Cell culturing device and cell culturing method

Abstract

The present invention prevents a decrease in cell culturing efficiency by preventing detachment of cells adhered to scaffolds. A cell culturing device (100) for culturing cells comprises: a culture tank (10) for retaining a culture solution (50); a partitioning means (20) for partitioning the culture tank into at least a first space (R1) and a second space (R2); and a stirring means (30) for stirring the culture solution. The first space (R1) accommodates scaffolds (40) to which cells adhere. The second space (R2) is where the stirring means (30) is provided. The partitioning means (20) is configured to allow the culture solution to pass therethrough, and to disable entry of the scaffolds to the second space from the first space.

Description

Cell culture device and cell culture method

[0001] The present invention relates to a cell culture device and a cell culture method.

[0002] In recent years, countries around the world have been developing truly sustainable food production systems in an effort to achieve the Sustainable Development Goals (SDGs), with the development of "cellular foods" in particular showing widespread growth. Furthermore, as background technology in this technical field, for example, Patent Document 1 describes a submerged culture method for tissue cell cultures that uses indirect aeration through a tubular gas-permeable membrane in a permeator. Patent Document 1 discloses a device for implementing this method, in which a tubular permeator forms a vertical central tube within the vessel of a cell culture device. An agitator is fixed to the bottom of the permeator, and the rotation of this agitator diffuses gas into the liquid.

[0003] Special Publication No. 03-021149

[0004] However, in the cell culture device described in Patent Document 1, the top of the tubular permeator is open, so there is a possibility that the cell culture may enter the interior from the top of the permeator. If the cell culture bound to the microcarriers enters the interior of the permeator, the cell culture will be sheared by the agitator. As a result, the cells may detach from the microcarriers or be destroyed, which may lead to a decrease in cell culture efficiency.

[0005] The present invention has been made in view of the above circumstances, and its object is to prevent cells attached to a scaffold from detaching, thereby suppressing a decrease in cell culture efficiency.

[0006] In order to achieve the above-mentioned object, a first aspect of the present invention is a cell culture device for culturing cells, comprising a culture tank for storing a culture solution, a partition means for dividing the culture tank into at least a first space and a second space, and an agitation means for agitating the culture solution, wherein the first space is a space for accommodating a scaffold to which the cells adhere, and the second space is a space in which the agitation means is provided, and the partition means is configured to allow the culture solution to pass through but to prevent the scaffold from entering the second space from the first space.

[0007] In addition, in order to achieve the above-mentioned object, a second aspect of the present invention is a cell culture method using the cell culture device described in the first aspect above, characterized in that it includes the steps of: introducing a scaffold into the first space and supplying the culture medium to the culture tank; driving the stirring means to stir the culture medium; removing a part of the scaffold from the first space; and introducing new scaffold into the first space and mixing it with the scaffold remaining in the first space.

[0008] According to the present invention, it is possible to prevent cells attached to a scaffold from detaching, thereby suppressing a decrease in cell culture efficiency. Note that problems, configurations, and effects other than those described above will become clear from the description of the following embodiments.

[0009] 10A is a diagram schematically showing the overall configuration of a cell culture device according to a first embodiment of the present invention and a longitudinal cross section of a culture tank. FIG. 11B is a diagram schematically showing a cross section of a culture tank. FIG. 11C is a perspective view of a cylindrical member provided in a culture tank. FIG. 11D is a flowchart showing the procedure for cell culture. FIG. 11E is a flowchart showing the procedure for scaffold recovery and replenishment. FIG. 11F is a diagram schematically showing the overall configuration of a cell culture device according to a second embodiment of the present invention and a longitudinal cross section of a culture tank. FIG. 11G is a perspective view of a cylindrical member provided in a culture tank. FIG. 11H is a diagram showing a state in which the cylindrical member is expanded in diameter. FIG. 11I is a diagram showing a state in which the cylindrical member is contracted in diameter. FIG. 11I is a flowchart showing the procedure for scaffold recovery and replenishment in the second embodiment. FIG. 11F is a diagram schematically showing the longitudinal cross section of a cell culture device according to a first embodiment of the present invention. FIG. 11H is a diagram schematically showing the cross section of the culture tank shown in FIG. 11A. FIG. 11H is a diagram showing variations in the shape of the culture tank. FIG. 11I is a diagram showing variations in the shape of the culture tank. FIG. 11I is a diagram showing variations in the shape of the culture tank. 16A is a diagram schematically showing a longitudinal section of a cell culture device according to Modification 5. FIG. 16B is a plan view of a basket. FIG. 16C is a longitudinal section of a basket. FIG. 16D is a plan view of a basket according to Modification 6. FIG. 16B is a cross-sectional view taken along the line XVIB-XVIB of FIG. 16A.

[0010] Hereinafter, an embodiment of the present invention will be described with reference to the drawings, taking the case of culturing animal cells as an example.

[0011] (First embodiment) Fig. 1 is a diagram schematically showing the overall configuration of a cell culture device 100 according to a first embodiment of the present invention and a longitudinal cross section of a culture tank 10, Fig. 2 is a diagram schematically showing a transverse cross section of the culture tank 10, and Fig. 3 is a perspective view of a cylindrical member 20 provided in the culture tank 10. Note that in Fig. 2, the stirring means 30 shown in Fig. 1 is omitted.

[0012] As shown in FIG. 1, the cell culture device 100 according to this embodiment mainly includes a culture vessel 10, a cylindrical member 20, and an agitation means 30.

[0013] The culture tank 10 includes a cylindrical container with a bottom formed by a body 11 and a bottom 12, and a disk-shaped lid 13 that covers the top of the body 11. The culture tank 10 stores a culture solution 50 therein. The culture tank 10 is made of, for example, stainless steel, but may also be made of other materials (glass, silicone resin, etc.). When the culture tank 10 is made of silicone resin, the culture tank 10 is sometimes referred to as a culture bag.

[0014] The lid 13 is tightly attached to the body 11 via a packing (not shown). This seals the inside of the culture tank 10. The lid 13 is provided with supply pipes 15 and 16. A scaffold 40 (described below) and a culture solution 50 supplied from a 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 lid 13 is also provided with various pipes, hoses, nozzles, etc. (not shown). These are used for various purposes, such as connecting various sensors, supplying oxygen, etc., or sampling.

[0015] One solenoid valve 60 is provided at the bottom 12 of the culture tank 10. Of course, there may be more than one solenoid valve 60. Furthermore, level switches 65, 66 for detecting the liquid level of the culture solution 50 are provided at the body 11 of the culture tank 10. The level switch 65 detects whether the liquid level of the culture solution 50 is at an appropriate level PL. The level switch 65 outputs a high level signal when the liquid level of the culture solution 50 exceeds the appropriate level PL. Here, the appropriate level PL is the position at which the culture tank 10 is substantially filled with the culture solution 50.

[0016] Meanwhile, the level switch 66 detects whether the liquid level of the culture solution 50 is at a specific level SL. The level switch 66 outputs a low level signal when the liquid level of the culture solution 50 falls below the specific level SL. Here, in this embodiment, the specific level SL is set to a height position that is approximately half the appropriate level PL, but the specific level SL can be set arbitrarily to a position that is suitable for recovering and replenishing the scaffold 40.

[0017] The solenoid valve 60 is connected to the controller 70 via electrical wiring E1, and the level switches 65 and 66 are connected to the controller 70 via electrical wiring E3. The controller 70 controls the opening and closing operation of the solenoid valve 60 based on inputs (low level signal / high level signal) from the level switches 65 and 66. When the solenoid valve 60 opens, a given amount of the scaffolds 40 and culture solution 50 in the culture tank 10 is recovered (discharged) into a recovery line, and when the solenoid valve 60 closes, the remaining scaffolds 40 and culture solution 50 are stored in the culture tank 10, after which more scaffolds 40 and culture solution 50 are replenished, causing animal cells to attach to the replenished scaffolds 40.

[0018] The cylindrical member (partition means) 20 is composed of a cylindrical member made of, for example, stainless steel. As shown in FIG. 2 , the cylindrical member 20 is provided coaxially (on the central axis O) with the culture tank 10, and concentrically divides the internal space of the culture tank 10 into two spaces. Specifically, the culture tank 10 is partitioned into a second space R2, which is the internal space of the cylindrical member 20, and a first space R1, which is an annular space formed between the culture tank 10 and the cylindrical member 20. A scaffold 40 is housed in the first space R1. An agitation means 30 is provided in the second space R2, but no scaffold 40 is present (see FIG. 1 ).

[0019] As shown in FIG. 3 , the cylindrical member 20 has a large number of holes 21 formed around its entire circumference. The diameter of the holes 21 is determined to be large enough to prevent the passage of the scaffolds 40. For example, the diameter of the holes 21 is approximately 500 microns. By making the holes 21 of this size, the first space R1 and the second space R2 are connected by the large number of 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, because the diameter of the scaffolds 40 is larger than the diameter of the holes 21, the scaffolds 40 cannot enter the second space R2 from the first space R1. In other words, the cylindrical member 20 is configured to allow the passage of the culture medium 50 but prevent the passage of the scaffolds 40.

[0020] 1, the cylindrical member 20 is configured to be at approximately the same height as the body 11 of the culture tank 10, and therefore the scaffolding 40 cannot go around and invade inside the cylindrical member 20. In this way, the cylindrical member 20 completely separates the culture tank 10 into two spaces R1 and R2, completely preventing the scaffolding 40 from invading or going around from the first space R1 to the second space R2.

[0021] Here, the cylindrical member 20 need only be able to separate the first space R1, R2 so that the scaffolding 40 housed in the first space R1 does not intrude into the second space R2, and does not necessarily have to completely isolate the culture tank 10 into two spaces. Even if there is a gap between the cylindrical member 20 and the lid 13, if the scaffolding 40 does not intrude into the second space R2 through the gap, such a cylindrical member 20 is included in the partitioning means of the present invention.

[0022] The cylindrical member 20 may be made of a wire mesh with a mesh size of 14 to 100 meshes (14 to 100 meshes per inch), for example. Even with this configuration, the same effects as those of the cylindrical member 20 shown in FIG. 3 can be achieved.

[0023] Next, the scaffold 40 will be described. The scaffold 40 is a material for attaching animal cells and is composed of a sheet or three-dimensional porous or gel-like structure. The material of the scaffold 40 is not particularly limited, but includes resins such as PET and PP, as well as plant- and animal-derived materials. In this embodiment, the scaffold 40 is an edible scaffold derived from plants or animals. The scaffold 40 includes, for example, proteins and polysaccharides. Here, "edible" refers to a composition that can be safely ingested into the body. Cellular food is produced by the attachment and proliferation of animal cells using the scaffold 40 as a substrate. Examples of animal cells that can be attached to the scaffold 40 include muscle-derived cells, skin-derived cells, intestinal-derived cells, heart-derived cells, brain-derived cells, stomach-derived cells, embryonic membrane-derived cells, liver-derived cells, kidney-derived cells, and lung-derived cells. As shown in FIGS. 1 and 2 , the scaffold 40 is packed evenly and densely in the first space R1, suppressing flow within the first space R1.

[0024] The stirring means 30 is used to stir the culture solution 50 and, as shown in FIG. 1 , includes a motor 31 serving as a drive source, a rotating shaft 32, and stirring blades 33. That is, in this embodiment, a propeller-type stirring means 30 is used. By arranging the stirring blades 33 in multiple stages in the height direction, the culture solution 50 is more evenly distributed in the height direction from the center of the culture tank 10 to the first space R1. Furthermore, this can also be applied to large culture tanks 10. In this embodiment, the stirring blades 33 are arranged in multiple stages, but the stirring blades 33 may be a single stirring blade that is continuous in the height direction, or a device that pumps liquid in a circumferential direction, such as a centrifugal pump, may be used.

[0025] The motor 31 is fixed to the cover 13. The output shaft of the motor 31 and the rotating shaft 32 are connected via a coupling (not shown). The rotating shaft 32 is provided with a plurality of (e.g., three) agitating blades 33 arranged in two rows, one above the other. When the motor 31 rotates, the rotating shaft 32 rotates in response to the rotation of the motor 31, and the pair of upper and lower agitating blades 33 rotate horizontally around the rotating shaft 32. In this way, the culture solution 50 in the second space R2 is agitated. The culture solution 50 is then evenly introduced into the first space R1 through the numerous holes 21 formed in the cylindrical member 20. The motor 31 is connected to the controller 70 via electrical wiring E2, and its rotation is controlled in accordance with commands from the controller 70.

[0026] The controller 70 is configured to include an arithmetic processing unit having a CPU, memory devices such as ROM and RAM, and other peripheral circuits. A control program is stored in the ROM, and the CPU reads the control program and executes various processes. Various sensors (not shown) attached to the cell culture device 100 are electrically connected to the controller 70. Based on the input sensor data, the controller 70 controls, for example, the rotation speed of the motor 31 and the opening and closing operation of the solenoid valve 60, as will be described later.

[0027] (Cell Culture Method) Next, a cell culture method using the cell culture device 100 will be described. FIG. 4 is a flowchart showing the cell culture procedure. Each step shown in FIG. 4 is controlled by the controller 70. First, as a preliminary step, an operator sterilizes the cell culture device 100 to remove microorganisms and prevent contamination. Next, in accordance with a command from the controller 70, the scaffold 40 is introduced into the first space R1 via the supply line and the supply pipes 15 and 16 (S1). Next, the culture solution 50 is supplied to the second space R2 of the culture tank 10 until the liquid level of the culture solution 50 reaches an appropriate level PL (S2). At this time, nutrients such as oxygen are also appropriately supplied to the culture tank 10. Then, according to a program pre-stored in the controller 70, the controller 70 periodically or continuously drives the motor 31 of the stirring means 30 (S3). In this way, the culture solution 50 is stirred, promoting the culture of animal cells. That is, the animal cells efficiently grow using the scaffold 40 as a substrate. After a sufficient number of animal cells have attached to the scaffold 40, a portion of the scaffold 40 is collected and a new scaffold 40 is replenished (S4). These steps S1 to S4 are repeated until the desired scaffold 40 is collected (S5).

[0028] Next, the procedure for recovering and replenishing the scaffold 40 shown in step S4 will be described in detail. Fig. 5 is a flowchart showing the procedure for recovering and replenishing the scaffold. As shown in Fig. 5, the solenoid valve 60 opens in response to a command from the controller 70 (S41), and the scaffold 40 is discharged together with the culture medium 50 into the recovery line (S42). That is, a portion of the scaffold 40 is recovered together with the culture medium 50 from the first space R1 of the culture tank 10, and the liquid level of the culture medium 50 gradually drops.

[0029] Next, the controller 70 closes the solenoid valve 60 based on the input of a low level signal from the level switch 66 (S43 / YES) (S44). Then, in response to a command from the controller 70, new scaffolds 40 are replenished into the first space R1 and the culture medium 50 is replenished into the second space R2 via the supply lines 15 and 16 (S45). Specifically, the same amount of new scaffolds 40 as the scaffolds 40 collected in S42 is replenished into the first space R1. Thus, in S45, the old scaffolds 40 (scaffolds 40 with cells attached) and the new scaffolds 40 (scaffolds 40 without cells attached) are mixed in the first space R1 at a ratio of approximately 1:1.

[0030] Then, as the culture solution 50 is supplied, the liquid level of the culture solution 50 rises, and when the liquid level of the culture solution 50 reaches the appropriate level PL, a high level signal is input from the level switch 65 to the controller 70. Based on the high level signal, the controller 70 stops the supply of the culture solution 50 and ends the process (S46 / YES). In this way, approximately half of the scaffold 40 to which the animal cells have attached is replaced with a new scaffold 40.

[0031] According to the present embodiment described above, the following advantageous effects can be achieved.

[0032] 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 configured to prevent the scaffold 40 from passing through. Therefore, the scaffold 40 cannot enter the second space R2. As a result, the scaffold 40 can be prevented from colliding with the stirring blades 33, and animal cells attached to the scaffold 40 can be prevented from peeling off. Therefore, according to this embodiment, cell culture efficiency can be improved. In addition, according to this embodiment, it also contributes to the achievement of the Sustainable Development Goals (SDGs).

[0033] Furthermore, the culture medium 50 is stirred by the stirring blades 33 and spreads evenly over the scaffold 40, allowing animal cells to attach uniformly to the scaffold 40. Furthermore, the numerous holes 21 buffer vortices during stirring, significantly reducing stress on the animal cells. This allows for the production of a good cellular food product.

[0034] Furthermore, by including the step (S4) of replacing only about half of the scaffolds 40, cell culture efficiency is improved compared to replacing all of the scaffolds 40. To explain in more detail, when all of the scaffolds 40 to which animal cells have attached are harvested, the state in which a large number of cells have been multiplied is reset, and when the next production is started, cells must be multiplied again from the initial state in which the cell amount is small.

[0035] Since cells generally double in number per day, if culture is initiated with an initial cell mass of 1 kg, the cell mass will reach 100 kg over a 7-day culture period, which translates to a daily production volume of approximately 14 kg / day. On the other hand, if production is initiated with an initial cell mass of 50 kg, the cells generally double in number per day, which translates to a daily production volume of approximately 50 kg / day.

[0036] In other words, by leaving about half of the old scaffold 40 and mixing it with the new scaffold 40, it is possible to start culture with a large initial cell amount by using the remaining cells instead of using the cells that should be added when starting the next production run, thereby improving the daily production volume.

[0037] If it is desired to change the replacement ratio between the old scaffolding 40 and the new scaffolding 40, the position of the level switch 66 can be changed.

[0038] Furthermore, the cell culture device 100 according to this embodiment has a simple configuration that includes only a culture tank 10 for storing the culture solution 50, a cylindrical member 20 provided in the culture tank 10, and a stirring means 30 for stirring the culture solution 50, and therefore has the advantage that the manufacturing equipment can be realized at low cost.

[0039] Second Embodiment Next, a cell culture device according to a second embodiment of the present invention will be described. Fig. 6 is a diagram schematically illustrating the overall configuration of a cell culture device 200 according to a second embodiment of the present invention and a longitudinal cross section of a culture tank 10. Fig. 7 is a perspective view of a cylindrical member 20-1 provided in the culture tank 10. Figs. 8A and 8B are diagrams illustrating the displacement state of the cylindrical member 20-1. Fig. 8A shows a state in which the cylindrical member 20-1 has expanded in diameter, and Fig. 8B shows a state in which the cylindrical member 20-1 has contracted in diameter. Note that the scaffolding 40 is omitted from Figs. 8A and 8B.

[0040] The cell culture device 200 according to the second embodiment is characterized in that the cylindrical member 20-1 is configured to expand and contract in diameter. As shown in FIGS. 6 and 7 , an expansion / contraction means 25 is provided at the upper end of the cylindrical member 20-1. The expansion / contraction means 25 is connected to the controller 70 via electrical wiring E4. The expansion / contraction means 25 expands or contracts the cylindrical member 20-1 in the axial direction in accordance with commands from the controller 70, thereby expanding or contracting the outer diameter of the cylindrical member 20-1. Note that any type of structure can be used for the cylindrical member 20-1 as long as it is configured to expand or contract in diameter in cooperation with the expansion / contraction means 25, including a stent type, a hinge type, a screw type, and an air pressure expansion type.

[0041] The volume ratio of the first space R1 to the second space R2 changes between the expanded diameter state of the cylindrical member 20-1 shown in Fig. 8A and the contracted diameter state of the cylindrical member 20-1 shown in Fig. 8B. Specifically, the volume ratio of the first space R1 is greater when the cylindrical member 20-1 is contracted than when the cylindrical member 20-1 is expanded. In other words, when the cylindrical member 20-1 contracts in diameter, the storage space for the scaffolding 40 expands. In this embodiment, the cylindrical member 20-1 is normally expanded in diameter, and contracts in diameter when the scaffolding 40 is retrieved or replenished, as described below.

[0042] Next, the processing procedure for recovering and replenishing the scaffolding 40 in the second embodiment will be described in detail. Figure 9 is a flowchart showing the processing procedure for recovering and replenishing the scaffolding in the second embodiment. The characteristic processes in the second embodiment are S44-1, S46-1, and S47-1. Therefore, these processes will be described in detail, and a description of the same processes as in the first embodiment will be omitted.

[0043] As shown in FIG. 9, when the solenoid valve 60 closes (S44), the controller 70 drives the expansion / contraction means 25 to contract the diameter of the cylindrical member 20-1 (S44-1), as shown in FIG. 8B. As the diameter of the cylindrical member 20-1 contracts, the volume of the first space R1 increases. Then, when new scaffolds 40 are replenished and the culture solution 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 has increased, allowing the scaffolds 40 to move relatively freely within the first space R1 compared to when the cylindrical member 20-1 is expanded. Therefore, compared to the first embodiment, the culture solution 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 means 25 to expand the diameter of the cylindrical member 20-1 (S47-1).

[0044] As described above, the second embodiment can achieve the same effects as the first embodiment. Furthermore, in the second embodiment, because the cylindrical member 20-1 is configured to expand or contract in diameter, when scaffolds 40 are replenished, the volume of the first space R1 increases, allowing the culture medium 50 to be uniformly supplied to all scaffolds 40. Furthermore, if the culture medium 50 is stirred by the stirring means 30 while the first space R1 is expanding, the scaffolds 40 to which cells have attached can be more uniformly mixed with the newly replenished scaffolds 40. As a result, the probability that cells cultured using the old scaffolds 40 as a substrate will migrate to and attach to the new scaffolds 40 increases, and the number of cells that grow using the new scaffolds 40 as a substrate increases, further improving cell culture efficiency.

[0045] The present invention is not limited to the above-described embodiments, and various modifications are possible within the scope of the gist of the present invention. The present invention covers all technical matters included in the technical ideas described in the claims. The above-described embodiments are preferred examples, but a person skilled in the art can realize various alternatives, modifications, variations, or improvements from the contents disclosed in this specification, and these are included in the technical scope described in the appended claims.

[0046] For example, the above embodiment can be modified as follows.

[0047] (Modification 1) FIG. 10A is a diagram schematically illustrating a longitudinal section of a cell culture device 100-1 according to Modification 1. FIG. 10B is a diagram schematically illustrating a transverse section of the culture tank 10 shown in FIG. 10A. As shown in FIGS. 10A and 10B, the cell culture device 100-1 according to Modification 1 has a configuration in which the culture tank 10 is concentrically partitioned into three spaces using two cylindrical members 20, 20-2. Note that the cylindrical member 20-2 (second cylindrical member) has the same configuration as the cylindrical member 20 except for its diameter. Similar to the second space R2, only the culture medium 50 is stored in the third space R3 formed on the outer periphery of the culture tank 10 (in other words, the third space R3 formed radially outward of the first space R1), and the scaffold 40 does not enter (is not present).

[0048] In this first modification, a circulation flow path C1 is provided for circulating the culture solution 50 between the third space R3 and the second space R2. A circulation pump 68 is provided in the circulation flow path C1, and when the circulation pump 68 is driven, the culture solution 50 in the third space R3 is sucked by the circulation pump 68 and discharged (returned) into the second space R2 via the circulation flow path C1. The circulation flow path C1 and the circulation pump 68 correspond to the culture solution circulating means of the present invention.

[0049] When the culture solution 50 in the third space R3 is sucked by the circulation pump 68, the fluid pressure in the third space R3 drops slightly, causing the fluid pressure in the second space R2 to increase relatively. As a result, the culture solution 50 flows from the second space R2 through the holes 21 in the cylindrical members 20 and 20-2 into the third space R3, and the culture solution 50 is circulated efficiently.

[0050] This allows the supply of culture medium 50 to scaffold 40 to be more uniform, further improving cell culture efficiency.

[0051] In this modified example 1, the culture solution 50 is sucked from the third space R3 and returned to the second space R2, but the culture solution 50 may be sucked from the second space R2 and returned to the third space R3. Even with this configuration, the culture solution 50 can be circulated to evenly supply the culture solution 50 to the scaffold 40 in the first space R1.

[0052] (Variation 2) Figure 11A is a diagram schematically showing a cross section of a cell culture device 100-2 according to Variation 2, and Figure 11B is an enlarged view of part B shown in Figure 11A. As shown in Figure 11A, the cell culture device 100-2 according to Variation 2 is configured in the same way as Variation 1, with the culture tank 10 being concentrically partitioned into three spaces using two cylindrical members 20, 20-2. Furthermore, the first space R1 is partitioned circumferentially into four equal parts by a plurality of rectifying plates 45, and is divided into four spaces, namely, a first division space R1-1, a second division space R1-2, a third division space R1-3, and a fourth division space R1-4.

[0053] The second space R2 and the third space R3 are connected to each other via a flow path 46 formed between a pair of straightening plates 45. In this embodiment, as shown in Fig. 11A, four pairs of straightening plates 45 are provided at 90-degree intervals in the circumferential direction, and a flow path 46 is formed between each pair of straightening plates 45. Although not shown, the straightening plates 45 are provided with a large number of holes or slits. Of course, the number and shape of the holes or slits are arbitrary.

[0054] Next, the effects of Modification 2 will be described. When the culture solution 50 flows from the second space R2 to the first space R1 as described above, turbulence (not shown) may occur if a scaffold 40 with an uneven shape is used. Because controlling turbulence is difficult, molecular uniformity of nutrients, oxygen, etc. within the culture tank 10 may be reduced, potentially adversely affecting culture efficiency. Therefore, as shown in Modification 2, by forming a flow path 46 connecting the second space R2 and the third space R3 between a pair of straightening plates 45, it is possible to create an area where laminar flow F1 is always present. In this way, by providing a flow path 46 for creating a laminar flow F1 that can control the flow of the culture solution 50, the occurrence of turbulence in the first space R1 is reduced, molecular uniformity within the culture tank 10 is improved, and culture efficiency is improved.

[0055] The number of rectifying plates 45, i.e., the number of flow paths 46, may be any number. An optimal number of flow paths 46 may be provided depending on conditions such as the size of the culture tank 10 and the shape of the scaffold 40.

[0056] Furthermore, as shown in FIG. 11B, by providing an R-shape 47 at the inlet of the flow path 46, the inlet loss (resistance) of the flow path 46 can be reduced, thereby allowing the culture medium 50 to flow smoothly from the second space R2 to the third space R3, and maintaining a good culture environment.

[0057] (Variation 3) Figures 12A to 12D show variations in the shape of the fermenter 10. Figure 12A shows an example of a configuration in which the internal space of a cylindrical fermenter 10 is partitioned concentrically, corresponding to the first embodiment. Figure 12B shows an example of a configuration in which the internal space of a cylindrical fermenter 10-1 is partitioned in the vertical direction. Figure 12C shows an example of a configuration in which the internal space of a rectangular box-shaped fermenter 10-2 is partitioned in the horizontal direction. Figure 12D shows an example of a configuration in which the internal space of a rectangular box-shaped fermenter 10-3 is partitioned in the vertical direction. As shown in these configuration examples, the fermenter according to the present invention can have any shape, and the internal space can be partitioned in any direction. Furthermore, the number of partitioned spaces does not have to be two. It may be partitioned into three or more spaces (see Figures 10A and 10B).

[0058] 12B, 12C, and 12D, the volume ratio between the first space R1 and the second space R2 can be changed by moving the partition plates 23-1, 23-2, and 23-3. In this case, the partition plates 23-1, 23-2, and 23-3 can be connected to a slide mechanism, for example, and the slide mechanism can be driven by the controller 70 to change the volume ratio. Alternatively, a configuration in which the partition plates 23-1, 23-2, and 23-3 are operated like diaphragms to change the volume ratio can also be applied.

[0059] (Modification 4) Figure 13 is a diagram schematically illustrating a longitudinal cross section of a cell culture device 100-3 according to Modification 4. As shown in Figure 13, the cell culture device 100-3 according to Modification 4 has a configuration in which the culture tank 10 is concentrically divided into three spaces using two cylindrical members 20, 20-2, and further divided into two spaces in the vertical direction using a disk member 80. The cylindrical member 20-2 (second cylindrical member) has the same configuration as the cylindrical member 20 except for its diameter. The disk member 80 also has a large number of holes or is formed in a mesh-like shape, and these large number of holes or meshes allow the culture solution 50 to pass through but prevent the scaffold 40 from passing through.

[0060] Therefore, in the third space R3 formed on the outer periphery of the culture tank 10, only the culture solution 50 is stored, similar to the second space R2, and the scaffolding 40 does not enter (is not present). Similarly, in the fourth space R4 formed in the lower part of the culture tank 10 separated by the disk member 80, only the culture solution 50 is stored, and the scaffolding 40 does not enter.

[0061] The cell culture device 100-3 is provided with a circulation flow path C1 through which the culture medium 50 circulates between the third space R3, the second space R2, and the fourth space R4. A circulation pump 68 is provided in the circulation flow path C1, and when the circulation pump 68 is driven, the culture medium 50 in the third space R3 and the fourth space R4 is sucked in by the circulation pump 68 and discharged (returned) into the second space R2 via the circulation flow path C1. The circulation flow path C1 and the circulation pump 68 correspond to the culture medium circulating means of the present invention.

[0062] When the culture solution 50 in the third space R3 and the fourth space R4 is sucked by the circulation pump 68, the fluid pressure in the third space R3 and the fourth space R4 drops slightly, causing the fluid pressure in the second space R2 to increase relatively. This causes the culture solution 50 to flow from the second space R2 to the third space R3 and the fourth space R4 through the holes 21 in the cylindrical members 20 and 20-2, respectively, and the culture solution 50 is circulated efficiently. This further uniforms the supply of the culture solution 50 to the scaffold 40, further improving cell culture efficiency.

[0063] Furthermore, in Modification 4, a shelf member 81 is provided at a position that divides the culture tank 10 into approximately two equal parts in the vertical direction. Like the disk member 80, this shelf member 81 also has a large number of holes or is formed in a mesh shape, allowing the culture solution 50 to pass through but not the scaffolds 40. Therefore, a flow space T through which the culture solution 50 can flow can be formed directly below the shelf member 81. The culture solution 50 passes through this flow space T, thereby further improving the culture efficiency of cells attached to the scaffolds 40.

[0064] Here, the shelf member 81 may be attached to the inner peripheral surface of the cylindrical member 20-2 by any suitable means. For example, if the shelf member 81 is configured to be engaged with a protrusion (not shown) provided on the cylindrical member 20-2, the shelf member 81 can be easily attached and detached, which makes it easy to replace the scaffolding 40.

[0065] In this fourth modification, the culture solution 50 is sucked from the third space R3 and the fourth space R4 and returned to the second space R2, but the culture solution 50 may be sucked from the second space R2 and returned to the third space R3 and the fourth space R4. The disk member 80 and the shelf member 81 may be made of the same material, or may have holes or meshes of different sizes.

[0066] (Variation 5) Figure 14 is a diagram schematically illustrating a longitudinal cross section of a cell culture device 100-4 according to Variation 5. As shown in Figure 14, the cell culture device 100-4 according to Variation 5 has four baskets 85-1, 85-2, 85-3, and 85-4 stacked coaxially inside the culture tank 10 as partition means for dividing the internal space. The lowest basket 85-4 is supported via a support member 87, and the four baskets 85-1, 85-2, 85-3, and 85-4 are disposed at a distance equal to the height of the support member 87 from the bottom 12 of the culture tank 10. In other words, a space (fourth space R4) is formed between the lowest basket 85-4 and the bottom 12 of the culture tank 10.

[0067] Next, the details of the structure of the baskets 85-1, 85-2, 85-3, and 85-4 will be described. Figure 15A is a plan view of the basket 85-1, and Figure 15B is a vertical cross-sectional view of the basket 85-1. Since the four baskets 85-1, 85-2, 85-3, and 85-4 have the same configuration, only the basket 85-1 will be described here.

[0068] 15A and 15B, the basket 85-1 has an inner cylinder 85a-1, an outer cylinder 85b-1, and an annular bottom plate 85c-1 that connects the lower portions of the inner cylinder 85a-1 and the outer cylinder 85b-1. The inner cylinder 85a-1, the outer cylinder 85b-1, and the bottom plate 85c-1 all have a large number of holes or are formed in a mesh-like shape, and these holes or meshes allow the culture solution 50 to pass through but prevent the scaffold 40 from passing through. With this configuration, the scaffold 40 is placed on the bottom plate 85c-1 and is contained in the space between the inner cylinder 85a-1 and the outer cylinder 85b-1.

[0069] When the baskets 85-1, 85-2, 85-3, and 85-4 are stacked, as shown in FIG. 14, 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 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.

[0070] Furthermore, in the fifth modification, four agitating blades 33 are provided, which is the same number as the number of stages of the baskets 85-1, 85-2, 85-3, and 85-4. This allows the culture solution 50 to circulate efficiently within each basket by each agitating blade 33. Of course, the number of agitating blades 33 may be less than the number of stages of the baskets.

[0071] In this fifth modification, when scaffolds 40 are housed in baskets 85-1, 85-2, 85-3, and 85-4, a flow space T through which the culture medium 50 flows can be formed above each basket. Therefore, the culture medium 50 circulates through the flow space T of each basket as shown by the white arrows in the figure. This allows cells attached to the scaffolds 40 to be cultured efficiently.

[0072] In the fifth modified example, the shape of the basket does not necessarily have to be cylindrical. For example, a basket of any shape, such as a rectangular cylindrical basket with a bottom, can be used.

[0073] (Variation 6) Variation 6 is an improvement on the structure of the basket in the cell culture device 100-4 according to Variation 5. Fig. 16A is a plan view of baskets 85-1, 85-2, 85-3, and 85-4 according to Variation 6, and Fig. 16B is a longitudinal cross-sectional view of baskets 85-1, 85-2, 85-3, and 85-4 according to Variation 6 in a stacked arrangement, which is a cross-sectional view taken along XVIB-XVIB in Fig. 16A.

[0074] 16A and 16B, the top basket 85-1 has three passages 86-1a, 86-1b, and 86-1c. In this example, the three passages 86-1a, 86-1b, and 86-1c may have the same shape or different shapes. The following explanation will be given on the assumption that the three passages 86-1a, 86-1b, and 86-1c have the same shape.

[0075] The passage 86-1a is made of a cylinder that penetrates the bottom plate 85c-1 of the basket 85-1. The height (axial length) of the passage 86-1a is approximately 1 / 2 to 2 / 3 of the height of the basket 85-1. The passage 86-1a is formed with a diameter that allows the scaffolding 40 to pass through.

[0076] The basket 85-2, which is the second from the top, is provided with two passages 86-2a and 86-2b, each having the same shape as the passage 86-1a, and the passage 86-2a and the passage 86-2b are provided with the same center as the uppermost passage 86-1a and the passage 86-1b, respectively, in a plan view. The basket 85-3, which is the third from the top, is provided with one passage 86-3a, which is the same shape as the passage 86-1a, and the passage 86-3a is provided with the same center as the uppermost passage 86-1a and the second passage 86-2a, respectively, in a plan view. The lowest basket 85-4 is not provided with a passage.

[0077] When the baskets configured in this manner are stacked and arranged, as shown in Figure 16A, the passages 86-1a, 86-2a, and 86-3a are aligned in a planar view (arranged on the Z1 axis), so when the scaffolding 40 is inserted into the passage 86-1a along the Z1 axis, as shown in Figure 16B, the scaffolding 40 passes through the passages 86-1a, 86-2a, and 86-3a in that order, and falls into the space between the inner tube 85a-4 and outer tube 85b-4 of the lowest basket 85-4, where it is stored.

[0078] Similarly, since passage 86-1b and passage 86-2b are aligned in a plan view (positioned on the Z2 axis), when scaffolding 40 is inserted into passage 86-1b along the Z2 axis as shown in Figure 16B, the scaffolding 40 passes through passage 86-1b and passage 86-2b in that order, and falls into the space between the inner tube 85a-3 and outer tube 85b-3 of the third-tier basket 85-3, where it is stored.

[0079] Furthermore, when storing the scaffolding 40 in the basket 85-2, which is the second from the top, the scaffolding 40 can be dropped into the passage 86-1c along the Z3 axis.

[0080] According to this sixth modification, the scaffolding 40 can be easily placed into the baskets 85-1, 85-2, 85-3, and 85-4, thereby improving work efficiency compared to the fifth modification.

[0081] For example, passages 86-1a, 86-2a, and 86-3a do not necessarily have to be arranged strictly on the Z1 axis, and as long as the scaffolding 40 can be smoothly dropped, all passages do not necessarily have to be arranged coaxially. In a plan view, it is sufficient that the passages at least overlap. The same applies to the arrangement of passages 86-1b and 86-2b.

[0082] Furthermore, it goes without saying that the above-described modifications 1, 2, 3, 4, 5, and 6 can also be applied to the cell culture device according to the second embodiment.

[0083] The present invention can also be applied to an apparatus for culturing cells other than animal-derived cells (e.g., plant-derived cells). Furthermore, when the culture vessel 10 is small, a magnetic stirrer, for example, may be used as the stirring means 30. This has the advantage of simplifying the structure.

[0084] The holes 21 formed in the cylindrical members 20, 20-1, and 20-2 may have any shape. For example, they may be round, rectangular, or elongated. The present invention can also be applied to cylindrical members having a large number of slits.

[0085] Furthermore, in the above-described embodiment, the scaffolding 40 is recovered and replenished using the level switches 65, 66. However, a weight sensor or the like may be used instead of the level switches 65, 66. For example, the weight of the culture tank 10 when the culture medium 50 is filled to the appropriate level PL and the scaffolding 40 is sufficiently introduced is set as the first threshold, and a weight that is approximately 50% of the first threshold is set as the second threshold. Then, if the solenoid valve 60 is closed when the weight sensor detects the second threshold (corresponding to S43 and S44 in FIG. 5), and the scaffolding 40 recovery and replenishment process is terminated when the weight sensor detects the first threshold (corresponding to S46 in FIG. 5), control similar to that of the first embodiment can be performed.

[0086] Of course, instead of using the controller 70, the procedure shown in FIG. 4 may be performed manually by an operator.

[0087] DESCRIPTION OF SYMBOLS 10 Culture tank 11 Body 12 Bottom 13 Lid 15, 16 Supply pipe 20, 20-1 Cylindrical member (partition means) 20-2 Cylindrical member (second cylindrical member / partition means) 21 Hole 23-1, 23-2, 23-3 Partition plate (partition means) 25 Expanding / contracting means 30 Stirring means 31 Motor 32 Rotating shaft 33 Stirring blade 40 Scaffolding 45 Straightening plate 46 Flow path 50 Culture solution 60 Solenoid valve 65, 66 Level switch 68 Circulation pump (culture solution circulation means) 70 Controller 80 Disk member (partition means) 81 Shelf member 85-1, 85-2, 85-3, 85-4 Basket (partition means) 85a-1, 85a-2, 85a-3, 85a-4 Inner cylinder 85b-1, 85b-2, 85b-3, 85b-4 Outer cylinder 85c-1, 85c-2, 85c-3, 85c-4 Bottom plate 86-1a, 86-1b, 86-1c Passage path 86-2a, 86-2b Passage path 86-3a Passage path 87 Support member 100, 100-1, 100-2, 200 Cell culture device C1 Circulation channel (culture solution circulation means) E1 to E4 Electrical wiring R1 First space R1-1, R1-2, R1-3, R1-4 First divided space (first space) R2 Second space R3 Third space R4 Fourth space T Flow space

Claims

1. A cell culture device for culturing cells, comprising: a culture tank for storing a culture solution; a partition means for partitioning the culture tank into at least a first space and a second space; and an agitation means for agitating the culture solution, wherein the first space is a space for accommodating a scaffold to which the cells adhere, and the second space is a space in which the agitation means is provided, and the partition means is configured to allow the culture solution to pass through but not allow the scaffold to enter the second space from the first space.

2. The cell culture device according to claim 1, wherein the volume ratio between the first space and the second space is changed by the operation of the partitioning means.

3. A cell culture device as described in claim 1, characterized in that the partition means comprises a cylindrical member having a large number of holes or a mesh-like structure, the annular space between the culture tank and the cylindrical member is the first space, and the internal space of the cylindrical member is the second space.

4. A cell culture device as described in claim 3, characterized in that the cylindrical member is configured to be capable of shrinking and expanding in diameter within the culture tank, and the volume ratio between the first space and the second space changes as the cylindrical member shrinks or expands in diameter.

5. A cell culture device according to claim 1, further comprising a culture medium circulating means for sucking the culture medium stored in the first space and returning it to the culture tank.

6. A cell culture method using the cell culture device described in claim 1, comprising the steps of: introducing a scaffold into the first space and supplying the culture medium to the culture tank; driving the stirring means to stir the culture medium; recovering a portion of the scaffold from the first space; and introducing new scaffold into the first space and mixing it with the scaffold remaining in the first space.

7. A cell culture method using the cell culture apparatus of claim 2, comprising the steps of: introducing a scaffold into the first space and supplying the culture medium to the culture tank; driving the stirring means to stir the culture medium; recovering a portion of the scaffold from the first space; expanding the volume of the first space by operating the partitioning means; introducing new scaffold into the first space whose volume has been expanded and mixing it with the scaffold remaining in the first space; and returning the first space whose volume has been expanded to its original volume by operating the partitioning means.

8. A cell culture device as described in claim 3, characterized in that the culture tank further has a circular third space which is a space radially outside the first space and is separated from the first space via a second cylindrical member having a large number of holes or a mesh-like shape, and the second cylindrical member is configured to allow the culture medium to pass through but not allow the culture medium to enter the third space from the first space of the scaffold.

9. A cell culture device as described in claim 8, wherein the culture tank further has a fourth space which is a space below the first space and the second space and is separated from the first space and the second space by a disk member having a large number of holes or a mesh-like shape, and the disk member is configured to allow the culture medium to pass through but not allow the culture medium to enter the fourth space from the first space of the scaffold.

10. A cell culture device as described in claim 8 or 9, further comprising at least one shelf member on which the scaffolding is placed, the at least one shelf member being arranged to divide the first space in the vertical direction, and the at least one shelf member having a large number of holes or formed in a mesh shape, and configured to allow the culture medium to pass through but not allow the scaffolding to pass through.

11. A cell culture device as described in claim 1, wherein the culture tank is partitioned by the partitioning means into three spaces concentrically from the center, in the order of the second space, the first space, and the third space; the cell culture device further comprises a plurality of baskets as the partitioning means; each of the plurality of baskets comprises an inner tube having a large number of holes or formed in a mesh-like shape, an outer tube arranged concentrically with the inner tube and having a large number of holes or formed in a mesh-like shape, and an annular bottom plate having a large number of holes or formed in a mesh-like shape that connects the bottom between the inner tube and the outer tube, and is configured to be able to accommodate the scaffolding between the inner tube and the outer tube; the plurality of baskets are arranged in a stacked manner on the central axis of the culture tank; and in the culture tank, when the plurality of baskets are arranged in a stacked manner, the first space and the third space are partitioned by the plurality of outer tubes, and the first space and the second space are partitioned by the plurality of inner tubes.

12. A cell culture device as described in claim 11, wherein all of the plurality of baskets except for the lowest basket have at least one cylindrical passageway penetrating their respective bottom plates and through which the scaffold can pass, and the number of passageways increases from the lower to the upper rows of the plurality of baskets.

13. The cell culture device according to claim 12, wherein the central positions of the passages coincide when the stacked baskets are viewed from the axial direction of the culture tank.

14. The cell culture device according to claim 11, wherein the basket located at the lowest level among the plurality of baskets is supported at a distance from the bottom of the culture tank.

Images