Cell culture method, cell culture kit, and cell culture system

Abstract

To provide a cell culture method, capable of, in cell culturing using a culture vessel, suitably controlling an exchange rate of a medium in the vessel.SOLUTION: A cell culture method uses: a culture vessel which is filled with cells and a medium and in which cell culture is carried out; and a medium supplying vessel which is filled with a new medium and which supplies the medium to the culture vessel. The culture vessel has at least a first port which is used for medium supply, and a second port which is used for medium discharge. The medium supplying vessel serves as a mixing vessel for adjusting concentration of a new medium to be supplied to the culture vessel. A portion of a medium which has been used, in the culture vessel is sent from the culture vessel to the mixing vessel. In the mixing vessel, a new medium and the portion of the medium which has been used, are mixed to prepare a concentration-adjusted medium. While the concentration-adjusted medium is transferred from the mixing vessel to the culture vessel, the remaining of the medium which has been used is discharged from the culture vessel, and the culture vessel is filled with the concentration-adjusted medium.SELECTED DRAWING: Figure 7

Description

[Technical Field]

[0001] The present invention relates to cell culture technology, and more particularly to medium exchange in cell culture using a culture vessel. [Background technology]

[0002] In recent years, there has been a demand for efficient mass cultivation of cells and tissues in an artificial environment in the fields of pharmaceutical production, gene therapy, regenerative medicine, immunotherapy, and the like. In this situation, various techniques have been developed for automatically culturing cells in culture vessels in order to culture cells in large quantities.

[0003] When cell culture is performed using a culture vessel, a medium that has been used in the vessel for a certain period of time must be replaced with a new medium at a predetermined timing. Methods for replacing the culture medium in a container mainly include a circulation method in which the culture medium is replaced by circulating it in the container, and a non-circulation method in which the culture medium is replaced by sending it in one direction without circulating it in the container. The circulation method here refers to a method in which a certain percentage of used culture medium is discharged from the culture vessel, new culture medium is supplied, and then the culture medium is circulated using a circulation path to uniformly mix these culture media in the culture vessel. [Prior art documents] [Patent documents]

[0004] [Patent Document 1] Japanese Patent Application Publication No. 2020-188691 Summary of the Invention [Problem to be solved by the invention]

[0005] However, the circulation method has the problem that extremely complicated work is required to increase the medium exchange rate to nearly 100%. In other words, this circulation method is actually carried out as follows.

[0006] First, when discharging spent culture medium from a culture vessel, there is a problem that if all of the culture medium is discharged, the cells will be discharged along with the culture medium. For this reason, it is necessary to discharge the culture medium while maintaining a certain level of liquid thickness in the vessel, so that some of the spent culture medium remains in the vessel. In particular, when culturing spheres (cell masses) made from iPS cells, organoids created by aggregating several types of cells, or floating cells, these are easily expelled when the medium is drained, so it is necessary to maintain a thicker liquid thickness than when culturing adhesive cells by adhering them to the inner surface of the container, which poses the problem of a low medium exchange rate.

[0007] Furthermore, when a used culture medium is discharged from a culture vessel and then new culture medium is supplied into the vessel, the culture medium in the vessel becomes non-uniform, resulting in a problem of reduced culture efficiency. For this reason, it was necessary to implement a circulation method in which two ports were provided in the culture vessel, these ports were connected with a tube to form a circular flow path, and the culture medium was circulated within the vessel to homogenize the culture medium, or to agitate and homogenize the culture medium by applying vibration to the culture vessel, for example.

[0008] Thus, when implementing the circulation method, in order to increase the medium exchange rate to nearly 100%, it was necessary to repeatedly discharge the used medium from the culture vessel, supply new medium into the vessel, and form a circular flow path to implement the circulation method, which required cumbersome work.Furthermore, when applying vibration to the culture vessel to homogenize the medium, it was necessary to stir it manually or use a stirring means, which was also cumbersome.

[0009] On the other hand, in the non-circulation method, although it is possible to perform a 100% medium exchange rate, there is a problem in that it is difficult to control the medium exchange rate. That is, according to the non-circulation method, a culture vessel is provided with two ports, and a tube for supplying and discharging medium is connected to each port. By discharging used medium from the tube for discharging medium while supplying new medium from the tube for supplying medium, 100% medium exchange is possible.

[0010] However, it has been experimentally shown that, for example, when inducing differentiation of spheres into mesoderm, carrying out a medium exchange close to 100% actually inhibits the cell culture efficiency (see Reference Example 1). Therefore, it is desirable to be able to control the medium exchange rate to a desired level, rather than just 100%, but this is difficult to achieve with non-circulating methods. Another problem with non-circulating methods is that a large amount of medium is required for medium exchange.

[0011] Therefore, the inventors conducted extensive research and succeeded in developing a method that can appropriately control the medium exchange rate within a culture vessel without requiring the formation of an annular flow path or stirring of the culture vessel to homogenize the medium within the vessel, thereby completing the present invention. Patent Document 1 discloses a cell culture device for continuous cell culture, and describes a configuration for controlling the supply of culture medium. However, this cell culture device does not enable suitable control of the culture medium exchange rate.

[0012] The present invention has been made in consideration of the above circumstances, and aims to provide a cell culture method, a cell culture kit, and a cell culture system that are capable of appropriately controlling the exchange rate of the culture medium in a culture vessel when culturing cells using the vessel. [Means for solving the problem]

[0013] In order to achieve the above-mentioned object, the cell culture method of the present invention is a cell culture method using a culture vessel filled with cells and culture medium and in which cell culture is performed, and a culture medium supply vessel filled with new culture medium and supplying the culture medium to the culture vessel, the culture vessel having at least a first port used for supplying the culture medium and a second port used for discharging the culture medium, the culture medium supply vessel being a mixing vessel for adjusting the concentration of the new culture medium to be supplied to the culture vessel, a portion of the used culture medium in the culture vessel being transferred from the culture vessel to the mixing vessel, the new culture medium and a portion of the used culture medium being mixed in the mixing vessel to prepare a concentration-adjusted culture medium, the remainder of the used culture medium being discharged from the culture vessel while the concentration-adjusted culture medium is transferred from the mixing vessel to the culture vessel, and the culture vessel is filled with the concentration-adjusted culture medium.

[0014] Furthermore, the cell culture method of the present invention is preferably a method in which, when the concentration-adjusted culture medium is transferred from the mixing vessel to the culture vessel while the remainder of the used culture medium is discharged from the culture vessel, the amount of culture medium in the culture vessel is not changed, and after the remainder of the used culture medium in the culture vessel has been replaced with the concentration-adjusted culture medium, the discharge from the culture vessel is stopped and an amount of the concentration-adjusted culture medium equivalent to a portion of the used culture medium is transferred from the mixing vessel to the culture vessel.

[0015] Furthermore, the cell culture method of the present invention is preferably a method in which, before discharging the remainder of the used culture medium from the culture vessel while transferring the concentration-adjusted culture medium from the mixing vessel to the culture vessel, a portion of the remainder of the used culture medium in the culture vessel is discharged from the culture vessel to reduce the liquid thickness of the culture medium in the culture vessel, and while maintaining the reduced liquid thickness of the culture medium in the culture vessel, the entire remainder of the used culture medium in the culture vessel is discharged from the culture vessel while transferring the concentration-adjusted culture medium from the mixing vessel to the culture vessel, and the culture vessel is filled with the concentration-adjusted culture medium.

[0016] Furthermore, the cell culture method of the present invention is preferably a method in which, after the entire remaining portion of the used culture medium in the culture vessel has been replaced with the concentration-adjusted culture medium, discharge from the culture vessel is stopped, and an amount of the concentration-adjusted culture medium equivalent to a portion of the used culture medium and a portion of the remaining portion of the used culture medium is sent from the mixing vessel to the culture vessel.

[0017] Furthermore, the cell culture method of the present invention is preferably a method for controlling the medium exchange rate of the culture vessel by adjusting the mixing ratio between the amount of new medium in the mixing vessel and the amount of used medium transferred from the culture vessel to the mixing vessel.

[0018] Furthermore, the cell culture method of the present invention is preferably a method in which, before adjusting the mixing ratio between the amount of new medium in the mixing vessel and the amount of used medium to be transferred from the culture vessel to the mixing vessel, the amount of fluid to be transferred necessary for medium replacement in the culture vessel is measured and a desired concentration of the new medium to be supplied to the culture vessel is determined, and the amount of new medium in the mixing vessel and the amount of used medium to be transferred from the culture vessel to the mixing vessel are determined based on the required amount of fluid to be transferred and the desired concentration.

[0019] Furthermore, the cell culture method of the present invention is preferably a method in which the medium exchange rate in the culture vessel is controlled to 50% or more and less than 100%.

[0020] Furthermore, the cell culture kit of the present invention includes at least a first port used for supplying culture medium and a second port used for discharging culture medium, and includes a culture vessel filled with cells and culture medium for cell culture; a mixing vessel that can communicate with the first port via a tubular member and is filled with new culture medium and supplies the culture medium to the culture vessel; and a waste liquid container that can communicate with the second port via a tubular member and into which used culture medium flows, wherein the culture vessel is configured to transfer a portion of the used culture medium in the culture vessel from the culture vessel to the mixing vessel, and while receiving a concentration-adjusted culture medium prepared in the mixing vessel by mixing the new culture medium and a portion of the used culture medium from the mixing vessel, the remaining portion of the used culture medium is discharged from the culture vessel to the waste liquid container, and the culture vessel is filled with the concentration-adjusted culture medium.

[0021] The cell culture system of the present invention includes at least a first port used for supplying a culture medium and a second port used for discharging the culture medium, and includes a culture vessel filled with cells and a culture medium for cell culture; a mixing vessel connected to the first port via a first tubular member and filled with new culture medium to supply the culture medium to the culture vessel; a waste liquid vessel connected to the second port via a second tubular member and into which used culture medium flows; a first liquid delivery means disposed in the first tubular member; and a control device that controls the operation of the second liquid transfer means to control the transfer of culture medium between the culture vessel, the mixing vessel, and the waste liquid container, and the control device is configured to transfer a portion of the used culture medium in the culture vessel from the culture vessel to the mixing vessel, transfer a concentration-adjusted culture medium created in the mixing vessel by mixing the new culture medium with a portion of the used culture medium from the mixing vessel to the culture vessel, and transfer the remainder of the used culture medium from the culture vessel to the waste liquid container.

[0022] Furthermore, it is preferable that the cell culture system of the present invention further comprises a detection unit that measures changes in the liquid thickness of the culture medium, and that the control device controls the transfer of culture medium between the culture vessel, the mixing vessel, and the waste liquid vessel based on the detection information input from the detection unit. [Effects of the Invention]

[0023] According to the present invention, it is possible to provide a cell culture method, a cell culture kit, and a cell culture system that are capable of suitably controlling the exchange rate of the culture medium in a culture vessel when culturing cells using the vessel. [Brief explanation of the drawings]

[0024] [Figure 1] 1 is a schematic diagram showing the configuration of a culture vessel and a mixing vessel that can be used in a cell culture method according to an embodiment of the present invention. FIG. [Figure 2] FIG. 1 is a schematic diagram showing the configuration of a cell culture kit that can be used in a cell culture method according to an embodiment of the present invention. [Figure 3] 1 is a schematic diagram showing the configuration of a cell culture system that can be used in a cell culture method according to an embodiment of the present invention. FIG. [Figure 4] FIG. 1 is a graph showing a calibration curve prepared in Experiment 1 for determining the amount of medium required for medium exchange used in a cell culture method according to an embodiment of the present invention. [Figure 5] FIG. 1 is a diagram showing the procedure of Experiment 1 for determining the amount of medium required for medium exchange used in a cell culture method according to an embodiment of the present invention. [Figure 6] FIG. 1 is a graph showing the results of Experiment 1 for determining the amount of medium required for medium exchange used in the cell culture method according to an embodiment of the present invention. [Figure 7] FIG. 1 is a diagram showing the procedure of Example 1 for confirming the effects of a cell culture method, a cell culture kit, and a cell culture system according to an embodiment of the present invention. [Figure 8] FIG. 1 is a diagram showing the procedure of Example 2 for confirming the effects of the cell culture method, cell culture kit, and cell culture system according to an embodiment of the present invention. [Figure 9] FIG. 10 is a diagram showing the procedure of Comparative Example 2 for confirming the effects of the cell culture method, cell culture kit, and cell culture system according to the embodiment of the present invention. DETAILED DESCRIPTION OF THE INVENTION

[0025] Hereinafter, embodiments of the cell culture method, cell culture kit, and cell culture system of the present invention will be described in detail. However, the present invention is not limited to the specific contents of the following embodiments and examples described later.

[0026] The cell culture method of this embodiment is a cell culture method that uses a culture vessel filled with cells and culture medium and in which cell culture is performed, and a culture medium supply vessel filled with new culture medium (additional culture medium to the culture vessel) and that supplies the culture medium to the culture vessel. The culture vessel has a first port used for supplying the culture medium and a second port used for discharging the culture medium, and may have three or more ports. In addition, in the cell culture method of this embodiment, the medium supply container is used as a mixing container for adjusting the concentration of the new medium in the culture container.

[0027] The culture vessel and mixing vessel used in the cell culture method of this embodiment are shown in FIG. As shown in the figure, a tube 41 is connected to a first port 11 of the culture vessel 10, and this tube 41 is connected to a port of the mixing vessel, thereby communicating the culture vessel 10 with the mixing vessel 20. In addition, a tube 42 is connected to a second port 12 of the culture vessel 10, and used culture medium can be discharged to the outside of the culture vessel via this tube 42. FIG. 1 shows a case where closed culture bags made of soft packaging material are used as the culture vessel 10 and the mixing vessel 20, but these vessels are not limited to bag-shaped ones, and the cell culture method of this embodiment can be applied to rigid open vessels as well.

[0028] In the cell culture method of this embodiment, first, a part of the used culture medium in the culture vessel 10 is transferred from the culture vessel 10 to the mixing vessel 20 via the port 11. Next, in the mixing container 20, the new medium and a portion of the used medium are mixed to prepare a concentration-adjusted medium, and while the concentration-adjusted medium is transferred from the mixing container 20 to the culture container 10, the remainder of the used medium is discharged from the culture container 10, and the culture container 10 is filled with the concentration-adjusted medium. At this time, the transfer of the concentration-adjusted culture medium from the mixing container 20 to the culture container 10 and the discharge of the remaining used culture medium into the culture container 10 do not necessarily have to be carried out simultaneously, and may be carried out intermittently.

[0029] Furthermore, when the concentration-adjusted culture medium is being transferred from the mixing container 20 to the culture container 10 while the remainder of the used culture medium is being discharged from the culture container 10, it is preferable to leave the amount of culture medium in the culture container 10 unchanged, and after the remainder of the used culture medium in the culture container 10 has been replaced with the concentration-adjusted culture medium, stop the discharge from the culture container 10, and transfer a quantity of new culture medium equivalent to a portion of the used culture medium from the mixing container 20 to the culture container 10.

[0030] Specifically, for example, the culture vessel 10 is filled in advance with 20 ml of new medium and cells, and the mixing vessel 20 is filled with 40 ml of new medium, and the cells are cultured in the culture vessel 10. After culturing for a predetermined period of time, 4.4 ml of the spent medium is transferred from the culture vessel 10 to the mixing vessel 20. At this time, the amount of the medium in the culture vessel 10 becomes 15.6 ml (= 20 ml - 4.4 ml). Next, in the mixing container 20, 40 ml of new medium is mixed with 4.4 ml of a portion of the used medium to prepare a concentration-adjusted medium in which the concentration of the new medium is 90% (volume %) (= 40 / (40 + 4.4) × 100).

[0031] Then, without changing the amount of culture medium in the culture vessel 10 (maintaining it at 15.6 ml), the concentration-adjusted culture medium is transferred from the mixing vessel 20 to the culture vessel 10, while the remainder of the used culture medium is discharged from the culture vessel 10 and the concentration-adjusted culture medium is filled into the culture vessel 10. Also, after the remainder of the used culture medium in the culture vessel 10 has been replaced with concentration-adjusted culture medium, discharge from the culture vessel 10 is stopped and an amount of new culture medium (4.4 ml) equivalent to a portion of the used culture medium is sent from the mixing vessel 20 to the culture vessel 10.

[0032] This allows 20 ml of concentration-adjusted medium with a new medium concentration of 90% to be filled into the culture vessel 10. In other words, the exchange rate of the medium in the culture vessel 10 can be controlled to 90%. By using this method for the cell culture method of this embodiment, it is possible to control the exchange rate of the culture medium in the container to any value without forming a circular flow path or stirring the container to homogenize the culture medium.

[0033] In addition, in the cell culture method of this embodiment, it is preferable to discharge a portion of the remaining used culture medium in the culture vessel 10 from the culture vessel 10 before discharging the remaining used culture medium from the culture vessel 10 while transferring the concentration-adjusted culture medium from the mixing vessel 20 to the culture vessel 10, thereby reducing the liquid thickness of the culture medium in the culture vessel 10.

[0034] The liquid thickness of the culture medium in the culture vessel 10 is the height from the bottom surface of the vessel to the liquid surface of the culture medium, and when the culture vessel 10 is a bag, it corresponds to the height from the bottom surface to the top surface of the vessel. At this time, the liquid thickness of the culture medium in the culture vessel 10 should be such that the cells do not flow out of the culture vessel; if the liquid thickness is up to about 1 mm, it is possible to change the culture medium without causing the cells to flow out of the culture vessel.

[0035] Furthermore, it is preferable to maintain the reduced liquid thickness of the culture medium in the culture vessel 10, while transferring the concentration-adjusted culture medium from the mixing vessel 20 to the culture vessel 10, and then discharge all of the remaining used culture medium in the culture vessel 10 from the culture vessel 10, and fill the culture vessel 10 with the concentration-adjusted culture medium.

[0036] Furthermore, in the cell culture method of this embodiment, after the entire remaining portion of the used culture medium in the culture vessel 10 has been replaced with concentration-adjusted culture medium, it is preferable to stop discharge from the culture vessel 10 and transfer a portion of the used culture medium and an amount of concentration-adjusted culture medium equivalent to a portion of the remaining portion of the used culture medium from the mixing vessel 20 to the culture vessel 10.

[0037] Specifically, for example, the culture vessel 10 has a bottom area of ​​50 cm 2 Let's assume that a bag with a maximum capacity of 50 ml is used. The culture vessel 10 is filled in advance with 20 ml of new medium and cells, and the mixing vessel 20 is filled with 22.5 ml of new medium, and the cells are cultured in the culture vessel 10.

[0038] As will be described later, when the liquid thickness of the culture medium in the culture vessel 10 is reduced and the liquid is fed, the amount of new medium used can be reduced, and therefore the amount of new medium pre-filled in the mixing vessel 20 can be reduced compared to when the liquid thickness of the culture medium is not reduced. This makes it possible to further reduce the amount of new medium required for medium replacement.

[0039] Next, after culturing for a predetermined period of time, 2.5 ml of the spent medium is transferred from the culture vessel 10 to the mixing vessel 20. At this time, the amount of the medium in the culture vessel 10 becomes 17.5 ml (= 20 ml - 2.5 ml). Next, in the mixing container 20, 22.5 ml of new medium is mixed with 2.5 ml of a portion of the used medium to prepare a concentration-adjusted medium in which the concentration of the new medium is 90% (=22.5 / (22.5+2.5)×100).

[0040] Next, while the concentration-adjusted medium is being sent from the mixing container 20 to the culture container 10, before the remainder of the used medium is discharged from the culture container 10, a part of the remainder of the used medium in the culture container 10 is discharged from the culture container 10, and the liquid thickness of the medium in the culture container 10 is reduced to 1 mm. 2 In this case, if 12.5 ml of used medium in the culture vessel 10 is discharged from the culture vessel 10 and the amount of used medium in the culture vessel 10 is set to 5 ml, the liquid thickness of the medium will be 1 mm.

[0041] Furthermore, while maintaining the liquid thickness of the culture medium in the culture vessel 10 at 1 mm, the concentration-adjusted culture medium is transferred from the mixing vessel 20 to the culture vessel 10, and all of the remaining used culture medium in the culture vessel 10 is discharged from the culture vessel 10, and the concentration-adjusted culture medium is filled into the culture vessel 10.

[0042] Furthermore, after the entire remaining portion of the used culture medium in the culture vessel 10 has been replaced with concentration-adjusted culture medium, discharge from the culture vessel 10 is stopped, and 15 ml (= 2.5 ml + 12.5 ml) of concentration-adjusted culture medium, an amount equivalent to a portion of the used culture medium and a portion of the remaining used culture medium, is transferred from the mixing vessel 20 to the culture vessel 10.

[0043] This allows 20 ml of concentration-adjusted medium with a new medium concentration of 90% to be filled into the culture vessel 10. In other words, the exchange rate of the medium in the culture vessel 10 can be controlled to 90%. In addition, it is possible to further reduce the amount of new medium required for medium exchange.

[0044] Furthermore, in the cell culture method of this embodiment, the culture medium exchange rate of the culture vessel 10 can be controlled by adjusting the mixing ratio between the amount of new culture medium in the mixing vessel 20 and the amount of used culture medium transferred from the culture vessel 10 to the mixing vessel 20. That is, in the above example, the amount of new culture medium in the mixing container 20 is set to 22.5 ml, and the amount of used culture medium sent from the culture container 10 to the mixing container 20 is set to 2.5 ml, so that the concentration of the new culture medium in the concentration-adjusted culture medium is set to 90%, and the exchange rate of the culture medium in the culture container 10 is controlled to 90%.

[0045] Furthermore, according to the cell culture method of this embodiment, it is possible to control the exchange rate of the culture medium in the culture vessel 10 to any value other than 90%. Furthermore, in the cell culture method of this embodiment, the medium exchange rate in the culture vessel is preferably controlled to 50% or more and less than 100%, more preferably 50% or more and less than 99%, from the viewpoint of cell culture efficiency.

[0046] Furthermore, in the cell culture method of this embodiment, before adjusting the mixing ratio between the amount of new medium in the mixing vessel 20 and the amount of used medium to be sent from the culture vessel 10 to the mixing vessel 20, it is preferable to measure the amount of fluid sent necessary for medium replacement in the culture vessel 10 and determine the desired concentration of the new medium to be supplied to the culture vessel 10, and then determine the amount of new medium in the mixing vessel 20 and the amount of used medium to be sent from the culture vessel 10 to the mixing vessel 20 based on the required amount of fluid sent and the desired concentration.

[0047] Specifically, as will be explained in detail in Experiment 1 below, for example, clear water is filled into the culture vessel 10, and while maintaining the liquid volume constant, red water is sent from the mixing vessel 20 to the culture vessel 10, and the clear water is then discharged from the culture vessel 10. By measuring the amount of liquid sent until the clear water in the culture vessel is replaced with red water, the amount of liquid sent required to replace the culture medium in the culture vessel 10 can be measured.

[0048] Furthermore, as will be described in detail in Examples 1 and 2 below, by taking into consideration the desired concentration of the new medium in the culture vessel 10, the amount of liquid in the mixing vessel 20, the amount of liquid to be flowed from the culture vessel into the mixing vessel, the amount of liquid sent necessary to replace the medium in the culture vessel 10, and the amount of liquid needed to adjust the amount of liquid in the culture vessel 10 to the original amount, it is possible to determine the amount of new medium in the mixing vessel 20 and the amount of used medium to be sent from the culture vessel 10 to the mixing vessel 20.

[0049] In the cell culture method of this embodiment, when a closed-system culture bag is used as the culture vessel 10, resin films and the like can be suitably used as the material, and polyolefin resins such as polyethylene and polypropylene can be used. Examples include polyethylene, copolymers of ethylene and α-olefins, copolymers of ethylene and vinyl acetate, and ionomers using ethylene, acrylic acid or methacrylic acid copolymers, and metal ions. Polyolefins, styrene-based elastomers, and polyester-based thermoplastic elastomers can also be used. Furthermore, soft vinyl chloride resins, polybutadiene resins, ethylene-vinyl acetate copolymers, chlorinated polyethylene resins, polyurethane-based thermoplastic elastomers, polyester-based thermoplastic elastomers, silicone-based thermoplastic elastomers, styrene-based elastomers such as SBS (styrene-butadiene-styrene), SIS (styrene-isoprene-styrene), SEBS (styrene-ethylene-butylene-styrene), SEPS (styrene-ethylene-propylene-styrene), polyolefin resins, and fluorine-based resins can also be used.

[0050] Furthermore, in the cell culture method of this embodiment, when a rigid open container is used as the culture container 10, the material thereof is not particularly limited, but may be, for example, a plastic such as polystyrene, PET, PETG, or TPX, a metal such as stainless steel, or glass, or a combination of these.

[0051] When a closed-system culture bag is used as the culture vessel 10, the upper surface of the culture bag follows the change in the amount of filling liquid (liquid thickness), making it easier to control the liquid thickness compared to when a rigid open-system container is used. Furthermore, the upper surface of the culture bag prevents the filling medium from rippling, improving the stability of liquid delivery. Furthermore, a liquid thickness detector can be attached to the upper surface of the culture bag, making it easy to measure the liquid thickness in the bag and enabling accurate control of the liquid thickness.

[0052] In the cell culture method of this embodiment, the cells cultured using the culture vessel 10 are not particularly limited, and may be floating cells, adhesive cells, spheres (cell clumps), or organoids created by aggregating several types of cells. Specific examples include nerve cells, pancreatic islet cells, kidney cells, hepatic cells, muscle cells, cardiac muscle cells, corneal endothelial cells, vascular endothelial cells, mesenchymal stem cells, lymphocytes, etc. Other examples include induced pluripotent stem cells (iPS cells), embryonic stem cells (ES cells), and cells induced to differentiate from these stem cells into the above-mentioned various cells. Furthermore, it is also possible to culture intermediates such as progenitor cells generated during the differentiation induction process. Furthermore, the cell culture method of this embodiment can be applied to differentiation-inducing culture in any culture of floating cells, adhesive cells, spheres, or organoids.

[0053] As shown in FIG. 2, the cell culture kit of this embodiment includes at least a first port 11 used for supplying culture medium and a second port 12 used for discharging culture medium, and includes a culture vessel 10 filled with cells and culture medium and used for cell culture, a mixing vessel 20 that can be connected to the first port 11 via a tube 41 and filled with new culture medium to supply the culture vessel 10, and a waste liquid container 30 that can be connected to the second port 12 via a tube 42 and into which used culture medium flows.

[0054] The culture vessel 10 is used to transfer a portion of the used culture medium from the culture vessel 10 to the mixing vessel 20, and while a concentration-adjusted culture medium created by mixing new culture medium and a portion of the used culture medium in the mixing vessel 20 is supplied from the mixing vessel 20, the remainder of the used culture medium is discharged from the culture vessel 10 to the waste liquid container 30, and the concentration-adjusted culture medium is filled into the culture vessel 10.

[0055] By using such a cell culture kit of this embodiment, the culture medium in the culture vessel 10 can be exchanged uniformly, and the exchange rate of the culture medium can be controlled to an arbitrary value.

[0056] As shown in FIG. 3 , the cell culture system of this embodiment includes at least a first port 11 used for supplying culture medium and a second port 12 used for discharging culture medium, and includes a culture vessel 10 filled with cells and culture medium and used for cell culture; a mixing vessel 20 connected to the first port 11 via a tube 41, filled with new culture medium, and supplying the culture medium to the culture vessel 10; a waste liquid container 30 connected to the second port 12 via a tube 42, into which used culture medium flows; and a control device 60 that controls the operation of a first pump 51 disposed in the tube 41 and a second pump 52 disposed in the tube 42, thereby controlling the transfer of culture medium between the culture vessel 10, the mixing vessel 20, and the waste liquid container 30.

[0057] Then, the control device 60 transfers a portion of the used culture medium in the culture vessel 10 from the culture vessel 10 to the mixing vessel 20, transfers a concentration-adjusted culture medium created by mixing new culture medium and a portion of the used culture medium in the mixing vessel 20 from the mixing vessel 20 to the culture vessel 10, and transfers the remainder of the used culture medium from the culture vessel 10 to the waste liquid container 20.

[0058] In addition, it is also preferable that the cell culture system of this embodiment further includes a detection unit 70 that measures changes in the liquid thickness of the culture medium, and that the control device 60 controls the transfer of culture medium between the culture vessel 10, the mixing vessel 20, and the waste liquid vessel 30 based on the detection information input from the detection unit 70.

[0059] Specifically, the control device 60 (control unit) may include an input / output section 61, a control section 62, an operation section 63, and a power supply section 64. The input / output unit 61 is connected to a first pump 51 disposed between the culture vessel 10 and the mixing vessel 20 and a second pump 52 disposed between the culture vessel 10 and the waste liquid container 30, and controls the operation of these pumps based on input information from the control unit 62. As a result, the culture vessel 10 is supplied with a concentration-adjusted culture medium from the mixing vessel 20, and the used culture medium is discharged from the culture vessel 10 to the waste liquid container 30.

[0060] The control unit 62 is configured with a PLC (programmable logic controller) or the like, and can pre-program and store desired control content, and control the operation of each unit based on this. That is, the control unit 62 transmits information for controlling the pump to the input / output unit 61 at a predetermined timing.

[0061] The operation unit 63 includes a display unit such as a touch panel, and transmits information input by a user to the control unit 62 to execute PLC settings, etc. The operation unit 63 also displays the information input from the control unit 62. The power supply unit 64 (such as a stabilized power supply) supplies electricity to each unit in the control device 60. Although not shown, the control device 60 may further include a relay and a circuit breaker. A part or all of the components of the control device 60, such as the control unit 62 and the operation unit 63, may be realized by a microcomputer or a computer.

[0062] In the cell culture system of this embodiment, a length measuring sensor, for example, can be suitably used as the detection unit 70 that measures changes in the liquid thickness of the culture medium. The control unit 62 can then calculate the liquid thickness of the culture medium based on the detection information, and control the operations of the first pump 51 and the second pump 52 based on this liquid thickness. For example, it is possible to control the discharge of used culture medium from the culture vessel 10 so that the liquid thickness of the culture medium in the culture vessel 10 becomes 1 mm.

[0063] According to the cell culture system of this embodiment, the culture medium in the culture vessel 10 can be uniformly replaced, and the culture medium replacement rate can be controlled to any value, and these can be performed automatically.

[0064] As described above, according to the cell culture method, cell culture kit, and cell culture system of this embodiment, it is possible to suitably control the exchange rate of the culture medium in the container when culturing cells using a culture container. Furthermore, according to this embodiment, the culture medium in the culture vessel can be exchanged uniformly, and the amount of culture medium used can be reduced. Furthermore, such culture medium exchange can be performed automatically. [Example]

[0065] Hereinafter, various experiments conducted to confirm the effects of the cell culture method, cell culture kit, and cell culture system according to the embodiments of the present invention will be described. [Experiment 1] First, in Examples 1 and 2 and Comparative Example 2 described below, experiments were carried out to determine the amount of medium required for replacing the medium in the culture vessel.

[0066] The culture vessel used was a closed culture bag with 18,000 hemispherical wells with a diameter of 0.5 mm molded on the bottom (culture surface) of the vessel and coated with a polymer with low cell adhesion. The bottom area of ​​the bag was 50 cm. 2The maximum liquid volume was 50 mL, the material of the bag was LLDPE (linear low-density polyethylene), and the culture vessel had two ports. The mixing container used was a bag with a flat bottom without a molded well. The bottom area of ​​the bag was 50 cm. 2 The maximum liquid volume was 50 mL, and the bag material was LLDPE. The mixing vessel had one port.

[0067] The culture vessel was filled with clear water, and while maintaining the liquid volume constant, red water was transferred from the mixing vessel to the culture vessel and the clear water was then discharged from the culture vessel, and the amount of liquid transferred until the clear water in the culture bag was replaced with red water was measured. Specifically, the amount of liquid delivered was measured when the culture vessel was filled with 15.6 mL of clear water (liquid thickness 3.1 mm) and when the culture vessel was filled with 5 mL of clear water (liquid thickness 1 mm).

[0068] In addition, as a preliminary step to the experiment, a calibration curve was created based on the relationship between red water concentration and absorbance. Specifically, we prepared undiluted red water (100%), 75%, 50%, and 25% red water, and clear water (0%, control), and placed 200 μL of each solution in a 96-well plate. Absorbance measurements (490 nm wavelength) were then performed using an absorbance plate reader (Corona Electric, SH-1000), and a calibration curve was created based on the relationship between red water concentration and absorbance. The results are shown in Figure 4. In the following experiments, this calibration curve was used to calculate the red water concentration from the absorbance of the red water.

[0069] As shown in Figure 5, the experimental method involved filling the culture vessel with 15.6 mL or 5 mL of clear water, and filling the mixing vessel with a sufficient amount (50 mL) of red water concentrate (100%). A tube was then connected to one port of the culture vessel, connecting the mixing vessel to the culture vessel. A tube was also connected to the other port of the culture vessel, allowing for sampling of wastewater. A tube pump was also installed on each tube.

[0070] Both tube pumps were operated to simultaneously pump the liquid toward the waste direction at a rate of 1 mL / min, thereby maintaining a constant liquid volume and thickness in the culture vessel. Waste liquid sampling was performed every 5 minutes. Next, the absorbance of the sampled waste liquid was measured, and the red water and clear water concentrations in the waste liquid were calculated. Furthermore, based on the clear water concentration and the waste liquid volume, the amount of clear water discharged per 5 minutes of liquid transfer was calculated, and the liquid exchange rate (proportion of red water in the container) relative to the amount of red water used was calculated. The results are shown in Figure 6.

[0071] Furthermore, the amount of liquid used when 99% of the liquid in the container was replaced with red water was confirmed in Figure 6. As a result, when the liquid volume in the culture container was set to 15.6 mL (liquid thickness 3.1 mL) and liquid was transferred while maintaining a constant liquid volume and liquid thickness in the culture container, it was found that the amount of liquid transferred to replace the liquid in the culture container with the liquid filled in the mixing container was 40 mL. Similarly, when the liquid volume in the culture vessel is 5 mL (liquid thickness 1 mL) and the liquid is transferred while maintaining the liquid volume and liquid thickness in the culture vessel constant, it was found that the amount of liquid required to transfer to replace the liquid in the culture vessel with the liquid filled in the mixing vessel is 7.5 mL.

[0072] [Example 1] An experiment was conducted to confirm the effectiveness of a method in which a portion of the used medium in a culture vessel is transferred from the culture vessel to a mixing vessel, new medium is mixed with a portion of the used medium in the mixing vessel to create a concentration-adjusted medium, the remainder of the used medium is discharged from the culture vessel while the concentration-adjusted medium is transferred from the mixing vessel to the culture vessel, and the culture vessel is filled with the concentration-adjusted medium. The culture vessel and mixing vessel were the same as those used in Experiment 1. The waste liquid vessel was the same as the mixing vessel.

[0073] In the experimental method, 20 mL of clear water was filled into the culture vessel, and 40 mL of red water concentrate (100%) was filled into the mixing vessel, as shown in Figure 7. A tube was then connected to one port of the culture vessel to connect the mixing vessel to the culture vessel, and a tube was connected to the other port of the culture vessel to connect the waste liquid container to the culture vessel. In addition, a tube pump 1 was installed on the tube connecting the culture vessel and the mixing vessel, and a tube pump 2 was installed on the tube connecting the culture vessel and the waste liquid container. The clear water in the culture vessel was replaced with red water with a concentration of 90%.

[0074] Then, by operating the tube pump 1, 4.4 mL of clear water from the culture vessel was flowed into the mixing vessel. This resulted in the preparation of red water with a concentration of 90% in the mixing vessel. The liquid delivery rate was 1 mL / min, and the delivery time was 4.4 minutes. Note that the liquid delivery rate was also 1 mL / min in all of the following experiments.

[0075] The reason why the filling volume into the mixing vessel was set to 40 mL is that the volume of liquid in the mixing vessel becomes +4.4 mL when 4.4 mL is flowed into the mixing vessel from the culture vessel, and from the results of Experiment 1, it becomes -40 mL when the liquid in the culture vessel is replaced with red water, and -4.4 mL when the volume of liquid in the culture vessel is increased to 20 mL, so liquid can be delivered if the filling volume is 40 mL or more.

[0076] Next, both tube pumps were operated to simultaneously pump the liquid toward the wastewater direction, maintaining a constant liquid volume and thickness in the culture vessel, and uniformly replacing the clear water in the culture vessel with 90% red water. The liquid pumping time was 40 minutes.

[0077] Furthermore, tube pump 2 was stopped to stop the discharge from the culture vessel, and tube pump 1 was operated to flow 4.4 mL of 90% red water from the mixing vessel into the culture vessel, filling the culture vessel with 20 mL of 90% red water. The liquid transfer time at this time was 4.4 minutes. This allowed the culture vessel to be replaced with red water with a concentration of 90%, and the medium replacement rate in the culture vessel could be controlled.

[0078] That is, according to the cell culture method of this embodiment shown in Example 1, medium exchange was performed uniformly and the medium exchange rate of the culture vessel was appropriately controlled. Furthermore, the medium exchange time was 48.8 minutes (= 4.4 + 40 + 4.4), which was shorter than the conventional circulation method (Comparative Example 3).

[0079] [Example 2] An experiment was conducted to confirm the effectiveness of a method in which, before discharging the remainder of the used culture medium from the culture vessel while transferring the concentration-adjusted culture medium from the mixing vessel to the culture vessel, a portion of the remainder of the used culture medium in the culture vessel is discharged from the culture vessel to reduce the liquid thickness of the culture medium in the culture vessel, and while maintaining the reduced liquid thickness of the culture medium in the culture vessel, the entire remainder of the used culture medium in the culture vessel is discharged from the culture vessel while transferring the concentration-adjusted culture medium from the mixing vessel to the culture vessel, and the culture vessel is filled with the concentration-adjusted culture medium. The culture vessel and mixing vessel were the same as those used in Experiment 1. The waste liquid vessel was the same as the mixing vessel.

[0080] In the experimental method, 20 mL of clear water was filled into the culture vessel, and 22.5 mL of red water concentrate (100%) was filled into the mixing vessel, as shown in Figure 8. Then, a tube was connected to one port of the culture vessel to connect the mixing vessel to the culture vessel, and a tube was connected to the other port of the culture vessel to connect the waste liquid container to the culture vessel. In addition, a tube pump 1 was installed on the tube connecting the culture vessel and the mixing vessel, and a tube pump 2 was installed on the tube connecting the culture vessel and the waste liquid container. The clear water in the culture vessel was replaced with red water with a concentration of 90%.

[0081] Then, the tube pump 1 was operated to flow 2.5 mL of clear water from the culture vessel into the mixing vessel. This resulted in the preparation of red water with a concentration of 90% in the mixing vessel. The liquid transfer time was 2.5 minutes. Next, tube pump 1 was stopped and tube pump 2 was operated to pump 12.5 mL of clear water from the culture vessel into a waste container, reducing the liquid volume in the culture vessel to 5 mL (liquid thickness 1 mm). The liquid pumping time was 12.5 minutes.

[0082] The reason why the filling volume into the mixing vessel was set to 22.5 mL is that the volume of liquid in the mixing vessel becomes +2.5 mL when 2.5 mL is flowed into the mixing vessel from the culture vessel, and from the results of Experiment 1, becomes -7.5 mL when the liquid in the culture vessel is replaced with red water, and becomes -15 mL when the volume of liquid in the culture vessel is increased to 20 mL, so liquid can be delivered if the filling volume is 20 mL or more.

[0083] Next, both tube pumps were operated to simultaneously pump the liquid toward the wastewater direction, maintaining a constant liquid volume and thickness in the culture vessel (reducing the thickness of the culture medium in the culture vessel), and the clear water in the culture vessel was uniformly replaced with 90% red water. The liquid pumping time was 7.5 minutes.

[0084] Furthermore, tube pump 2 was stopped to stop the discharge from the culture vessel, and tube pump 1 was operated to flow 15 mL of 90% red water from the mixing vessel into the culture vessel, filling the culture vessel with 20 mL of 90% red water. The liquid transfer time was 15 minutes. This allowed the culture vessel to be replaced with red water with a concentration of 90%, and the medium replacement rate in the culture vessel could be controlled.

[0085] That is, according to the cell culture method of this embodiment shown in Example 2, the amount of medium used was very small at 22.5 mL, and medium exchange was performed uniformly and the medium exchange rate of the culture vessel was appropriately controlled. Furthermore, the medium exchange time was 37.5 minutes (= 2.5 + 7.5 + 15), which was shorter than the conventional circulation method (Comparative Example 3).

[0086] [Comparative Example 1] An experiment was conducted to confirm the effectiveness of a method in which the culture vessel and the medium supply vessel are connected by a tube and new medium is supplied from the medium supply vessel to the culture vessel.

[0087] The culture vessel used was a closed culture bag with 18,000 hemispherical wells with a diameter of 0.5 mm molded on the bottom (culture surface) of the vessel and coated with a polymer with low cell adhesion. The bottom area of ​​the bag was 50 cm. 2 The maximum liquid volume was 50 mL, the material of the bag was LLDPE, and the culture vessel had one port. A bag with a flat bottom and no wells was used as the medium supply container. The bag bottom area was 50 cm. 2 The maximum liquid volume was 50 mL, and the bag material was LLDPE. The medium supply container had one port.

[0088] The experimental method was as follows: 20 mL of clear water was filled into the culture vessel, and 18 mL of red water concentrate (100%) was filled into the medium supply vessel. A tube was then connected to the port of the culture vessel, and a tube pump was attached to the tube and operated to discharge 18 mL of clear water from the culture vessel. The liquid transfer time was 18 minutes. Next, the medium supply container was connected to a tube, and the tube pump was operated to send 18 mL of red water concentrate (100%) from the medium supply container to the culture container for 18 minutes.

[0089] As a result, it was found that although the clear water in the culture vessel could be replaced with red water of 90% concentration, the red water in the culture vessel was not uniform, and this was not an appropriate method for medium replacement.

[0090] Comparative Example 2 Unlike Example 1, an experiment was conducted to confirm the effectiveness of a method in which new medium is delivered from a medium supply container to a culture vessel while the used medium is discharged from the culture vessel, without preparing a concentration-adjusted medium by mixing a part of the used medium with the new medium in a mixing container. Also, as in Example 2, the liquid thickness in the culture vessel was reduced before delivery. The culture vessel used was the same as in Experiment 1. The medium supply vessel and waste liquid vessel were the same as the mixing vessel in Experiment 1.

[0091] In the experimental method, 20 mL of clear water was filled into the culture vessel, and 25 mL of red water concentrate (100%) was filled into the culture medium supply container, as shown in Figure 9. Then, a tube was connected to one port of the culture vessel to connect the culture medium supply container to the culture vessel, and a tube was connected to the other port of the culture vessel to connect the waste liquid container to the culture vessel. Furthermore, a tube pump 1 was disposed on the tube connecting the culture vessel and the medium supply vessel, and a tube pump 2 was disposed on the tube connecting the culture vessel and the waste liquid vessel.

[0092] Then, by operating the tube pump 2, 15 mL of the clear water in the culture vessel was pumped to the waste liquid container, and the liquid volume in the culture vessel was reduced to 5 mL (liquid thickness 1 mm). At this time, the liquid pumping time was 15 minutes.

[0093] The reason why the filling volume into the mixing container was set to 25 mL is that, based on the results of Experiment 1, the volume of liquid in the mixing container is -7.5 mL when the liquid in the culture container is replaced with red water, and is -15 mL when the volume of liquid in the culture container is increased to 20 mL, so a filling volume of 22.5 mL or more is sufficient to transfer the liquid.

[0094] Next, both tube pumps were operated to simultaneously pump the liquid toward the wastewater direction, maintaining a constant liquid volume and thickness in the culture vessel (reducing the thickness of the culture medium in the culture vessel), and the clear water in the culture vessel was uniformly replaced with 100% red water. The liquid pumping time was 7.5 minutes.

[0095] Furthermore, tube pump 2 was stopped to stop the discharge from the culture vessel, and tube pump 1 was operated to flow 15 mL of 100% concentration red water from the culture medium supply container into the culture vessel, filling the culture vessel with 20 mL of 100% concentration red water. The liquid transfer time at this time was 15 minutes.

[0096] As a result, it was found that the method of Comparative Example 2 could exchange the culture medium in the culture vessel at a 100% exchange rate, but could not arbitrarily control the medium exchange rate, and therefore could not achieve the object of the present invention.

[0097] Comparative Example 3 An experiment was conducted to confirm the effectiveness of the conventional method of replacing the culture medium, which is to replace the culture medium by circulating it in a container. The same culture vessels as those used in Experiment 1 were used.

[0098] The experimental method was as follows: First, 20 mL of clear water was filled into the culture vessel. Then, a tube was connected to one port of the culture vessel, and a tube pump was placed on the tube. The tube pump was operated to discharge 14 mL of clear water from the culture vessel. The liquid transfer time was 14 minutes. The tube pump was then operated to supply 14 ml of red water to the culture vessel through the tube, for a period of 14 minutes. At this time, the concentration of the red water in the culture vessel was 70%, and the red water in the culture vessel was not uniform.

[0099] Here, the reason why 70% (14 ml) of the clear water was discharged to achieve a 70% medium exchange rate, rather than 90% (18 ml) of the clear water in the culture vessel to achieve a 90% medium exchange rate, is that with this circulation method, if 90% of the clear water was discharged, the cells would also be discharged along with the medium.

[0100] Next, a tube was connected to two ports of the culture vessel to form a circular flow channel, and a tube pump was placed on the tube. The tube pump was then operated to circulate the 70% red water in the culture vessel, thereby equalizing the uneven distribution of the liquid. After 40 minutes of circulation, the 70% red water in the culture vessel was homogenized.

[0101] Next, by repeating the above-mentioned steps from discharging 14 mL of clear water from the culture vessel to circulating the red water in the culture vessel, the contents of the culture vessel were replaced with red water at a concentration of 91% (= (7 / 10) × (3 / 10) + 1 × (7 / 10) × 100).

[0102] That is, according to the method of Comparative Example 3, although it is possible to control the efficiency of culture medium exchange, it takes a long time of 136 minutes (=14+14+40+14+14+40) to exchange the culture medium, and there is also the problem that it is complicated because it requires repeated circulation of the liquid.

[0103] [Reference example 1] An experiment was carried out to confirm whether or not nearly 100% medium exchange inhibits cell culture efficiency when inducing differentiation of spheres into mesoderm. Specifically, iPS cells (1231A3 strain, Center for iPS Cell Research and Application, Kyoto University) were used as the cells to be differentiated, and ultra-low attachment surface 6-well plates (Corning) were used as the culture vessels.

[0104] Furthermore, the culture medium used on day 1 was StemFit AK02N medium (Ajinomoto Healthy Supply Co., Ltd.) + 10 μM Y-27623 (Fujifilm Wako Pure Chemical Corporation), and on day 2 was RPMI Medium 1640 (Thermo Fisher Scientific) + B-27 Minus Insulin (Thermo Fisher Scientific) + 6 μM CHIR99021 (Fujifilm Wako Pure Chemical Corporation). Accumax solution (Sigma-Aldrich) was used as a reagent to break down cell aggregates into single cells.

[0105] On day 1, iPS cells were seeded at a density of 6.0 x 10 cells / well onto five 6-well plates with an ultra-low attachment surface. On day 2, medium exchange rates were performed for each plate at 90%, 95%, 99%, 99.75%, and 100% exchange rates. On day 3, cell clumps were collected in a 15 mL tube and washed. Then, 1 mL of Accumax solution was added and the plate was heated at 37°C for 3 minutes. The spheres were then broken down into single cells by pipetting. The number of single cells was then counted using a hemocytometer.

[0106] As a result, as shown in the table below, the number of cells after culture decreased at medium exchange rates of 90% or more, with a particularly rapid decrease observed between 99% and 99.75%. [Table 1]

[0107] This confirmed that when inducing differentiation of spheres into mesoderm, if nearly 100% medium exchange is performed, the cell culture efficiency is inhibited. That is, it was confirmed that it is extremely important that the cell culture method of this embodiment can control the medium exchange rate to less than 100%, particularly less than 99%.

[0108] The present invention is not limited to the above-described embodiments and examples, and various modifications are possible within the scope of the present invention. For example, the volumes of the culture vessels and mixing vessels used are not limited to those used in the examples, and the present invention can be applied to medium exchange using culture vessels of various volumes and shapes. [Industrial Applicability]

[0109] The present invention can be suitably used in cell culture using a culture vessel when it is desired to control the exchange rate of the medium in the vessel to a desired value. [Explanation of symbols]

[0110] 10 Culture vessel 11,12 ports 20 Mixing container (medium supply container) 30 Waste liquid container 41, 42 Tube (tubular member) 51, 52 Pump (liquid delivery means) 60 Control device 61 Input / output section 62 Control Unit 63 Operation section 64 Power supply section 70 Detector

Claims

1. A cell culture method using a culture vessel filled with cells and a medium for cell culture, and a medium supply vessel filled with new medium for supplying the medium to the culture vessel, comprising: the culture vessel has at least a first port used for supplying a culture medium and a second port used for discharging the culture medium, the culture medium supply vessel is a mixing vessel for adjusting the concentration of a new culture medium to be supplied to the culture vessel, A portion of the used culture medium in the culture vessel is transferred from the culture vessel to the mixing vessel; The new medium and a portion of the used medium are mixed in the mixing container to prepare a concentration-adjusted medium; While transferring the concentration-adjusted medium from the mixing vessel to the culture vessel, the remainder of the used medium is discharged from the culture vessel; When discharging the remainder of the used culture medium from the culture vessel while feeding the concentration-adjusted culture medium from the mixing vessel to the culture vessel, the amount of culture medium in the culture vessel is not changed, and after the remainder of the used culture medium in the culture vessel is replaced with the concentration-adjusted culture medium, the discharge from the culture vessel is stopped, and the concentration-adjusted culture medium in an amount equivalent to a part of the used culture medium is fed from the mixing vessel to the culture vessel, thereby filling the culture vessel with the concentration-adjusted culture medium. A cell culture method characterized by:

2. A cell culture method using a culture vessel filled with cells and a culture medium for cell culture, and a culture medium supply vessel filled with new culture medium and supplying the culture medium to the culture vessel, the culture vessel has at least a first port used for supplying a culture medium and a second port used for discharging the culture medium, the culture medium supply vessel is a mixing vessel for adjusting the concentration of a new culture medium to be supplied to the culture vessel, A portion of the used culture medium in the culture vessel is transferred from the culture vessel to the mixing vessel; The new medium and a portion of the used medium are mixed in the mixing container to prepare a concentration-adjusted medium; before discharging the remainder of the used medium from the culture vessel while feeding the concentration-adjusted medium from the mixing vessel to the culture vessel, discharging a portion of the remainder of the used medium in the culture vessel from the culture vessel to reduce the liquid thickness of the medium in the culture vessel; While maintaining the reduced liquid thickness of the culture medium in the culture vessel, the concentration-adjusted culture medium is sent from the mixing vessel to the culture vessel, and the remaining part of the used culture medium in the culture vessel is discharged from the culture vessel, and the concentration-adjusted culture medium is filled into the culture vessel. A cell culture method characterized by:

3. After the entire remaining portion of the used culture medium in the culture vessel has been replaced with the concentration-adjusted culture medium, discharge from the culture vessel is stopped, and a portion of the used culture medium and an amount of the concentration-adjusted culture medium equivalent to a portion of the remaining portion of the used culture medium are sent from the mixing vessel to the culture vessel. The cell culture method according to claim 2 .

4. adjusting a mixing ratio between the amount of new medium in the mixing vessel and the amount of used medium transferred from the culture vessel to the mixing vessel; Controlling the medium exchange rate of the culture vessel The cell culture method according to any one of claims 1 to 3.

5. Before adjusting the mixing ratio between the amount of new medium in the mixing vessel and the amount of used medium to be transferred from the culture vessel to the mixing vessel, The amount of fluid to be sent necessary for replacing the culture medium in the culture vessel is measured, and a desired concentration of a new culture medium to be supplied to the culture vessel is determined. Based on the necessary amount of fluid to be sent and the desired concentration, the amount of new culture medium in the mixing vessel and the amount of used culture medium to be sent from the culture vessel to the mixing vessel are determined. The cell culture method according to claim 4 .

6. The medium exchange rate of the culture vessel is controlled to be 50% or more and less than 100%.

6. The cell culture method according to claim 4 or 5.

7. a culture vessel that includes at least a first port used for supplying a culture medium and a second port used for discharging the culture medium, and that is filled with cells and a culture medium and used for cell culture; a mixing vessel that is connected to the first port via a first tubular member, is filled with a new medium, and supplies the medium to the culture vessel; a waste liquid container that is connected to the second port via a second tubular member and into which the used culture medium flows; a control device that controls the operation of a first liquid-transfer means disposed in the first tubular member and a second liquid-transfer means disposed in the second tubular member to control the transfer of culture medium between the culture vessel, the mixing vessel, and the waste liquid vessel; the control device transfers a portion of the used medium in the culture vessel from the culture vessel to the mixing vessel, transfers a concentration-adjusted medium prepared by mixing the new medium and a portion of the used medium in the mixing vessel from the mixing vessel to the culture vessel while transferring the remainder of the used medium from the culture vessel to the waste liquid container, and transfers the concentration-adjusted medium from the mixing vessel to the culture vessel while transferring the remainder of the used medium from the culture vessel to the waste liquid container, without changing the amount of medium in the culture vessel, and after the remainder of the used medium in the culture vessel is replaced with the concentration-adjusted medium, stops transferring the concentration-adjusted medium from the culture vessel to the waste liquid container, and transfers the concentration-adjusted medium in an amount equivalent to a portion of the used medium from the mixing vessel to the culture vessel, thereby filling the culture vessel with the concentration-adjusted medium. A cell culture system characterized by:

8. A culture vessel having at least a first port used for supplying a culture medium and a second port used for discharging the culture medium, the culture vessel being filled with cells and a culture medium and used for cell culture; a mixing vessel that is connected to the first port via a first tubular member, is filled with a new medium, and supplies the medium to the culture vessel; a waste liquid container that is connected to the second port via a second tubular member and into which the used culture medium flows; a control device that controls the operation of a first liquid-transfer means disposed in the first tubular member and a second liquid-transfer means disposed in the second tubular member to control the transfer of culture medium between the culture vessel, the mixing vessel, and the waste liquid vessel; The control device transfers a portion of the used medium in the culture vessel from the culture vessel to the mixing vessel, and transfers a concentration-adjusted medium prepared by mixing the new medium and a portion of the used medium in the mixing vessel from the mixing vessel to the culture vessel while transferring the remainder of the used medium from the culture vessel to the waste liquid container, thereby reducing the liquid thickness of the medium in the culture vessel, and while maintaining the reduced liquid thickness of the medium in the culture vessel, transfers the entire remaining portion of the used medium in the culture vessel from the culture vessel to the waste liquid container, thereby filling the culture vessel with the concentration-adjusted medium. A cell culture system characterized by:

9. Further provided is a detection unit for measuring a change in the liquid thickness of the culture medium, The control device controls the transfer of culture medium between the culture vessel, the mixing vessel, and the waste liquid vessel based on the detection information input from the detection unit.

9. The cell culture system according to claim 7 or 8.

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