Cell culture system, cultured meat, and culture supernatant

Abstract

Provided is a novel cell culture system that makes the culture of cells more efficient. The present invention relates to a cell culture system (S1) in which a plurality of cell culture devices (100, 200, 300) are connected and a culture solution (50) moves among the plurality of cell culture devices, and the cell culture system (S1) controls the liquid level of the culture solution stored in each of the plurality of cell culture devices so that the liquid level of the culture solution in each of the plurality of cell culture devices is adjusted to the liquid level of the culture solution in each of the plurality of cell culture devices. The cells of at least one cell culture device are in a first state in which the cells are fully covered with the culture solution, and the cells of the remaining cell culture devices are in a second state in which the cells are not fully covered with the culture solution.

Description

Technical Field

[0001] The present invention relates to a cell culture system formed by connecting a plurality of cell culture devices, and cultured meat and culture supernatant produced by the cell culture system. Background Art

[0002] In recent years, in order to achieve the "United Nations Sustainable Development Goals (SDGs)", countries around the world have been developing truly sustainable food production systems, among which development related to "cultured meat" is expanding. In addition, as background technology in this technical field, for example, Patent Document 1 describes a cell culture device having a first chamber and a second chamber. In Patent Document 1, the second chamber is, for example, formed in a bellows shape, and the second chamber is expanded and contracted by a volume adjustment unit, thereby forming a structure in which liquid and air can flow between the two chambers.

[0003] Furthermore, the cell culture device of Patent Document 1 has a growth substrate unit for cell attachment and growth in the first chamber. The growth substrate unit receives oxygen through the expansion and contraction of the second chamber, or is immersed in the culture medium, and is intermittently and periodically exposed to the gas environment indirectly through the rarefied gas / growth substrate interface. In this way, cell growth and the production of cell products are promoted.

[0004] Prior art literature

[0005] Patent Literature

[0006] Patent document 1: Japanese Patent No. 4430317. Summary of the invention

[0007] Problem that the invention aims to solve

[0008] However, Patent Document 1 discloses only the structure and use method (control method) of a cell culture device alone, and does not disclose any specific structure of a cell culture system combining a plurality of cell culture devices and its control method.

[0009] The present invention is completed in view of such actual conditions, and its main purpose is to provide a new cell culture system that makes cell culture more efficient. In addition, another purpose of the present invention is to provide cultured meat and culture supernatant produced by the cell culture system.

[0010] Solutions for solving problems

[0011] In order to achieve the above-mentioned purpose, the first aspect of the present invention is a cell culture system, characterized in that it is connected to multiple cell culture devices, and the culture fluid moves between the multiple cell culture devices. The cell culture system controls the liquid level of the culture fluid stored in each of the multiple cell culture devices so that among the multiple cell culture devices, the cells of at least one of the cell culture devices are in a first state of being fully covered with the culture fluid, while the cells of the remaining cell culture devices are in a second state of not being fully covered with the culture fluid.

[0012] A second aspect of the present invention is a culture supernatant produced by the above-mentioned cell culture system.

[0013] A third aspect of the present invention is cultured meat produced by the above-mentioned cell culture system.

[0014] Effects of the Invention

[0015] According to the present invention, a new cell culture system that makes cell culture more efficient can be provided. In addition, cultured meat and culture supernatant produced by the cell culture system can be provided. In addition, the above-mentioned problems, structures and effects become clearer through the description of the following embodiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 This is an overall structural diagram of the cell culture system according to the first embodiment.

[0017] Figure 2 It is a diagram schematically showing a longitudinal section of a cell culture device.

[0018] Figure 3 It is a diagram schematically showing a top view of a cell culture device.

[0019] Figure 4 A top view showing details of the carrier.

[0020] Figure 5 The graphs show the results of cell culture experiments.

[0021] Figure 6 The graphs are graphs showing the experimental results of cell numbers and glucose consumption.

[0022] Figure 7 This is an overall structural diagram of a cell culture system according to a second embodiment. DETAILED DESCRIPTION

[0023] 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.

[0024] (First embodiment)

[0025] A cell culture system S1 according to a first embodiment of the present invention will be described. Figure 1 It is a diagram showing the overall structure of the cell culture system S1 according to the first embodiment. Figure 1 The cell culture system S1 shown is formed by, for example, three cell culture apparatuses 100, 200, 300 and one buffer tank 400 connected in a ring shape (in series) via flow paths L1 to L4. Here, the flow paths L1 to L4 are, for example, tubes or hoses.

[0026] The cell culture apparatuses 100, 200, and 300 are placed on the balances 80, 81, and 82, respectively. Then, different animal cells are supplied to the cell culture apparatuses 100, 200, and 300. Examples of animal cells 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. In this embodiment, for example, lung-derived cells are supplied to the cell culture apparatus 100, liver-derived cells are supplied to the cell culture apparatus 200, and muscle-derived cells are supplied to the cell culture apparatus 300.

[0027] The buffer tank 400 is a stainless steel or glass container for storing a culture supernatant, and the interior is sealed by closing the lid 405 .

[0028] Pumps 65 to 68 are provided in the respective flow paths L1 to L4. The pumps 65 to 68 are driven by electric motors (not shown). The flow rates of the pumps 65 to 68 are changed by changing the rotation speed of the electric motors.

[0029] Pump 65 sucks the culture solution 50 in the buffer tank 400 and discharges it to the cell culture device 100. Pump 66 sucks the culture solution 50 in the cell culture device 100 and discharges it to the cell culture device 200. Pump 67 sucks the culture solution 50 in the cell culture device 200 and discharges it to the cell culture device 300. Pump 68 sucks the culture solution 50 in the cell culture device 300 and discharges it to the buffer tank 400. With this structure, when all pumps 65 to 68 are driven, the culture solution 50 flows along Figure 1 Circulate in the direction of the arrow.

[0030] The controller 70 is connected to the pumps 65 to 68 (more specifically, the motors driving the pumps 65 to 68) via the electrical wirings E1 to E4, and is connected to the scales 80, 81, and 82 via the electrical wirings E5 to E7. The weight data of the cell culture apparatuses 100, 200, and 300 detected by the scales 80, 81, and 82 is input to the controller 70 via the electrical wirings E5 to E7. In addition, the controller 70 is electrically connected to various sensors (not shown) respectively installed in the cell culture apparatuses 100, 200, and 300, and the sensor data (such as the temperature and pH value of the culture solution) detected by the various sensors is input to the controller 70.

[0031] The controller 70 is configured to include a processing unit having a CPU, a ROM and a RAM as storage devices, and other peripheral circuits. The ROM stores a control program, and the CPU reads the control program to execute various processes. The controller 70 controls, for example, the driving of the pumps 65 to 68 based on various input information (details will be described later).

[0032] Next, the structures of the cell culture apparatuses 100, 200, and 300 will be described in detail. The cell culture apparatus 100 and the cell culture apparatuses 200 and 300 have the same structure except that the types of animal cells to be cultured are different, and thus the description of the cell culture apparatuses 200 and 300 will be omitted.

[0033] Figure 2 2 is a diagram schematically showing a longitudinal section of the cell culture apparatus 100. Figure 3 Schematic diagram of a top view of the cell culture device 100. Figure 2 and Figure 3 As shown, the cell culture apparatus 100 includes a culture tank 1 , a mounting rack 20 , and a stirring unit 30 .

[0034] The culture tank 1 has a cylindrical container with a bottom formed by a body portion 3 and a bottom portion 4, and a disc-shaped cover 5 covering the upper portion of the body portion 3. The culture tank 1 stores a culture solution 50 therein. The culture tank 1 is made of, for example, stainless steel. A flange 2 protruding radially outward is provided at the upper portion of the body portion 3, and a disc-shaped cover 5 is mounted on the flange 2 via a sealing member such as an O-ring not shown. As a result, the interior of the culture tank 1 is sealed. In addition, in the present embodiment, the flange 2 and the cover 5 are fixed using butterfly screws, but the fixing method is not limited to this.

[0035] A waste liquid nozzle 8 serving as a drain pipe is provided at the bottom 4 of the culture tank 1. A flange 9 protruding radially outward is provided at the lower end of the waste liquid nozzle 8, and a blind flange 10 is installed on the flange 9 via a sealing member such as an O-ring. Therefore, under normal conditions, the waste liquid nozzle 8 is closed by the blind flange 10. Moreover, by removing the blind flange 10, the culture solution 50 stored in the culture tank 1 can be discharged. Of course, a valve such as an isolation valve can be provided instead of the blind flange 10, and the culture solution 50 can be discharged by opening and closing the valve.

[0036] An elongated observation window 7 is provided in the body portion 3 of the culture tank 1 in the vertical direction. The observation window 7 is formed of, for example, a transparent glass plate, and the interior of the culture tank 1 can be viewed through the observation window 7. For example, the user can check the liquid level of the culture solution 50 through the observation window 7, or can visually observe the state of the carrier 40 (the growth state of the animal cell) described later.

[0037] Furthermore, a plurality of (e.g., four) protrusions (holding portions) 12 are provided at the same height along the circumferential direction at the lower portion of the inner peripheral surface of the body portion 3. These protrusions 12 are formed to a size that can support the carrier rack 20 described later from below. That is, the protrusions 12 function as stoppers for limiting the position of the carrier rack 20 in the vertical direction. In addition, as long as it is a structure that can hold the carrier rack 20 at a predetermined height position, a structure in which, for example, legs are provided on the carrier rack 20, a structure in which the carrier rack 20 is suspended from the cover body 5, etc., can be applied instead of the protrusions 12.

[0038] Furthermore, a plurality of (for example, four) support legs 6 are provided at the lower portion of the outer peripheral surface of the carcass portion 3. The culture tank 1 is supported by these support legs 6. In addition, when the culture tank 1 is large, a skirt plate may be used instead of the support legs 6.

[0039] The cover 5 is described. Figure 3 As shown in FIG. 1 , the cover body 5 is provided with a plurality of (e.g., eight) mounting holes 5a to 5h along the circumferential direction. Various sensors, tubes, etc. are mounted in these mounting holes 5a to 5h. Figure 2 As shown, for example, a culture solution supply pipe 16 for supplying a culture solution 50 to the culture tank 1 is provided in the mounting hole 5a, and a culture solution delivery pipe 17 for delivering the culture solution 50 in the culture tank 1 to the outside (specifically, the cell culture device 200) is provided in the mounting hole 5e. In addition, the culture solution supply pipe 16 is connected to the flow path L1, and the culture solution delivery pipe 17 is connected to the flow path L2. Figure 1 As shown, the culture solution 50 discharged by the pump 65 flows through the flow path L1 and is supplied to the culture tank 1 (cell culture device 100) from the culture solution supply pipe 16. When the culture solution 50 in the culture tank 1 is sucked by the pump 66, it flows through the flow path L2 via the culture solution delivery pipe 17 and is delivered to the cell culture device 200.

[0040] Here, if Figure 2 As shown, the lower end of the culture solution supply pipe 16 extends to a position lower than the mounting rack 20. Therefore, when the culture solution 50 is supplied to the culture tank 1, the liquid level of the culture solution 50 does not shake too much, and the culture solution 50 can be stored in the culture tank 1. In addition, the lower end of the culture solution delivery pipe 17 also extends to a position lower than the mounting rack 20. Therefore, the culture solution 50 can be delivered to the outside until the liquid level of the culture solution 50 becomes a position lower than the mounting rack 20. Moreover, when the liquid level of the culture solution 50 becomes a position lower than the mounting rack 20, the mounting rack 20 (the carrier 40 mounted on the mounting rack 20) ​​is not covered with the culture solution 50 and is exposed to the air.

[0041] In addition, in the mounting holes 5a to 5h, in addition to the culture solution supply tube 16 and the culture solution delivery tube 17, sensors for detecting required physical quantities such as the composition, temperature, pH, and protein content of the culture solution 50; tubes such as a sampling tube, an animal cell supply tube, an alkali supply tube, and an oxygen supply tube; sensors and tubes required for culturing animal cells; or plugs for ventilation, etc. can be arbitrarily mounted. In addition, a motor 31 of a stirring unit 30 described later is fixed to the central portion of the cover 5. In addition, although not shown, a tube connected to the outside air is provided in the cell culture device 100, and a filter is provided in the tube to prevent microorganisms and microscopic objects from mixing in from the outside air.

[0042] Next, the carrier rack 20 will be described. The carrier rack 20 is a disc-shaped component that can carry the carrier 40 described later. The carrier rack 20 is supported by four protrusions 12, is maintained at a position of a predetermined height from the bottom 4 of the culture tank 1, and divides the space of the culture tank 1 into two in the height direction (up and down direction). In addition, the predetermined height position can be appropriately determined in consideration of the volume of the culture tank 1, the amount of the culture solution 50, etc., but in practical application, a height position of about 1 / 3 of the length of the carcass 3 from the bottom 4 of the culture tank 1 is preferred.

[0043] Figure 4 2 is a top view showing details of the carrier 20. Figure 4 As shown, the carrier 20 has a ring 21 forming an outer frame and a metal mesh 22 formed by weaving, for example, stainless steel wires into a grid shape. The metal mesh 22 has a plurality of meshes (openings) 23 of a size through which the culture solution 50 can pass. The mesh size (that is, the size of the mesh) of the metal mesh 22 is appropriately determined based on the shape of the carrier 40, the size of the animal cells attached to the carrier 40, the viscosity and specific gravity of the culture solution 50, etc. In the present embodiment, for example, a metal mesh 22 of 100 to 150 meshes (the number of meshes per inch is 100 to 150) is used.

[0044] The metal mesh 22 is provided with a central hole 25 through which a rotating shaft 32 of a stirring unit 30 described later can be inserted, and small holes 24a to 24d through which a culture solution supply tube 16 , a culture solution transport tube 17 , and the like can be inserted.

[0045] A carrier 40 is placed on the carrier 20. The carrier 40 is used to attach animal cells and is composed of a three-dimensional porous structure. In this embodiment, the carrier 40 is edible. The carrier 40 contains, for example, protein and polysaccharides. Here, edible refers to a composition that can be safely ingested into the body. Cultured meat is produced by attaching animal cells to the carrier 40.

[0046] Next, the stirring unit 30 is described. The stirring unit 30 is used to stir the culture solution 50. Figure 2 As shown in FIG. 1 , the stirring unit 30 includes a motor 31 as a driving source, a rotating shaft 32, and a stirring blade (stirring portion) 33. That is, in the present embodiment, a propeller-type stirring unit 30 is used.

[0047] The motor 31 is fixed to the cover 5. The output shaft of the motor 31 is connected to the rotating shaft 32 via a coupling not shown. A plurality of (for example, three) stirring blades 33 are provided at the top of the rotating shaft 32. When the motor 31 rotates, the rotating shaft 32 rotates along with the rotation of the motor 31, and the three stirring blades 33 rotate in the horizontal direction with the rotating shaft 32 as the center. In this way, the culture solution 50 is stirred.

[0048] Here, the stirring blade 33 is arranged in the space between the mounting frame 20 and the bottom 4. Therefore, when the stirring blade 33 rotates, the culture solution 50 is gradually stirred from the lower side of the culture tank 1. In addition, in this embodiment, the motor 31 can be rotated forward and reversely based on the controller 70 (see Figure 1 ) control signal, with a specified period and at a specified rotation speed forward or reverse. Of course, the rotation direction of the motor 31 can also be one direction.

[0049] Next, the operation mode of the cell culture system S1 will be described.

[0050] The cell culture system S1 has an operation mode 1 in which the pumps 65 to 68 are stopped one by one and the remaining pumps are driven at a predetermined flow rate at the same time, and operation modes 2 to 4 in which only specific pumps among the pumps 65 to 68 are temporarily stopped. The controller 70 controls the cell culture system S1 according to the operation modes 1 to 4. The operation modes 1 to 4 are described in detail below.

[0051] (Operation mode 1)

[0052] Operation mode 1 is an operation mode in which the culture solution 50 is circulated while the driving of the pumps 65, 66, 67, and 68 is repeatedly temporarily stopped in the order of the pumps. When the controller 70 temporarily stops the pump 65, the supply of the culture solution 50 from the buffer tank 400 to the cell culture apparatus 100 is stopped. During this period, the controller 70 drives the remaining pumps 66, 67, and 68, and the culture solution 50 of the cell culture apparatus 100 is sucked by the pump 66, and the liquid level of the culture solution 50 of the cell culture apparatus 100 decreases. Then, when the liquid level of the culture solution 50 of the cell culture apparatus 100 decreases to a position lower than the predetermined height of the mounting rack 20, the carrier 40 mounted on the mounting rack 20 is not covered with the culture solution 50 and is exposed to the air (second state). At this time, the liquid levels of the culture solutions 50 of the other cell culture apparatuses 200 and 300 are at a position higher than the mounting rack 20 (first state).

[0053] Next, when the controller 70 temporarily stops the pump 66 and drives the remaining pumps 65, 67, and 68, the supply of the culture solution 50 to the cell culture apparatus 100 is restarted. On the other hand, since the supply of the culture solution 50 to the cell culture apparatus 200 is stopped, the liquid level of the culture solution 50 in the cell culture apparatus 200 is lowered, and the carrier 40 placed on the carrier 20 is exposed to the air. In this way, the controller 70 repeatedly temporarily stops the pumps 65, 66, 67, and 68 in the order of the pumps, thereby successively lowering the liquid level of the culture solution 50 in each of the cell culture apparatuses 100, 200, and 300. That is, the operation mode 1 is a mode in which the carriers 40 in the cell culture apparatuses 100, 200, and 300 are successively exposed to the air by temporarily stopping the pumps 65, 66, 67, and 68 in order. In addition, in the present embodiment, the time for which the carrier 40 is exposed to the air is about 30 seconds to 180 seconds.

[0054] Here, the controller 70 determines that the liquid level of the culture solution 50 is at a predetermined position lower than the mounting rack 20 based on the weight data from the scales 80, 81, and 82. Specifically, the weight of the cell culture apparatus 100, 200, and 300 when the liquid level of the culture solution 50 is at a predetermined position is stored in the controller 70 as a predetermined threshold value, and when the input weight data becomes less than the threshold value, the controller 70 determines that the liquid level of the culture solution 50 of the cell culture apparatus 100, 200, and 300 has dropped to a position lower than the mounting rack 20. In addition, in the present embodiment, the scales 80, 81, and 82 are used to detect the height position of the liquid level, but instead of this structure, a level sensor, an ultrasonic liquid level sensor, or the like may be used to detect the height position of the liquid level of the culture solution 50.

[0055] (Operation mode 2)

[0056] Operation mode 2 is a mode in which only the liquid level of the culture solution 50 of the cell culture apparatus 100 is lowered to temporarily expose the carriers 40 to the air, while the carriers 40 of the other cell culture apparatuses 200 and 300 are always covered with the culture solution 50 .

[0057] Specifically, the controller 70 temporarily stops the pump 65 and drives the pumps 66, 67, and 68. Then, when the liquid level of the culture solution 50 of the cell culture apparatus 100 is at a height position lower than the mounting rack 20, the controller 70 stops all the pumps 65 to 68 for a predetermined time (e.g., 1 minute) to expose the carriers 40 of only the cell culture apparatus 100 to the air. At this time, the cell culture apparatuses 200 and 300 are in a state where the carriers 40 are covered with the culture solution 50. When the predetermined time has passed, the controller 70 controls in the following manner: first, the pump 65 is driven to fill the cell culture apparatus 100 with the culture solution 50, and then, the pumps 66, 67, and 68 are driven to circulate the culture solution 50 among the cell culture apparatuses 100, 200, and 300. In this way, the operation mode 2 is an operation mode in which only the cell culture apparatus 100 is in a state in which the carriers 40 are temporarily exposed to the air, and the carriers 40 of the other cell culture apparatuses 200 and 300 are always covered with the culture solution 50.

[0058] (Operation mode 3)

[0059] Operation mode 3 is a mode in which only the liquid level of the culture solution 50 of the cell culture apparatus 200 is lowered to temporarily expose the carriers 40 to the air, while the carriers 40 of the other cell culture apparatuses 100 and 300 are always covered with the culture solution 50 .

[0060] Specifically, the controller 70 temporarily stops the pumps 65 and 66 and drives the pumps 67 and 68. Then, when the liquid level of the culture solution 50 of the cell culture apparatus 200 is at a lower height than the mounting rack 20, the controller 70 stops all the pumps 65 to 68 for a predetermined time (e.g., 1 minute) and makes only the cell culture apparatus 200 in a state where the carriers 40 are exposed to the air. At this time, the cell culture apparatuses 100 and 300 are in a state where the carriers 40 are covered with the culture solution 50. When the predetermined time has passed, the controller 70 controls in the following manner: first, the pumps 65 and 66 are driven to fill the cell culture apparatus 200 with the culture solution 50, and then, the pumps 67 and 68 are driven to circulate the culture solution 50 among the cell culture apparatuses 100, 200, and 300. In this way, the operation mode 3 is an operation mode in which only the cell culture apparatus 200 is in a state in which the carriers 40 are temporarily exposed to the air, and the carriers 40 of the other cell culture apparatuses 100 and 300 are always covered with the culture solution 50.

[0061] (Operation mode 4)

[0062] Operation mode 4 is a mode in which only the liquid level of the culture solution 50 of the cell culture apparatus 300 is lowered to temporarily expose the carriers 40 to the air, while the carriers 40 of the other cell culture apparatuses 100 and 200 are always covered with the culture solution 50 .

[0063] Specifically, the controller 70 temporarily stops the pumps 65, 66, and 67 and drives the pump 68. Then, when the liquid level of the culture solution 50 of the cell culture apparatus 300 is at a height position lower than the mounting rack 20, the controller 70 stops all the pumps 65 to 68 for a predetermined time (e.g., 1 minute) and makes only the cell culture apparatus 300 in a state where the carriers 40 are exposed to the air. At this time, the cell culture apparatuses 100 and 200 are in a state where the carriers 40 are covered with the culture solution 50. When the predetermined time has passed, the controller 70 controls in the following manner: first, the pumps 65, 66, and 67 are driven to fill the cell culture apparatus 300 with the culture solution 50, and then, the pump 68 is driven to circulate the culture solution 50 among the cell culture apparatuses 100, 200, and 300. In this way, the operation mode 4 is an operation mode in which only the cell culture apparatus 300 is in a state in which the carriers 40 are temporarily exposed to the air, and the carriers 40 of the other cell culture apparatuses 100 and 200 are always covered with the culture solution 50.

[0064] In this embodiment, there are: a mode (operation mode 1) in which the liquid level of the culture solution 50 of each of the cell culture devices 100, 200, and 300 is sequentially lowered, and the carriers 40 of each of the cell culture devices 100, 200, and 300 are sequentially exposed to the air; and a mode (operation modes 2 to 4) in which only the carriers 40 of a specific cell culture device are exposed to the air. Thus, the operation mode can be appropriately switched according to the culture conditions to efficiently culture animal cells.

[0065] In addition, the time for which the carrier 40 is exposed to the air can be arbitrarily set by inputting it into the controller 70 by the operator. Specifically, in the present embodiment, the operator can change the time within the range of 30 seconds to 180 seconds according to the culture conditions. Thus, the controller 70 appropriately controls the pumps 65 to 68 to expose the carrier 40 of the cell culture apparatus 100, 200, 300 to the air for the set time. Of course, the flow rate of the pumps 65 to 68 can also be arbitrarily set by inputting it into the controller 70.

[0066] (experiment)

[0067] The inventors of the present invention have experimentally confirmed the extent of the difference in the culture efficiency of animal cells between the conventional operation mode and the operation mode 1 of the present embodiment.

[0068] First, the existing operation mode (hereinafter referred to as the existing mode) is described. In the existing mode, pumps 65 to 68 are driven simultaneously at a specified flow rate. Thus, for example, when the flow rate of pumps 65 to 68 is set to 150 ml / min, the controller 70 controls each pump 65 to 68 at a specified rotation speed. Therefore, the culture solution 50 circulates between the cell culture devices 100, 200, 300 and the buffer tank 400 at 150 ml / min. In this case, the liquid level of the cell culture devices 100, 200, 300 is higher than the mounting rack 20 and remains approximately fixed. That is, in the existing mode, the animal cells are always covered with the culture solution 50.

[0069] On the other hand, as described above, in the operation mode 1 of the present embodiment, the animal cells (carriers 40) in the cell culture apparatuses 100, 200, and 300 are sequentially switched between a state covered with the culture solution 50 (first state) and a state exposed to the air (second state). Therefore, by measuring and comparing the extent of protein production in the two operation modes, the difference in culture efficiency between the two operation modes can be known.

[0070] The experimental conditions are as follows.

[0071] Temperature of culture medium 50: 37°C

[0072] pH value of culture medium 50: 7.2

[0073] Volume of culture medium 50: 350ml each

[0074] Carrier mass: 5.5g

[0075] Experimental methods:

[0076] In the conventional mode, the culture solution 50 is replaced periodically while the carrier 40 is always covered with the culture solution 50, and the mass of the protein contained in the culture solution 50 in the buffer tank 400 is measured. On the other hand, in the operation mode 1, the culture solution 50 is replaced periodically while the carrier 40 is exposed to the air for 1 minute and the carrier 40 is covered with the culture solution 50 for 15 minutes. Similarly, the mass of the protein contained in the culture solution 50 in the buffer tank 400 is measured.

[0077] Figure 5 1 is a graph showing the experimental results of the above-mentioned cell culture, and is a graph comparing the amount of protein produced in the conventional mode and in the operation mode 1 of the present embodiment. Figure 5 Graph G1 shows the experimental results of the conventional mode, and graph G2 shows the experimental results of the operation mode 1. Figure 5The horizontal axis represents the number of days, and the vertical axis represents the mass per unit volume (g / l) of the protein contained in the culture solution 50. Figure 5 In the figure, "MC" indicates the time when the culture solution 50 is replaced.

[0078] like Figure 5 As shown, in the graph G1 (conventional mode), the mass of protein increases slowly from P7 to P8 between the 0th day and the 10th day. The culture solution 50 is replaced on the 10th day as the glucose contained in the culture solution 50 is consumed, and as a result, the mass of protein decreases sharply from P8 to P5. Thereafter, the mass of protein decreases slowly until the 15th day, and further, the mass of protein decreases sharply to P3 when the culture solution 50 is replaced on the 15th day as the glucose contained in the culture solution 50 is consumed. Thereafter, the mass of protein decreases to the minimum value P1 on the 17th day, but gradually increases until the 20th day, and the mass of protein is P2 on the 20th day. Then, the culture solution 50 is replaced on the 20th day and the 27th day as the glucose contained in the culture solution 50 is consumed, and thereafter, the mass of protein stabilizes in the range of approximately P1 to P2.

[0079] On the other hand, in graph G2 (operation mode 1), the mass of protein increases slowly from P8 to P9 between the 0th day and the 10th day, and reaches the maximum value of P10 on the 13th day. Then, the mass of protein decreases from the 13th day to the 16th day. As the glucose contained in the culture solution 50 is consumed, the culture solution 50 needs to be replaced, so the culture solution 50 is replaced on the 16th day. As a result, the mass of protein decreases sharply from P9 to P4 on the 16th day. Thereafter, the mass of protein increases from the 16th day to the 18th day, and the mass of protein is P6 on the 18th day. Then, after the 18th day, the culture solution 50 is not replaced, and the mass of protein is maintained at approximately P6.

[0080] By comparing Graph G1 and Graph G2, the following facts are deduced.

[0081] (1) From day 0 to day 10, there is almost no difference in the mass of protein contained in the culture solution 50 between the conventional mode and operation mode 1.

[0082] (2) On the 10th day, in the conventional mode, the culture solution 50 needs to be replaced as glucose is consumed, but in the operation mode 1, the consumption of glucose is small, and the culture solution 50 does not need to be replaced on the 10th day. In addition, in the operation mode 1, the mass of protein increases after the 10th day.

[0083] (3) In the conventional mode, the culture solution 50 was replaced for the second time on the 15th day, and in the operation mode 1, the culture solution 50 was replaced for the first time on the 16th day. The protein in both of them decreased sharply, and there was almost no difference in the quality of the protein between the two on the 16th day (the conventional mode was P2, and the operation mode 1 was P4). However, after the 16th day, the difference in the quality of the protein between the conventional mode and the operation mode 1 widened, and in the conventional mode, the culture solution 50 was replaced on the 20th day and the 27th day. In contrast, in the operation mode 1, the culture solution 50 was replaced for the first time only on the 16th day, and there was no need to replace the culture solution 50 thereafter.

[0084] Next, in the above-mentioned experiment, the consumption amount of glucose contained in the culture solution 50 was measured in the conventional mode and the operation mode 1, and the result will be described. Figure 6 This is a graph showing the measurement results of the number of cells in the cell culture apparatus 200 and the glucose consumption in the buffer tank 400 in the above-mentioned experiment. Figure 6 (a) is a graph showing the results of measuring the amount of cells produced (the number of cells grown) per unit of glucose consumed by the cell culture apparatus 200 , that is, the yield of cell growth, in the conventional mode and the operation mode 1 . Figure 6 (b) is a graph showing the measurement results of the number of liver cells and glucose consumption in the conventional model. Figure 6 (c) is a graph showing the measurement results of the number of liver cells and the glucose consumption in operation mode 1.

[0085] like Figure 6 As shown in (a), in the conventional mode, the cell proliferation yield is 4.18E+06 (cell / glu-g), whereas in operation mode 1, the cell proliferation yield increases to 1.90E+08 (cell / glu-g), which is about 45 times that of the conventional mode. Figure 6 As shown in (b), in the conventional model, 37.4 g of glucose is consumed to increase the number of liver cells from 3.36E+08 to 4.92E+09. Figure 6 As shown in (c), in operation mode 1, 17.7 g of glucose was consumed in order to increase the number of liver cells from 3.51E+08 to 3.72E+09.

[0086] according to Figure 6 (b) and Figure 6(c) shows that there is no big difference in the liver cells of the cell culture device 200 on the 30th day in both modes, but the consumption of glucose in operation mode 1 is half (17.7 g) compared with the consumption in the existing mode (37.4 g). It can be seen that operation mode 1 significantly promotes the yield of animal cell culture compared with the existing mode. In other words, it can be seen that compared with the state in which the animal cells (carrier 40) are always covered with the culture solution 50, the state in which the animal cells are exposed to the air for about 1 minute every 15 minutes makes the culture efficiency of the animal cells higher. It can be speculated that the change in the metabolic state of the animal cells caused by exposing them to the air may improve the culture efficiency of the animal cells.

[0087] In addition, although the illustration is omitted, the inventors of the present invention conducted the same experiment by changing the time for which the carrier 40 (animal cell) was exposed to the air in the range of 30 seconds to 180 seconds. As a result, it was confirmed that there was a similar tendency in the difference in protein quality between the existing mode and operation mode 1.

[0088] According to the present embodiment described above, the following effects can be achieved.

[0089] By exposing the animal cells to air for a predetermined time (30 to 180 seconds) (second state), the culture efficiency can be greatly improved (about 2 times). In addition, the cell culture devices 100, 200, and 300 in which different animal cells are placed are connected in a ring shape, and the culture solution 50 is circulated between each cell culture device 100, 200, and 300, so that high-quality culture supernatant can be stored in the buffer tank 400. In addition, the liquid level of the culture solution 50 is controlled in such a way that the animal cells of the cell culture devices 100, 200, and 300 are sequentially exposed to air, so that the cells of the cultured meat cultured in each cell culture device 100, 200, and 300 can be efficiently obtained.

[0090] In addition, the cell culture apparatus 100, 200, 300 of the present embodiment has a simple structure and only has: a culture tank 1 that stores a culture solution 50; a carrier 20 that is disposed in the culture tank 1; and a stirring unit 30 that stirs the culture solution 50. Therefore, the cell culture apparatus 100, 200, 300 can be manufactured at a low cost. In other words, the culture efficiency of animal cells can be improved at a low cost. In addition, since the cell culture apparatus 100, 200, 300 itself has a simple structure, it can be assembled in various devices to easily construct a culture system for animal cells.

[0091] Moreover, simply by placing the carrier 40 on the carrier 20 and lowering the liquid level of the culture solution 50 to below the carrier 20, the animal cells (carriers 40) can be easily placed in a state (second state) where they are not covered with the culture solution 50. Furthermore, by placing the carrier 40 on the carrier 20, the animal cells can be easily attached to the carrier 40, and the culture efficiency is high. In addition, since the stirring blade 33 of the stirring unit 30 is disposed in the space below the carrier 20, the influence of the rotation of the stirring blade 33 on the carrier 40 can be suppressed to a minimum.

[0092] To explain this in detail, a vortex is generated when the stirring blade 33 rotates. This vortex is generated at a position lower than the carrier 20, and is buffered by the mesh 23 of the carrier 20 (metal mesh 22), and becomes a gentle flow toward the carrier 40. Therefore, it is possible to prevent the animal cells attached to the carrier 40 from peeling off, and it is possible to alleviate the pressure on the animal cells. In addition, the vortex of the culture solution 50 generated below the carrier 20 is buffered by the metal mesh 22 and rises slowly, so that the nutrients contained in the culture solution 50 can be uniformly supplied to the animal cells attached to the carrier 40.

[0093] As a result, according to this embodiment, the production time of cultured meat can be significantly shortened, and further contributes to the realization of the United Nations Sustainable Development Goals (SDGs).

[0094] In addition, although the culture of animal cells is fully promoted when the stirring blade 33 is located above the carrier 20, a vortex is generated above the carrier 40 anyway, so the adhesion effect of animal cells to the carrier 40 is reduced compared with the case where the stirring blade 33 is located below the carrier 20. Therefore, the position of the stirring blade 33 is more preferably below the carrier 20. In addition, the following structure can also be adopted, in which the stirring unit 30 has two stirring blades 33, one stirring blade 33 is arranged in the space above the carrier 20, and the other stirring blade 33 is arranged in the space below.

[0095] Furthermore, since the observation window 7 is provided in the present embodiment, the state inside the culture tank 1 can be easily visually observed, which facilitates use.

[0096] (Second embodiment)

[0097] Next, a cell culture system S2 according to a second embodiment of the present invention will be described. Figure 7 FIG. 2 is an overall structural diagram of a cell culture system according to a second embodiment. Figure 7As shown, the cell culture system S2 of the second embodiment is characterized by adopting the following structure, the cell culture devices 100, 200, and 300 are respectively connected in parallel with the buffer tank 400, and the culture solution 50 moves between the cell culture device 100 and the buffer tank 400, between the cell culture device 200 and the buffer tank 400, and between the cell culture device 300 and the buffer tank 400.

[0098] Furthermore, the cell culture system S2 includes a pump 91 provided in the flow path L11 between the cell culture apparatus 100 and the buffer tank 400, a pump 92 provided in the flow path L12 between the cell culture apparatus 200 and the buffer tank 400, and a pump 93 provided in the flow path L13 between the cell culture apparatus 300 and the buffer tank 400. The controller 70 controls the driving of the pumps 91, 92, 93 to allow the culture solution 50 in the buffer tank 400 to flow in the flow paths L11, L12, L13, respectively.

[0099] In addition, the cell culture apparatuses 100, 200, 300 and the buffer tank 400 are connected via flow paths L14, L15, L16 as drain pipe flow paths, and electromagnetic valves 85, 86, 87 are installed in the flow paths L14, L15, L16, respectively. More specifically, the waste liquid nozzle 8 (see Figure 2 ) and the buffer tank 400 are connected by a pipe or a hose to form flow paths L14, L15, L16, and electromagnetic valves 85, 86, 87 are provided in these flow paths L14, L15, L16. In addition, the electromagnetic valves 85, 86, 87 are closed in a normal state, and when opened according to a command from the controller 70, the culture solution 50 stored in the cell culture apparatus 100, 200, 300 is discharged and flows back to the buffer tank 400.

[0100] Next, operation modes 5 to 7 of the cell culture system S2 will be described.

[0101] (Operation mode 5)

[0102] Operation mode 5 is a mode in which only the liquid level of the culture solution 50 of the cell culture apparatus 100 is lowered to temporarily expose the carriers 40 to the air, and the carriers 40 of the other cell culture apparatuses 200 and 300 are always covered with the culture solution 50 .

[0103] In operation mode 5, the controller 70 controls the pump 91 to be temporarily stopped once every 15 minutes and the electromagnetic valve 85 to be fully opened. Thus, the culture solution 50 of the cell culture device 100 is discharged into the buffer tank 400. The controller 70 controls the electromagnetic valve 85 to be closed when the culture solution 50 is detected to be at a predetermined position lower than the height of the carrier 20 by a level sensor (not shown) or the like. The controller 70 maintains this state for, for example, 1 minute. Thus, the carrier 40 is exposed to the air for 1 minute. In addition, the holding time can be arbitrarily changed by setting it in the controller 70 by the operator.

[0104] On the other hand, the controller 70 controls the opening of the electromagnetic valves 86 and 87 in a manner that is balanced with the flow rates of the pumps 92 and 93 while continuing to operate the pumps 92 and 93. In this way, the cell culture apparatuses 200 and 300 maintain the liquid level of the culture solution 50 at a predetermined position higher than the carrier 20, and the carriers 40 of the cell culture apparatuses 200 and 300 are always covered with the culture solution 50.

[0105] (Operation mode 6)

[0106] Operation mode 6 is a mode in which only the liquid level of the culture solution 50 of the cell culture apparatus 200 is lowered to temporarily expose the carriers 40 to the air, and the carriers 40 of the other cell culture apparatuses 100 and 300 are always covered with the culture solution 50 .

[0107] In operation mode 6, the controller 70 temporarily stops the pump 92 every 15 minutes and controls the electromagnetic valve 86 to be fully opened. Thereafter, when the culture solution 50 of the cell culture apparatus 200 is at a predetermined position lower than the height of the rack 20, the electromagnetic valve 86 is fully closed and this state is maintained for, for example, 1 minute.

[0108] On the other hand, the controller 70 controls the openings of the electromagnetic valves 85 and 87 while continuing to operate the pumps 91 and 93 , so that the carriers 40 of the cell culture apparatuses 100 and 300 are always covered with the culture solution 50 .

[0109] (Operation mode 7)

[0110] Operation mode 7 is a mode in which only the liquid level of the culture solution 50 of the cell culture apparatus 300 is lowered to temporarily expose the carriers 40 to the air, and the carriers 40 of the other cell culture apparatuses 100 and 200 are always covered with the culture solution 50 .

[0111] In operation mode 7, the controller 70 controls the pump 93 to be temporarily stopped once every 15 minutes and the electromagnetic valve 87 to be fully opened. Thereafter, when the culture solution 50 of the cell culture apparatus 300 is at a predetermined position lower than the height of the mounting rack 20, the electromagnetic valve 87 is fully closed and this state is maintained for, for example, 1 minute.

[0112] On the other hand, the controller 70 controls the openings of the electromagnetic valves 85 and 86 while continuing to operate the pumps 91 and 92 so that the carriers 40 of the cell culture apparatuses 100 and 200 are always covered with the culture solution 50 .

[0113] According to the cell culture system S2, since the cell culture apparatuses 100, 200, 300 and the buffer tank 400 are connected in parallel, the operation modes 5 to 7 can be arbitrarily selected to control the state in which only specific cell culture apparatuses 100, 200, 300 are exposed to the air. That is, only the animal cells of the specific cell culture apparatuses 100, 200, 300 can be exposed to the air, thereby improving the culture efficiency.

[0114] Furthermore, if the controller 70 controls the operation of the cell culture system S2 so as to sequentially repeat the operation modes 5 to 7, the cell culture apparatuses 100, 200, and 300 can be sequentially exposed to the air as in the first embodiment.

[0115] Furthermore, since the cell culture apparatuses 100, 200, and 300 are connected in parallel, in addition to operation modes 5 to 7, an operation mode can be set in which any two of the cell culture apparatuses 100, 200, and 300 are simultaneously exposed to air. Therefore, the culture of animal cells can be promoted by culture conditions and culture methods different from those in the first embodiment.

[0116] As described above, according to the second embodiment, similarly to the first embodiment, different types of animal cells can be cultured efficiently and a culture supernatant rich in effective ingredients can be obtained.

[0117] In addition, the present invention is not limited to the above-mentioned embodiments, and various modifications can be made within the scope of the main purpose of the present invention, and all technical matters included in the technical ideas recorded in the scope of the claims are the objects of the present invention. The above-mentioned embodiments are only preferred examples, and those skilled in the art can realize various alternative examples, modified examples, variations or improved examples based on the contents disclosed in this specification, which are included in the technical scope recorded in the scope of the attached claims.

[0118] For example, the present invention can also be used as a device for culturing cells other than cells from animals (e.g., cells from plants, etc.). In addition, when the culture tank 1 is small, a magnetic stirrer, for example, can also be used as the stirring unit 30. In this case, there is an advantage that the structure can be simplified.

[0119] In addition, in the carrier 20, a stamped plate provided with a plurality of holes can also be used instead of the metal mesh 22. The size of the hole in this case can be, for example, about 100 microns in diameter. In addition, the shape of the hole is arbitrary. For example, it can be a round hole, a square hole, or a long hole. In addition, a plate provided with a plurality of slits can also be used as the carrier 20. In addition, a plurality of carriers 20 can also be arranged in multiple sections in the culture tank 1. In this case, each carrier 20 can also adopt a different size of the mesh 23 (i.e., different mesh sizes) or different pore sizes.

[0120] In addition, the number of cell culture devices is not limited to three, and the number of buffer tanks 400 is not limited to one. The buffer tank 400 may not be assembled in the system. In addition, in each embodiment, not all of the above-mentioned operation modes may be provided. That is, the cell culture system may have a part of the operation modes according to the culture conditions.

[0121] Furthermore, the same animal cells may be supplied to all of the cell culture apparatuses 100 , 200 , and 300 , or different animal cells may be supplied to only a part of the cell culture apparatuses 100 , 200 , and 300 .

[0122] Description of Reference Numerals

[0123] 1: culture tank;

[0124] 2: Flange;

[0125] 3: Carcass part;

[0126] 4: bottom;

[0127] 5: Cover;

[0128] 5a~5h: mounting holes;

[0129] 6: Supporting legs;

[0130] 7: Observation window;

[0131] 8: Waste liquid nozzle;

[0132] 9: Flange;

[0133] 10: Blind flange;

[0134] 12: convex;

[0135] 16: culture medium supply tube;

[0136] 17: Culture medium delivery tube;

[0137] 20: loading rack;

[0138] 21: Ring;

[0139] 22: Metal mesh;

[0140] 23: Mesh;

[0141] 24a~24d: small holes;

[0142] 25: center hole;

[0143] 27: Legs;

[0144] 30: stirring unit;

[0145] 31: Electric motor;

[0146] 32: Rotation axis;

[0147] 33: stirring blade;

[0148] 40: carrier;

[0149] 50: culture medium;

[0150] 65-68: Pump;

[0151] 70: controller;

[0152] 80-82: Balance;

[0153] 85-87: solenoid valve;

[0154] 91-93: Pump;

[0155] 100, 200, 300: cell culture device;

[0156] S1, S2: cell culture system.

Claims

1. A cell culture system, characterized in that: It is connected to a plurality of cell culture devices, and the culture fluid moves between the plurality of cell culture devices. The cell culture system controls the liquid level of the culture solution stored in each of the multiple cell culture devices so that among the multiple cell culture devices, the cells of at least one of the cell culture devices are in a first state of being fully covered with the culture solution, while the cells of the remaining cell culture devices are in a second state of not being fully covered with the culture solution.

2. The cell culture system according to claim 1, characterized in that The multiple cell culture devices are connected in a ring shape, and the culture solution circulates among the multiple cell culture devices.

3. The cell culture system according to claim 2, characterized in that The device further comprises a buffer tank connected between the plurality of cell culture devices and used for storing the culture solution.

4. The cell culture system according to any one of claims 1 to 3, characterized in that: The at least one cell culture device has: a culture tank storing the culture solution; a loading rack which divides the culture tank in the height direction and has an opening through which the culture solution can pass; as well as a stirring unit for stirring the culture solution, The carrier can carry the carrier to which the cells are attached.

5. The cell culture system according to claim 4, characterized in that: When the at least one cell culture device is in the second state, the liquid level of the culture solution is maintained at a position lower than that of the mounting rack.

6. The cell culture system according to claim 5, characterized in that: The second state is maintained for 30 seconds to 180 seconds.

7. Cultured meat produced by the cell culture system according to any one of claims 1 to 3.

8. A culture supernatant produced by the cell culture system according to any one of claims 1 to 3.

Images