[0118] Additionally, for example, paths 86-1a, 86-2a, and 86-3a do not necessarily need to be strictly aligned on the Z1 axis. As long as the carrier 40 can fall smoothly, it is not necessary for all paths to be aligned on the same axis. When viewed from above, each path only needs to overlap at least slightly. Furthermore, the same applies to the alignment of paths 86-1b and 86-2b.

[0119] Furthermore, the aforementioned variations 1, 2, 3, 4, 5, and 6 can also be applied to the cell culture apparatus of the second embodiment.

[0120] Here, the present invention can also be used as an apparatus for culturing cells other than animal cells (e.g., plant cells). Furthermore, if the culture tank 10 is small, a magnetic stirrer, for example, can be used as the stirring unit 30. In this case, there is an advantage in simplifying the structure.

[0121] Furthermore, the shape of the holes 21 provided in the cylindrical components 20, 20-1, and 20-2 is arbitrary. For example, they can be round holes, square holes, or elongated holes. Moreover, the present invention can be applied to cylindrical components provided with multiple slits.

[0122] Furthermore, in the above embodiment, a structure using level switches 65 and 66 to recover and replenish the carrier 40 is employed. However, a weight sensor or similar device can be used instead of level switches 65 and 66. For example, the weight of the culture tank 10 when the culture medium 50 is filled to the appropriate level PL and the carrier 40 is fully added is set as a first threshold, and approximately 50% of the weight of the first threshold is set as a second threshold. Then, if configured such that the solenoid valve 60 is closed when the weight sensor detects the second threshold (equivalent to...), Figure 5 (S43, S44) When the weight sensor detects the first threshold, the recovery and replenishment of carrier 40 is terminated (equivalent to...) Figure 5 If S46 is used, then the same control as in the first embodiment can be performed.

[0123] Of course, it can also be replaced by manual operation by the operator, for example... Figure 4 The steps are shown.

[0124] Explanation of reference numerals in the attached figures

[0125] 10: Culture tank;

[0126] 11: Carcass part;

[0127] 12: Bottom;

[0128] 13: Cover;

[0129] 15, 16: Supply management;

[0130] 20, 20-1: Cylindrical components (dividing units);

[0131] 20-2: Cylindrical component (second cylindrical component / separation unit);

[0132] 21: Hole;

[0133] 23-1, 23-2, 23-3: Divider (divider unit);

[0134] 25: Expansion / Contraction Unit;

[0135] 30: Stirring unit;

[0136] 31: Electric motor;

[0137] 32: Rotation axis;

[0138] 33: Agitator blades;

[0139] 40: Carrier;

[0140] 45: Rectifier plate;

[0141] 46: Flow path;

[0142] 50: Culture medium;

[0143] 60: Solenoid valve;

[0144] 65, 66: Liquid level switches;

[0145] 68: Circulation pump (culture medium circulation unit);

[0146] 70: Controller;

[0147] 80: Circular plate component (divider unit);

[0148] 81: Partition components;

[0149] 85-1, 85-2, 85-3, 85-4: Baskets (dividing units);

[0150] 85a-1, 85a-2, 85a-3, 85a-4: Inner cylinder;

[0151] 85b-1, 85b-2, 85b-3, 85b-4: outer cylinder;

[0152] 85c-1, 85c-2, 85c-3, 85c-4: Base plate;

[0153] 86-1a, 86-1b, 86-1c: via path;

[0154] 86-2a, 86-2b: via path;

[0155] 86-3a: via path;

[0156] 87: Supporting components;

[0157] 100, 100-1, 100-2, 200: Cell culture apparatus;

[0158] C1: Circulation path (culture medium circulation unit);

[0159] E1~E4: Electrical wiring;

[0160] R1: First space;

[0161] R1-1, R1-2, R1-3, R1-4: First partition space (first space);

[0162] R2: Second space;

[0163] R3: Third Space;

[0164] R4: Fourth Space;

[0165] T: Flow space.

Claims

1. A cell culture device for culturing cells, characterized by, Having: a culture tank that stores a culture solution; a partition unit that partitions the culture tank into at least a first space and a second space; and a stirring unit that stirs the culture solution, the first space is a space that houses a carrier to which the cells adhere, the second space is a space provided with the stirring unit, the partition unit is configured such that the culture solution can pass through and the carrier cannot intrude from the first space into the second space.

2. The cell culture device according to claim 1, wherein the volume ratio of the first space to the second space is changed by the action of the partition unit.

3. The cell culture device according to claim 1, wherein the partition unit is configured from a cylindrical member having a plurality of holes or a mesh shape, the annular space between the culture tank and the cylindrical member is the first space, the inner space of the cylindrical member is the second space.

4. The cell culture device according to claim 3, wherein the cylindrical member is configured to be able to contract and expand in diameter within the culture tank, the volume ratio of the first space to the second space is changed by the contraction or expansion of the cylindrical member in diameter.

5. The cell culture device according to claim 1, wherein a culture solution circulating unit that sucks in the culture solution stored in the first space and returns the culture solution to the culture tank is provided.

6. A method of culturing cells, comprising, The cell culture method using the cell culture device according to claim 1 includes: a step of feeding a carrier to the first space and supplying the culture solution to the culture tank; a step of driving the stirring unit to stir the culture solution; a step of recovering a part of the carrier from the first space; and a step of feeding a new carrier to the first space and mixing it with the carrier remaining in the first space.

7. A method of culturing cells, comprising, The cell culture method using the cell culture device according to claim 2 includes: a step of feeding a carrier to the first space and supplying the culture solution to the culture tank; a step of driving the stirring unit to stir the culture solution; a step of recovering a part of the carrier from the first space; a step of expanding the volume of the first space by the action of the partition unit; a step of feeding a new carrier to the first space after the volume expansion and mixing it with the carrier remaining in the first space; and a step of returning the first space after the volume expansion to the original volume by the action of the partition unit.

8. The cell culture device according to claim 3, wherein the culture tank further has a third annular space that is a space radially outside the first space and is partitioned from the first space by a second cylindrical member having a plurality of holes or a mesh shape, the second cylindrical member is configured such that the culture solution can pass through and the carrier cannot intrude from the first space into the third space.

9. The cell culture device according to claim 8, wherein The culture tank also has a fourth space which is a space below the first and second spaces, is separated from the first and second spaces by a circular plate member having a plurality of holes or a mesh shape, The circular plate member is configured so that the culture solution can pass through and the carriers cannot intrude from the first space into the fourth space.

10. The cell culture device according to claim 8 or 9, characterized in that, It also has at least one partition member which holds the carriers, The at least one partition member is configured to separate the first space in the vertical direction, The at least one partition member has a plurality of holes or is formed in a mesh shape, while being configured so that the culture solution can pass through and the carriers cannot pass through.

11. The cell culture device according to claim 1, characterized in that, The culture tank is separated into the second space, the first space, and the third space in this order from the center by the partition unit in a concentric circular shape, The cell culture device also has a plurality of baskets as the partition unit, The plurality of baskets each has: an inner cylinder having a plurality of holes or being formed in a mesh shape, an outer cylinder which is arranged in a concentric circular shape with the inner cylinder and has a plurality of holes or is formed in a mesh shape, and a circular ring-shaped bottom plate which connects the bottom between the inner cylinder and the outer cylinder and has a plurality of holes or is formed in a mesh shape, the plurality of baskets each being configured to be able to accommodate the carriers between the inner cylinder and the outer cylinder, The plurality of baskets are arranged in a stacked manner on the central axis of the culture tank, In the state in which the plurality of baskets are arranged in a stacked manner, the culture tank is separated into the first space and the third space by the plurality of outer cylinders, and the culture tank is separated into the first space and the second space by the plurality of inner cylinders.

12. The cell culture device according to claim 11, characterized in that, All of the plurality of baskets except for the lowermost basket have at least one cylindrical passage which penetrates through the respective bottom plates and through which the carriers can pass, The number of the passages increases from the lower side to the upper side of the plurality of baskets.

13. The cell culture device according to claim 12, characterized in that, When the plurality of baskets arranged in a stacked manner are observed from the axial direction of the culture tank, the central positions of the passages coincide.

14. The cell culture device according to claim 11, characterized in that, The lowermost basket among the plurality of baskets is supported with a space from the bottom of the culture tank.

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