Culture medium regeneration system

Abstract

Provided is a culture medium regeneration system capable of appropriately adjusting components in the process of culturing a cell aggregate. This culture medium regeneration system comprises: a culture medium circulation path through which a culture medium accommodated in a culturing part for culturing a cell aggregate circulates; a nutrient component supply path through which a regeneration culture medium containing nutrient components for the cell aggregate flows; and a culture medium regeneration module that is connected to the culture medium circulation path and the nutrient component supply path and that accommodates a semipermeable membrane through which at least either of nutrient components or metabolites produced by the cell aggregate can pass. In the culture medium regeneration system, at least one of the occasion in which the nutrient components pass through the semipermeable membrane and make contact with the cell aggregate and the occasion in which the metabolites pass through the semipermeable membrane and make contact with the regeneration culture medium in the nutrient component supply path is adjusted.

Description

Culture medium regeneration system

[0001] The present invention relates to a culture medium regeneration system for regenerating a culture medium.

[0002] Generally, cell aggregates (cells) consume nutrients (e.g., glucose) and produce waste products (e.g., lactic acid) during the culture (growth) process. Therefore, it is necessary to replenish nutrients and remove waste products. A common method for this is to replace the culture medium at predetermined intervals (see, for example, Patent Documents 1, 2, and 3).

[0003] Japanese Patent Publication No. 2002-85049 Japanese Patent No. 6422221 Japanese Patent Publication No. 2003-521877

[0004] The conventional technology described above regenerates the culture medium by dialysis, and adjusts the components in multiple tanks by installing a separation membrane in part of the culture circuit. However, the performance (dialysis capacity) of the separation membrane cannot keep up with the fluctuations in the concentration of the components that occur during the culture process, making it impossible to adjust the components appropriately.

[0005] The present invention has been made in view of the above points, and an object of the present invention is to provide a medium regeneration system that can appropriately adjust the components during the culture process of cell aggregates.

[0006] The medium regeneration system according to the present invention is characterized in that it comprises a medium circulation path through which medium contained in a culture section for culturing cell aggregates circulates, a nutrient component supply path through which regeneration medium containing nutritional components for the cell aggregates flows, and a medium regeneration module connected to the medium circulation path and the nutrient component supply path and containing a semipermeable membrane that is permeable to at least one of the nutritional components and metabolic products of the cell aggregates, and at least one of the opportunities for the nutritional components to permeate the semipermeable membrane and come into contact with the cell aggregates, and the opportunities for the metabolic products to permeate the semipermeable membrane and come into contact with the regeneration medium in the nutrient component supply path is regulated.

[0007] The components can be adjusted appropriately during the process of culturing the cell aggregates.

[0008] 2A is a schematic diagram showing the configuration of a culture medium regeneration system 100 according to a first embodiment. 2B is a cross-sectional view (FIG. 2A) showing an outline of the movement of cell aggregates within a suction tube nozzle 130-1, and a cross-sectional view (FIG. 2B) showing the distribution of cell aggregates within the suction tube nozzle 130-1 at the II section shown in FIG. 2A. 2C is a graph showing the relationship between the inclination angle of the suction tube nozzle 130-1 and the circulation flow rate when the cell aggregates rise to a position midway between the suction opening 134-1 and the discharge opening 136-1. 2C is a schematic diagram showing the configuration of a culture medium regeneration system 200 according to a second embodiment. 2D is a schematic diagram showing the configuration of a culture medium regeneration system 300 according to a third embodiment. 2E is a schematic diagram showing the configuration of a culture medium regeneration system 400 according to a fourth embodiment. 2F is a schematic diagram showing the configuration of a culture medium regeneration system 500 according to a fifth embodiment. 2G is a schematic diagram showing the configuration of a culture medium regeneration system 600 according to a sixth embodiment. 2G is a schematic diagram showing the configuration of a culture medium regeneration system 700 according to a seventh embodiment. 2H is a table showing the conditions of a verification experiment for the culture medium regeneration system 700 according to the seventh embodiment. 10 is a table showing the conditions of a verification experiment for the culture medium regeneration system 700 according to the seventh embodiment. FIG. 11 is a graph showing the change in lactic acid concentration as a result of a verification experiment for the culture medium regeneration system 700 according to the seventh embodiment. FIG. 12 is a schematic diagram showing the configuration of a culture medium regeneration system 800 according to the eighth embodiment. FIG. 13 is a graph showing the flow rate at which cell aggregates are not sucked in by suction using inclined suction.

[0009] <<<<<Outline of Embodiments>>>>> In order to culture cell aggregates (cells (hereinafter mainly referred to as cell aggregates)), it is necessary to replenish nutrients and remove waste products. As a method for this, medium exchange is generally carried out.

[0010] However, the method of medium replacement is expected to have the following three problems. (1) Replacement in accordance with waste removal Even if nutrients remain in the medium, the medium needs to be replaced to remove waste, and replacing the medium more than necessary can result in excessive medium usage. (2) Volume fluctuation during medium replacement If the volume of the medium present in the culture vessel temporarily decreases when the medium is removed from the culture vessel during medium replacement, the density of the cell aggregates may increase, potentially leading to increased shear stress on the cell aggregates. (3) Fluctuations in medium concentration It is expected that the concentration of medium components may suddenly fluctuate before and after medium replacement when new medium is replenished into the culture vessel. The medium regeneration system according to this embodiment was made to solve the above-mentioned problems.

[0011] <<First Feature>> According to the first feature, there is provided a culture medium regeneration system comprising: a culture medium circulation path (for example, a culture medium circulation circuit described below) through which a culture medium contained in a culture section for culturing cell aggregates circulates; a nutrient component supply path (for example, a culture medium regeneration circuit described below) through which a regeneration medium containing nutritional components for the cell aggregates flows; and a culture medium regeneration module connected to the culture medium circulation path and the nutrient component supply path and containing a semipermeable membrane (for example, a culture medium regeneration membrane 179 described below) through which at least one of the nutritional components and metabolic products of the cell aggregates can permeate, wherein at least one of the opportunities for the nutritional components to permeate the semipermeable membrane and come into contact with the cell aggregates and the opportunities for the metabolic products to permeate the semipermeable membrane and come into contact with the regeneration medium in the nutrient component supply path is regulated.

[0012] The medium regeneration system includes a medium circulation path, a nutrient component supply path, and a medium regeneration module. The medium regeneration system includes a culture system that cultures cell aggregates in a culture section. The medium regeneration system is a system for regenerating a medium used for culturing cell aggregates in the culture system.

[0013] <Culture medium circulation path> The culture medium circulation path is a flow path through which the culture medium circulates. The culture medium is contained in a culture unit for culturing cell aggregates. Cell aggregates also flow through parts of the culture medium circulation path (for example, suction tube nozzles 130-1, 130-2, 130-3, etc., which will be described later), but the cell aggregates do not circulate throughout the culture medium circulation path. In contrast, the culture medium flows and circulates throughout the entire culture medium circulation path.

[0014] <Nutrient Component Supply Channel> The nutrient component supply channel allows a fluid containing nutrients for the cell aggregate, such as a regeneration medium, to flow.

[0015] <Culture medium regeneration module> The culture medium regeneration module is connected to both the culture medium circulation path and the nutrient component supply path. The culture medium regeneration module has a semipermeable membrane. The semipermeable membrane is permeable to at least one of nutrients and metabolic products produced by the cell aggregates. The nutrients permeate the semipermeable membrane and move from the nutrient component supply path to the culture medium circulation path. The metabolic products permeate the semipermeable membrane and move from the culture medium circulation path to the nutrient component supply path. The cell aggregates can be cultured by the exchange of nutrients and metabolic products via the semipermeable membrane.

[0016] <Adjustment of contact opportunity> Furthermore, the opportunity for nutritional components to permeate the semipermeable membrane and come into contact with the cell aggregate, and the opportunity for metabolic products from the cell aggregate to permeate the semipermeable membrane and come into contact with the fluid in the nutritional component supply path are adjusted.

[0017] By adjusting the contact opportunity, necessary nutrients can be supplied to the medium circulation path while unnecessary metabolic products can be removed from the medium circulation path, and the components can be appropriately adjusted during the culture process of the cell aggregate mass.

[0018] <<Second Feature>> The second feature is the first feature, in which the concentration of at least one of a nutrient component supplied to the cell aggregate via the semipermeable membrane and a metabolic product of the cell aggregate is adjusted.

[0019] Since the concentration of at least one of the nutritional components and the metabolic products of the cell aggregates is adjusted, an environment suited to the growth of the cell aggregates can be provided.

[0020] <<Third Feature>> The third feature is the first or second feature, further comprising a nutrient component storage tank (for example, a regeneration medium tank 180 described later) in which the nutrient components are stored, and the flow path between the nutrient component storage tank and the semipermeable membrane is a non-circulating path that includes the nutrient component supply path and does not include a flow path from the semipermeable membrane to the nutrient component storage tank.

[0021] By adopting a non-circulating route, it is possible to prevent the influence of metabolic products and to maintain the nutritional components at a desired concentration. For example, it is possible to maintain the nutritional components at a constant concentration. It is possible to always provide an environment suitable for the growth of cell aggregates.

[0022] <<Fourth Feature>> A fourth feature is the first to third features, further comprising a nutrient component storage tank in which the nutrient components are stored, and a nutrient component adding device that adds new nutrient components to the nutrient component storage tank.

[0023] Since new nutrients are added to the nutrient reservoir, the nutrients can be adjusted to a desired concentration. For example, the nutrients can be maintained at a constant concentration. By adjusting the concentration of the nutrients, it is possible to achieve a balance between the supply of nutrients and the removal of unnecessary metabolic products.

[0024] <<Fifth Feature>> The fifth feature is the first to fourth features, in which a flow rate of the culture medium flowing through the culture medium circulation path is adjusted.

[0025] It is possible to supply nutrients required in accordance with the growth of the cell aggregate, and to provide an environment suited to the growth of the cell aggregate.

[0026] <<Sixth Feature>> The sixth feature is the first to fifth features, further comprising a discharge nozzle (for example, suction tube nozzles 130-1, 130-2, 130-3, etc., described later) that discharges the culture medium from the culture unit toward the semipermeable membrane, and the discharge nozzle is provided at an angle to the vertical direction.

[0027] By arranging the outlet nozzle at an angle to the vertical, the flow rate of the culture medium flowing through the outlet nozzle can be adjusted to control the amount of nutrients and metabolic products that come into contact with the semipermeable membrane, thereby achieving a balance between the amount of nutrients supplied and the amount of metabolic products removed.

[0028] <<Seventh Feature>> The seventh feature is the first to sixth features, in which the regeneration medium contains an adsorbent that adsorbs the metabolic product.

[0029] The concentration of metabolic products in the regeneration medium can be kept low by permeating the semipermeable membrane.

[0030] <<Eighth Feature>> The eighth feature is any one of the first to seventh features, wherein the target of the adsorbent is at least one of lactic acid, ammonia, glutamic acid, isovaleric acid, butyric acid, and citric acid, which are waste products.

[0031] The concentration of metabolic products in the regeneration medium can be kept low by permeating the semipermeable membrane.

[0032] <<Ninth Feature>> According to the ninth feature, there is provided a culture medium regeneration system comprising: a culture medium circulation path (for example, a culture medium circulation circuit described later) through which the culture medium and metabolic products contained in a culture unit are circulated; a nutrient component supply path (for example, a culture medium regeneration circuit described later) through which a regeneration medium containing nutritional components is circulated; and a culture medium regeneration module (for example, a culture medium regeneration module 170 described later) connected to the culture medium circulation path and the nutrient component supply path and containing a semipermeable membrane (for example, a culture medium regeneration membrane 179 described later) through which the nutritional components and the metabolic products can permeate; and a control unit that adjusts the circulation flow rate of the culture medium circulating through the culture medium circulation path and the circulation flow rate of the regeneration medium circulating through the nutrient component supply path, wherein a rate of change of at least one of the concentration of the metabolic products in the culture unit and the concentration of the nutritional components is changed depending on the circulation flow rate of the culture medium circulating through the culture medium circulation path and the circulation flow rate of the regeneration medium circulating through the nutrient component supply path (for example, FIG. 11 described later).

[0033] <<Tenth Feature>> The tenth feature is the ninth feature, in which the circulation flow rate of the regeneration culture medium flowing through the nutrient component supply path and the circulation flow rate of the culture medium circulating in the culture medium circulation path are increased, thereby increasing at least one of the rate of decrease in concentration of the metabolic product in the culture section and the rate of increase in concentration of the nutrient component in the culture section.

[0034] <<Eleventh Feature>> The device comprises a culture medium circulation path through which the culture medium contained in the culture section circulates together with cell aggregates, and an outlet nozzle that discharges the culture medium from the culture section, wherein the outlet nozzle is disposed at an angle to the vertical direction, and the circulation flow rate is increased according to the inclination angle of the outlet nozzle while preventing the cell aggregates from being discharged from the culture section into the culture medium circulation path.

[0035] <<Twelfth Feature>> The twelfth feature is the ninth to eleventh features, wherein the circulation flow rate through the culture medium circulation path is increased to increase at least one of the rate of decrease in concentration of the metabolic product in the culture section and the rate of increase in concentration of the nutrient component in the culture section.

[0036] <<<<<Details of the Present Embodiment>>>> The present embodiment will be described below with reference to the drawings. The present embodiment refers to the first to eighth embodiments described below.

[0037] <<<<Cells>>> Cells are the subject of culture in a culture system. By culturing cells, they divide and increase in number. When cells come into contact with each other, they form cell aggregates (cell aggregates). A culture system is a system that grows cells and forms cell aggregates.

[0038] The cells are not particularly limited as long as they form cell aggregates in a culture medium. The cells are preferably derived from mammals, and preferably from species commonly used in research, such as humans, primates (e.g., monkeys), or mice. Examples of cells include cells used in research on regenerative medicine and cells used as cell preparations. Specific examples include pluripotent stem cells (ES cells and iPS cells), various progenitor cells (e.g., nephron progenitor cells, ureteric bud cells, and interstitial progenitor cells), and various stem cells (e.g., mesenchymal stem cells, neural stem cells, and adipose stem cells). The cells may be cells derived from pluripotent stem cells, immortalized cells, or established cell lines, or primary culture cells isolated from tissue. Depending on the purpose, the cells may be normal cells, cells with a disease, genetic abnormality, or transgene.

[0039] Cell aggregates also include those substituted with non-growing resins, etc. Those substituted with resins, etc. do not require nutritional components and do not produce waste products (metabolites). Those substituted with resins, etc. may be those whose fluidity in the culture medium, such as size, shape, and specific gravity, is similar to that of growing cell aggregates. In this specification, those substituted with resins, etc. may also be referred to as cell aggregates.

[0040] <<<Culture Medium>>> The culture medium used in the culture medium regeneration system is classified into new culture medium and return culture medium. New culture medium is a culture medium that is newly supplied to the culture vessels 110-1 (FIGS. 1, 4, 6, 7, and 8 described below) and 110-3 (FIG. 5 described below) (reactors), etc. The return culture medium is a culture medium that has already been supplied to the culture vessels 110-1 and 110-3, etc., and used for culture, and is then returned for circulation or disposal.

[0041] The medium can be selected and used appropriately depending on the cells, and is not particularly limited. Conventional materials and additives useful for cell culture can also be used as appropriate. Examples of basal media include DMEM, DMEMHG, EMEM, IMDM (Iscove's Modified Dulbecco's Medium), GMEM (Glasgow's MEM), RPMI-1640, α-MEM, Ham's Medium F-12, Ham's Medium F-10, Ham's Medium F12K, etc. Additives such as amino acids, vitamins, inorganic salts, proteins (growth factors), glucose, antibiotics, signal transduction inhibitors, reducing agents, and buffers may be added. Serum (preferably mammalian serum, e.g., fetal bovine serum, human serum, etc.) may also be added. Supplements can also be used as a substitute for serum.

[0042] In this specification, the term "medium" refers to a liquid that does not contain nutrients but has similar pH, osmotic pressure, viscosity, etc., and can be considered a medium from the perspective of fluidity. For example, phosphate-buffered saline (PBS) can be used as a substitute for a medium.

[0043] <<<Cell Suspension>>> A cell suspension is a system in which cells or cell aggregates are dispersed in a medium. Cells or cell aggregates and a medium are stored as a cell suspension in the culture vessels 110-1 and 110-3.

[0044] <<<<Waste Products (Metabolites)>>> Waste products are produced as the cell aggregates grow. Typical waste products include lactic acid, ammonia, glutamic acid, isovaleric acid, butyric acid, and citric acid. In this specification, the term "waste products" refers not only to waste products produced as the cell aggregates grow, but also to substances similar to these waste products. For example, lithium lactate, which is similar to lactic acid, an example of a waste product, is also referred to as a waste product.

[0045] <<<<Agitation>>> Agitation means displacing the cells, cell aggregates, and culture medium in a cell suspension. Agitation displaces the cells and cell aggregates along with the culture medium. The cells and cell aggregates are mainly displaced due to the displacement of the culture medium (convection). Note that the action on the cell suspension can be anything other than agitation as long as it can displace the cells, and can also be an action such as shaking.

[0046] <<<<Directions, conditions, etc.>>> <Horizontal direction> The horizontal direction is the direction that intersects at a right angle with the Earth's gravity.

[0047] <Vertical direction> The vertical direction refers to the direction of gravity. It is the direction indicated by the string that suspends an object. It refers to the direction perpendicular to the horizontal direction. Specifically, it refers to the direction in which the central axes CO of the culture vessels 110-1 and 110-3, the rotation axis RO of the stirring blade 124a, and the central axes AO of the aspirating tube nozzles 130-1, 130-2, and 130-3 (FIGS. 1, 2A, 4, 5, 6, 7, and 8) are vertical.

[0048] <Tilt Direction> The tilt direction refers to a state of tilt from the vertical or horizontal direction, but is a direction that is neither vertical nor horizontal. Specifically, it refers to a direction in which the central axis CO of the culture vessels 110-1 and 110-3, the rotation axis RO of the stirring blade 124a, or the central axis AO of the aspirating tube nozzles 130-1, 130-2, and 130-3 is tilted at a predetermined angle from the vertical or horizontal direction.

[0049] For convenience, the angle between the vertical direction and the inclined direction may also be referred to as the inclination angle. The inclination angle refers to the angle between the vertical direction and the reference direction. An acute inclination angle is preferable. For example, the inclination angle is preferably in the range of 5 to 30 degrees. For example, the inclination angle is more preferably in the range of 10 to 20 degrees. The inclination angle is not limited to these ranges, and a suitable range can be determined as appropriate depending on the circulating flow rate of the culture medium, the type and size of the cell aggregates, the desired treatment time, etc.

[0050] <Axial direction> This refers to the direction along the central axis CO of the culture vessels 110-1 and 110-3, the rotation axis RO of the stirring blade 124a, and the central axis AO of the aspirating tube nozzles 130-1, 130-2, and 130-3. The axial direction can be defined mainly for elongated shapes, cylindrical shapes, and the like. The axial direction is not limited to a direction along a straight line. It may be any direction along the central axis CO, rotation axis RO, or central axis AO.

[0051] <Vertical State> The vertical state refers to a state in which the direction of the central axis CO of the culture vessel 110-1 or 110-3, the direction of the rotation axis RO of the agitator blade 124a, and the direction of the central axis AO of the aspirating tube nozzle 130-1, 130-2, or 130-3 are parallel to the vertical direction. In other words, the vertical state refers to a state in which the central axis CO of the culture vessel 110-1 or 110-3, the rotation axis RO of the agitator blade 124a, and the central axis AO of the aspirating tube nozzle 130-1, 130-2, or 130-3 extend along the vertical direction.

[0052] <Tilt State> The tilt state refers to a state in which the direction of the central axis CO of the culture vessel 110-1 or 110-3, the rotation axis RO of the agitator blade 124a, or the central axis AO of the aspiration tube nozzle 130-1, 130-2, or 130-3 is tilted. A state in which the central axis CO of the culture vessel 110-1 or 110-3, the rotation axis RO of the agitator blade 124a, or the central axis AO of the aspiration tube nozzle 130-1, 130-2, or 130-3 is tilted with respect to the vertical direction is called a tilt state.

[0053] <Size of cell aggregates> The size of cell aggregates may be any value that indicates the degree of growth of the cell aggregates, and is not limited to diameter, but may be any value that indicates the approximate range in the growth process of the cell aggregates, such as the radius, density distribution of the cell aggregates, occupied area, etc. In this specification, the size may be referred to as the size of the cell aggregates, or the diameter of the cell aggregates.

[0054] <<<<<First Embodiment>>>>> FIG. 1 is a schematic diagram showing the configuration of a culture medium regeneration system 100 according to a first embodiment.

[0055] <<<<Main components of culture medium regeneration system 100>>> The culture medium regeneration system 100 mainly includes a culture vessel 110-1, an agitator 120, an aspirating tube nozzle 130-1, pumps 150a, 150b, and 150c, a culture medium regeneration module 170, and a regeneration culture medium tank 180.

[0056] <<<<Culture Medium Circulation Circuit and Culture Medium Regeneration Circuit>>> The culture medium regeneration system 100 has a culture medium circulation circuit and a culture medium regeneration circuit.

[0057] <<Culture Medium Circulation Circuit (Culture Medium Circulation Path)>> The culture medium circulation circuit is mainly formed by the culture vessel 110-1, the suction tube nozzle 130-1, the pump 150a, the culture medium regeneration module 170, the tube 160a, the tube 160b, the tube 160c, etc. The culture medium circulates through the culture medium circulation circuit by driving the pump 150a. The culture medium circulates in the following order: culture vessel 110-1, the suction tube nozzle 130-1, the tube 160a, the pump 150a, the tube 160b, the culture medium regeneration module 170, and the tube 160c.

[0058] <<Culture Medium Regeneration Circuit (Nutrient Component Supply Path)>> The culture medium regeneration circuit is mainly formed by pump 150b, pump 150c, culture medium regeneration module 170, regeneration medium tank 180, pipe 160d, pipe 160e, pipe 160f, pipe 160g, etc. By driving pump 150b and pump 150c, the regeneration medium stored in regeneration medium tank 180 circulates through the culture medium regeneration circuit. The regeneration medium circulates in the following order: regeneration medium tank 180, pipe 160f, pump 150c, pipe 160g, culture medium regeneration module 170, pipe 160d, pump 150b, and pipe 160e.

[0059] <<<Culture Vessel 110-1, Stirring Device 120, and Aspirating Tube Nozzle 130-1>>> As shown in FIG. 1, the culture vessel 110-1, the stirring device 120, and the aspirating tube nozzle 130-1 are fixedly arranged in an inclined state at a constant inclination angle θ.

[0060] <<Culture Vessel 110-1>> The culture medium regeneration system 100 has a culture vessel 110-1. During the cell culture process, a culture medium and cells (cell suspension) are contained in the culture vessel 110-1. The cells are cultured in the culture vessel 110-1 and grow as cell aggregates.

[0061] The culture vessel 110-1 has a substantially cylindrical shape. The culture vessel 110-1 has a substantially cylindrical side wall 112 and a substantially circular bottom 114. The shape of the culture vessel 110-1 is not limited to a cylindrical shape, and it may have any shape that allows the stirring blades 124a (described later) to be rotated to stir the cell suspension, smoothly culture the cells, and smoothly circulate the culture medium.

[0062] <Central Axis CO> The culture vessel 110-1 has a central axis CO that extends along the center of the side wall portion 112.

[0063] <<Stirring Device 120>> <Configuration of Stirring Device 120> The stirring device 120 stirs the cell suspension stored in the culture vessel 110-1. The stirring device 120 has a driving unit 122, a stirring bar 124, and a holding member 126. The driving unit 122 can be, for example, a magnetic stirrer. The driving unit 122 has a motor (not shown). When the motor rotates, the driving unit 122 uses magnetic force to rotate the stirring bar 124, which is disposed at a distance from the driving unit 122. The rotation speed of the motor may be determined appropriately depending on the amount of cell suspension, the number and size of cell aggregates, etc.

[0064] The rotation speed and direction of the motor are controlled by a control device (not shown). The control device has a processor (such as a CPU (Central Processing Unit)), ROM (Read Only Memory), RAM (Random Access Memory), an input / output interface, etc. The ROM, RAM, etc. store programs for controlling the rotation speed and direction of the motor. The processor reads and executes the programs for controlling the rotation speed and direction of the motor from the ROM, RAM, etc.

[0065] It is also possible to rotate the stirring bar 124 by directly connecting it to the shaft of a motor (not shown) without using a magnetic stirrer.

[0066] <Agitation by agitator 120> The cell suspension is agitated by rotation of agitator 124. Agitating the cell suspension can prevent cell aggregates from contacting each other or from contacting sidewall 112 or substantially circular bottom 114 of culture vessel 110-1 during culture or recovery of the cell aggregates.

[0067] <Stirring bar 124> The stirring bar 124 has a stirring blade 124a and an extension portion 124b. The stirring blade 124a has a thin plate-like shape that is approximately an isosceles triangle. The stirring blade 124a is located inside the culture vessel 110-1 and is separated from the drive unit 122. The bottom of the stirring blade 124a faces the bottom 114 of the culture vessel 110-1.

[0068] The shape of the stirring blades 124a is not limited to a substantially isosceles triangle, and may be any shape that can stir the cell suspension and rotate around the central axis CO. The shape of the stirring blades 124a may be any shape that can form a desired flow of the cell suspension.

[0069] The extension portion 124b has a generally rod-like shape. The extension portion 124b extends from the top of the agitator blade 124a (the top facing the bottom of the agitator blade 124a) in a direction away from the agitator blade 124a. The direction in which the extension portion 124b extends is the direction in which the rotation axis RO of the agitator bar 124 extends. It is preferable to position the agitator bar 124 so that the rotation axis RO roughly coincides with the central axis CO of the culture vessel 110-1. By positioning the agitator bar 124 in this manner, the cell suspension can be agitated throughout every corner of the culture vessel 110-1. Note that it is sufficient that the extension direction of the rotation axis RO and the extension direction of the central axis CO are parallel.

[0070] <Holding member 126> The holding member 126 rotatably holds the stirring bar 124. The holding member 126 rotatably holds the stirring bar 124 even if the rotation axis RO of the stirring bar 124 is tilted. By holding the stirring bar 124 with the holding member 126, the rotation axis RO and the central axis CO can be roughly aligned.

[0071] An example has been shown in which the rotation axis RO is roughly aligned with the central axis CO, but the rotation axis RO may be appropriately determined depending on the inclination angle, the amount of cell suspension, the number and size of cell aggregates, and the bias of the cell aggregates within the culture vessel 110-1.

[0072] <<Aspiration Tube Nozzle 130-1>> The aspiration tube nozzle 130-1 is used to separate the cell aggregates that are retained in the aspiration tube nozzle 130-1 from the culture medium that is discharged through the discharge opening 136-1 of the aspiration tube nozzle 130-1 and circulated. The cell aggregates cultured in the culture vessel 110-1 vary in size. The aspiration tube nozzle 130-1 is used so that the cell aggregates that are being cultured remain in the aspiration tube nozzle 130-1 and only the culture medium is discharged through the discharge opening 136-1 of the aspiration tube nozzle 130-1. The operation of the cell aggregates and culture medium within the aspiration tube nozzle 130-1 will be described later. The aspiration tube nozzle 130-1 is formed from glass, resin, stainless steel, or the like and has a fixed shape. The aspiration tube nozzle 130-1 is preferably made of a sterilizable material.

[0073] <Extended portion 132-1, suction opening 134-1, and discharge opening 136-1> The suction tube nozzle 130-1 has an extended portion 132-1, a suction opening 134-1, and a discharge opening 136-1.

[0074] <Extending portion 132-1> The extending portion 132-1 has an elongated shape. That is, the aspirating tube nozzle 130-1 has an elongated shape. The extending portion 132-1 extends linearly along the longitudinal direction. The extending portion 132-1 extends in an oblique direction. That is, the aspirating tube nozzle 130-1 extends in an oblique direction. In other words, the central axis AO of the aspirating tube nozzle 130-1 extends in an oblique direction.

[0075] As shown in FIG. 2, the extension portion 132-1 has a tubular shape. The extension portion 132-1 has an outer peripheral portion 132O-1 and an inner peripheral portion 132I-1. The outer peripheral portion 132O-1 forms the outer surface of the tube. The inner peripheral portion 132I-1 forms the inner surface of the tube. The outer peripheral portion 132O-1 and the inner peripheral portion 132I-1 are concentrically positioned. A long surrounding region SR, surrounded by the inner peripheral portion 132I-1 and extending along the longitudinal direction, functions as a hollow conduit. The culture medium and cell aggregates can flow through the surrounding region SR. Note that in FIG. 2A, the surrounding region SR is shown as a region surrounded by a square dashed line along the inner peripheral portion 132I-1. Also, in FIG. 2B, the surrounding region SR is shown as a region surrounded by a circular dashed line along the inner peripheral portion 132I-1. The extension 132-1 has a constant cross-sectional area A1, which is the area of ​​the inner diameter portion when the suction tube nozzle 130-1 is cut along a direction perpendicular to the central axis AO of the suction tube nozzle 130-1.

[0076] <Intake Opening 134-1 and Discharge Opening 136-1> The suction tube nozzle 130-1 has two ends in the longitudinal direction. The suction tube nozzle 130-1 has an intake opening 134-1 at one end and an exhaust opening 136-1 at the other end. The extension portion 132-1 extends between the intake opening 134-1 and the exhaust opening 136-1.

[0077] The intake opening 134-1 has a substantially circular opening. The discharge opening 136-1 has a substantially circular opening. The intake opening 134-1, the discharge opening 136-1, and the surrounding region SR are connected to each other. The culture medium and cell aggregates stored in the culture vessel 110-1 are sucked in through the intake opening 134-1 and guided to the surrounding region SR. The cell aggregates guided to the surrounding region SR flow through the surrounding region SR and remain there.

[0078] Note that some of the cell aggregates guided to the surrounding region SR flow through the surrounding region SR, are discharged from the intake opening 134-1, and return to the culture vessel 110-1 again. The culture medium guided to the surrounding region SR flows through the surrounding region SR and is discharged from the discharge opening 136-1.

[0079] The suction opening 134-1 and the discharge opening 136-1 have a constant opening area A1 that is the same as the cross-sectional area A1 of the extension portion 132-1. The suction tube nozzle 130-1 has a cross-sectional area A1.

[0080] <<<Pump 150a, Pipe 160a, Pipe 160b, Pipe 160c>>> <Pump 150a> The pump 150a may be any pump capable of adjusting the flow rate of the culture medium. The pump 150a may be, for example, a tube pump, a reciprocating pump, a centrifugal pump, a propeller pump, or the like. The pump 150a has a motor (not shown). The pump 150a can adjust the flow rate by the rotation speed of the motor, the frequency at which the motor is driven, or the like. Hereinafter, for simplicity, driving the motor of the pump 150a may be simply referred to as driving the pump 150a.

[0081] The rotation speed of the motor, the timing at which the motor is driven, and other parameters are controlled by a control device (not shown). The control device includes a processor (such as a CPU (Central Processing Unit)), a ROM (Read Only Memory), a RAM (Random Access Memory), and an input / output interface. The ROM and RAM store programs for controlling the rotation speed and timing at which the motor is driven. The processor reads and executes the programs for controlling the rotation speed and timing at which the motor is driven from the ROM and RAM.

[0082] The control device that controls the motor of the pump 150a may be the same as or different from the control device that controls the motor of the drive unit 122 of the agitator 120 described above.

[0083] <Driving the Pump 150a> By driving the pump 150a, cell aggregates can be discharged together with the culture medium from the culture vessel 110-1. The flow rate of the culture medium per unit time generated by driving the pump 150a is referred to as the circulation flow rate.

[0084] <<<Tubes 160a, 160b, 160c>>> The tubes 160a, 160b, 160c are made of flexible resin etc. For simplicity, the tubes 160a, 160b, 160c are shown in a linear shape in Fig. 1 .

[0085] Tube 160a, tube 160b, and tube 160c have an elongated shape. Tube 160a has a first end 162a and a second end 164a in the longitudinal direction. Tube 160b has a first end 162b and a second end 164b in the longitudinal direction. Tube 160c has a first end 162c and a second end 164c in the longitudinal direction.

[0086] <Tube 160a> A first end 162a of tube 160a is connected to the discharge opening 136-1 of the suction tube nozzle 130-1. A second end 164a of tube 160a is connected to the first end 152a of the pump 150a. The suction tube nozzle 130-1, tube 160a, and pump 150a are in communication with each other.

[0087] <Tube 160b> A first end 162b of the tube 160b is connected to a second end 154a of the pump 150a. A second end 164b of the tube 160b is connected to a medium inlet 172 of the medium regeneration module 170. The pump 150a, the tube 160b, and the medium regeneration module 170 are in communication with each other.

[0088] <Tube 160c> A first end 162c of the tube 160c is connected to a medium outlet 174 of the medium regeneration module 170. A second end 164c of the tube 160c is fixed so as to be disposed at a predetermined position inside the culture vessel 110-1. The tube 160c and the medium regeneration module 170 are in communication with each other.

[0089] <<<<Culture Medium Regeneration Circuit>>> The culture medium regeneration circuit mainly consists of pump 150b, pump 150c, culture medium regeneration module 170, regeneration culture medium tank 180, pipe 160d, pipe 160e, pipe 160f, pipe 160g, etc. The regeneration culture medium stored in regeneration culture medium tank 180 circulates through the culture medium regeneration circuit.

[0090] <Pump 150b, Pump 150c> Pump 150b delivers the liquid from which waste products and the like have been removed in medium regeneration module 170 to regeneration medium tank 180. Pump 150c supplies the regeneration medium stored in regeneration medium tank 180 to medium regeneration module 170. This allows components necessary for cell growth to be supplied to the medium.

[0091] The pumps 150b and 150c may be any pump capable of adjusting the flow rate of the regeneration medium. The pumps 150b and 150c may be, for example, a tube pump, a reciprocating pump, a centrifugal pump, or a propeller pump. The pumps 150b and 150c each have a motor (not shown). The flow rate of the pumps 150b and 150c can be adjusted by the rotational speed of the motor, the frequency at which the motor is driven, or the like. Hereinafter, for simplicity, the operation of the motors of the pumps 150b and 150c may be simply referred to as the operation of the pumps 150b and 150c. The pump 150b has a first end 152b on the inlet side and a second end 154b on the outlet side. The pump 150c has a first end 152c on the inlet side and a second end 154c on the outlet side.

[0092] The rotation speed of the motor, the timing at which the motor is driven, and other parameters are controlled by a control device (not shown). The control device includes a processor (such as a CPU (Central Processing Unit)), a ROM (Read Only Memory), a RAM (Random Access Memory), and an input / output interface. The ROM and RAM store programs for controlling the rotation speed and timing at which the motor is driven. The processor reads and executes the programs for controlling the rotation speed and timing at which the motor is driven from the ROM and RAM.

[0093] The control device that controls the motors of the pumps 150b and 150c may be the same as or different from the control device that controls the motor of the drive unit 122 of the agitator 120 described above.

[0094] <<<Tubes 160d, 160e, 160f, 160g>>> Tubes 160d, 160e, 160f, and 160g are made of flexible resin or the like. For simplicity, tubes 160d, 160e, 160f, and 160g are shown in Fig. 1 as being linear. Tubes 160d, 160e, 160f, and 160g have elongated shapes.

[0095] Tube 160d has a first longitudinal end 162d and a second longitudinal end 164d. Tube 160e has a first longitudinal end 162e and a second longitudinal end 164e. Tube 160f has a first longitudinal end 162f and a second longitudinal end 164f. Tube 160g has a first longitudinal end 162g and a second longitudinal end 164g.

[0096] <Culture medium regeneration module 170> The culture medium regeneration module 170 has a culture medium regeneration membrane 179 for regenerating the culture medium. The culture medium regeneration membrane 179 may be a hollow fiber type culture medium regeneration module or a flat membrane type culture medium regeneration module.

[0097] The medium regeneration module 170 has a medium inlet 172 and a medium outlet 174. The medium inlet 172 is an opening for introducing the medium flowing through the suction tube nozzle 130-1 into the medium regeneration module 170. The medium outlet 174 is an opening for discharging the medium introduced into the medium regeneration module 170.

[0098] The medium regeneration module 170 has a regeneration medium supply port 176 and a regeneration medium discharge port 178. The regeneration medium supply port 176 is an opening for supplying the regeneration medium from the regeneration medium tank 180 to the medium regeneration module 170. The regeneration medium discharge port 178 is an opening for discharging the regeneration medium supplied from the regeneration medium supply port 176 to the medium regeneration module 170.

[0099] <Tube 160d> A first end 162d of tube 160d is connected to the regeneration medium outlet 178 of the medium regeneration module 170. A second end 164d of tube 160d is connected to a first end 152b of the pump 150b.

[0100] <Tube 160e> A first end 162e of tube 160e is connected to a second end 154b of pump 150b. A second end 164e of tube 160e is fixed so as to be positioned at a predetermined position inside regeneration medium tank 180.

[0101] <Removal of waste products> Pipes 160d and 160e are used to guide metabolic products (waste products such as lactic acid, ammonia, glutamic acid, isovaleric acid, butyric acid, and citric acid) from the cell aggregates from the medium regeneration module 170 to the regeneration medium tank 180. This allows the metabolic products to be removed from the medium circulation circuit. Pump 150b can adjust the flow rate of the liquid guided from the medium regeneration module 170 to the regeneration medium tank 180.

[0102] <Tube 160f> A first end 162f of tube 160f is fixed so as to be positioned at a predetermined position inside regeneration medium tank 180. A second end 164f of tube 160f is connected to a first end 152c of pump 150c.

[0103] <Tube 160g> A first end 162g of the tube 160g is connected to the second end 154c of the pump 150c. A second end 164g of the tube 160g is connected to a regeneration medium supply port 176 of the medium regeneration module 170.

[0104] <Supply of Nutrients> Nutrients for the cell aggregates are delivered from the regeneration medium tank 180 to the medium regeneration module 170 via pipes 160f and 160g. This allows the nutrients to be supplied to the medium circulation circuit. The flow rate delivered from the regeneration medium tank 180 to the medium regeneration module 170 can be adjusted by pump 150c.

[0105] <Operation of medium regeneration module 170> Nutrients for the cell aggregates are supplied from the regeneration medium tank 180 to the regeneration medium supply port 176 of the medium regeneration module 170 via pipes 160f and 160g. The supplied nutrients permeate the medium regeneration membrane 179 and move to the medium circulation circuit. In this way, the nutrients are supplied to the medium circulation circuit.

[0106] On the other hand, metabolic products (waste products such as lactic acid, ammonia, glutamic acid, isovaleric acid, butyric acid, and citric acid) from the cell aggregates permeate through the medium regeneration membrane 179 from the medium circulation circuit and are discharged from the regeneration medium outlet 178 of the medium regeneration module 170 through pipes 160d and 160e to the regeneration medium tank 180. In this way, the metabolic products are removed from the medium circulation circuit.

[0107] In this way, the culture medium regeneration module 170 regenerates the culture medium by bringing the regeneration medium supplied from the regeneration medium supply port 176 into contact with the culture medium introduced from the culture medium inlet 172 via the culture medium regeneration membrane 179. The regenerated culture medium is discharged from the culture medium outlet 174 and guided to the culture vessel 110-1 via the pipe 160c. The regeneration medium that has come into contact with the culture medium via the culture medium regeneration membrane 179 is discharged from the regeneration medium outlet 178 and returned to the regeneration medium tank 180.

[0108] The culture medium regeneration module 170 removes waste products and the like discharged as the cell aggregates grow within the culture vessel 110-1 by filtration, and supplies the culture medium with components necessary for the growth of the cell aggregates (e.g., glucose, amino acids, vitamins, inorganic salts, etc.).

[0109] <Regeneration Medium Tank 180> The regeneration medium tank 180 stores regeneration medium. The regeneration medium contains sufficient amounts of glucose, amino acids, vitamins, inorganic salts, and the like. For example, the regeneration medium may be the aforementioned basal medium such as DMEM, DMEMHG, EMEM, IMDM (Iscove's Modified Dulbecco's Medium), GMEM (Glasgow's MEM), RPMI-1640, α-MEM, Ham's Medium F-12, Ham's Medium F-10, Ham's Medium F12K, and the like. Furthermore, additives such as amino acids, vitamins, inorganic salts, proteins (growth factors), sugars including glucose, antibiotics, signal transduction inhibitors, reducing agents, buffers, and the like may be added.

[0110] <Creating Non-Circulation> In the example described above, a configuration was shown in which the metabolic products discharged from the medium regeneration module 170 were returned to the regeneration medium tank 180 via pipes 160d and 160e. That is, a circulation path was formed by pipes 160d, 160e, 160f, and 160g. Because the metabolic products are returned to the regeneration medium tank 180, the concentration of the nutrient components in the regeneration medium tank 180 gradually changes as the metabolic products increase. For this reason, it is expected that an appropriate amount of nutrient components cannot be supplied to the culture vessel 110-1.

[0111] From this perspective, a non-circulating system may be configured. For example, the metabolic products discharged from the medium regeneration module 170 may be returned to a storage tank (not shown) different from the regeneration medium tank 180. In this way, the concentration of the nutrient components in the regeneration medium tank 180 can be maintained, and an appropriate amount of nutrient components can always be supplied to the culture vessel 110-1.

[0112] Furthermore, a nutrient component storage tank in which the nutrient components are stored and a supply pump (not shown) may be provided, and by driving the supply pump, the nutrient components may be appropriately supplied from the nutrient component storage tank to the regeneration medium tank 180. The concentration of the nutrient components in the regeneration medium tank 180 can be maintained at a desired concentration, and an appropriate amount of nutrient components can always be supplied to the culture vessel 110-1.

[0113] <Capacity and Number of Regeneration Medium Tanks 180> The capacity of the regeneration medium tanks 180 can be set to a desired size. By increasing the capacity of the regeneration medium tanks 180, the concentration of the regeneration medium stored in the regeneration medium tanks 180 can be made less susceptible to the influence of the regeneration medium returned from the medium regeneration module 170. The capacity of the regeneration medium tanks 180 can be determined appropriately depending on the flow rate per unit time of the regeneration medium returning from the medium regeneration module 170, etc.

[0114] Furthermore, the number of regeneration medium tanks 180 is not limited to one. The number of regeneration medium tanks 180 may be plural.

[0115] For example, regeneration medium may be supplied to the medium regeneration module 170 from a first regeneration medium tank 180a, and the regeneration medium from the medium regeneration module 170 may be returned to a second regeneration medium tank 180b, which is different from the first regeneration medium tank 180a.

[0116] A tank for storing spare regeneration medium may also be provided to maintain the state of the regeneration medium tank 180. When multiple regeneration medium tanks 180 are used, they may be connected to each other with pipes that communicate with each other, or the flow may be controlled by a pump or valve. When multiple regeneration medium tanks 180 are connected to each other, at least one medium regeneration module may be provided between the regeneration medium tanks 180, separate from the medium regeneration module 170. Any module may be provided that can prevent the concentration of the regeneration medium from changing until it is supplied to the medium regeneration module 170.

[0117] <Removal by Adsorbent> In the example described above, a configuration was shown in which the metabolic products discharged from the medium regeneration module 170 were returned to the regeneration medium tank 180 via pipes 160d and 160e. That is, a circulation path was formed by pipes 160d, 160e, 160f, and 160g. In this configuration, the metabolic products flow into the regeneration medium tank 180 along with the regeneration medium, and therefore, waste products may gradually accumulate in the regeneration medium tank 180 as the metabolic products in the regeneration medium tank 180 increase. For this reason, the difference in concentration of waste components between the culture vessel 110-1 and the regeneration medium tank 180 becomes smaller, and it is expected that the solute removal ability will decrease.

[0118] From this perspective, an adsorbent or the like may be used to selectively remove metabolic products (waste products such as lactic acid, ammonia, glutamic acid, isovaleric acid, butyric acid, and citric acid) produced by the cell aggregates.

[0119] 1 , an adsorbent column 190 filled with an adsorbent 192 for adsorbing waste metabolic products can be provided midway along the tube 160g. In this way, during the process of discharging the regeneration medium from the regeneration medium tank 180 to the medium regeneration module 170, the metabolic products contained in the regeneration medium can be adsorbed onto the adsorbent 192 and removed while the regeneration medium flows through the adsorbent column 190. With this configuration, the regeneration medium from which the metabolic products have been removed can be discharged to the medium regeneration module 170.

[0120] The adsorbent 192 packed in a single adsorbent column 190 may be a mixture of multiple types of adsorbents for adsorbing target substances such as lactic acid and ammonia.

[0121] 1, the adsorbent column 190 is provided in the middle of the tube 160g, but the adsorbent column 190 may be provided in the middle of the tube 160d, the tube 160e, or the tube 160f. The adsorbent column 190 may be provided at any position in at least one of the tubes 160d, 160e, 160f, and 160g.

[0122] Furthermore, although the example shown in FIG. 1 illustrates an example in which a single adsorbent column 190 is provided, multiple adsorbent columns 190 may be provided in series or in parallel in at least one of tubes 160d, 160e, 160f, and 160g.

[0123] In addition, an adsorbent column may be provided in communication between the pipe 160d or pipe 160e for leading the medium from the medium regeneration module 170 to the regeneration medium tank 180 and the pipe 160f or pipe 160g for supplying nutrients from the regeneration medium tank 180 to the medium regeneration module 170. A new flow path for the adsorbent column can be formed in parallel with the flow path to the medium regeneration module 170, allowing the adsorption of metabolic products contained in the regeneration medium while balancing the flow rate with the medium regeneration module 170.

[0124] In this case, a separate electromagnetic valve may be provided to switch the communication state. Furthermore, a separate pump may be provided to adjust the flow rate of the regeneration medium. Furthermore, a concentration sensor may be provided. The concentration of metabolites contained in the regeneration medium can be detected, and the electromagnetic valve or pump can be controlled according to the detection result.

[0125] Furthermore, instead of using the pipes 160d, 160e, 160f, and 160g, the metabolic products may be adsorbed in the regeneration medium tank 180. Specifically, the regeneration medium tank 180 may be configured to have an adsorbent provided therein.

[0126] By placing an adsorbent in the regeneration medium tank 180, it is possible to adsorb metabolic products accumulated in the regeneration medium tank 180 and maintain a low concentration of waste products in the regeneration medium tank 180. As a result, the difference in concentration of waste products between the culture vessel 110-1 and the regeneration medium tank 180 can be maintained, and the solute removal ability can be fully exerted.

[0127] <Aspect 2 of Removal Using Adsorbent> For example, by holding an adsorbent along the wall surface of the regeneration medium tank 180, metabolic products approaching the wall surface of the regeneration medium tank 180 can be adsorbed.

[0128] <Aspect 3 of removal using an adsorbent> Alternatively, a regeneration medium tank 180 having a structure separated into an upper region and a lower region by a diaphragm (not shown) such as a porous membrane can be used. The upper region stores the regeneration medium and metabolites. The lower region stores the regeneration medium and metabolites via the diaphragm, and also contains an adsorbent. The regeneration medium and metabolites can pass through the diaphragm and move between the upper and lower regions. In contrast, the adsorbent cannot pass through the diaphragm and remains only in the lower region. With this configuration, the metabolites are adsorbed by the adsorbent when they pass through the diaphragm from the upper region to the lower region.

[0129] <Fourth mode of removal using adsorbent> Furthermore, a particulate adsorbent may be dispersed, precipitated, or suspended in the regeneration medium in the regeneration medium tank 180. By configuring in this way, the metabolic products can be adsorbed onto the adsorbent in the regeneration medium tank 180.

[0130] Furthermore, in the case of a circuit comprising a plurality of regeneration medium tanks, the adsorbent may be added to each of the regeneration medium tanks.

[0131] Furthermore, in addition to placing the adsorbent in the regeneration medium tank, a method of adding new regeneration medium from a tank storing spare regeneration medium may be combined.

[0132] The glucose concentration, lactic acid concentration, and pH may be monitored to operate the pumps 150b and 150c. For example, the pumps 150b and 150c may be operated according to the flow rate of the pump 150a, the glucose concentration, the lactic acid concentration, and the pH. In this way, glucose, amino acids, vitamins, inorganic salts, and the like can be added to the culture medium according to the flow rate of the culture medium.

[0133] <<<<Movement of Cell Aggregates Within the Aspirate Tube Nozzle 130-1>>> Figure 2A is a cross-sectional view showing an outline of the movement of cell aggregates within the aspirate tube nozzle 130-1. In Figure 2A, multiple white circular regions represent cell aggregates.

[0134] <Top Generator UG and Bottom Generator BG> The suction tube nozzle 130-1 has an extension 132-1, which has a tubular shape. The outer periphery 132O-1 of the extension 132-1 has multiple generators along the longitudinal direction. Generators refer to the straight lines at each position when a curved surface (tubular (cylindrical) surface) is drawn (formed) by moving a straight line. By tilting the suction tube nozzle 130-1, the cross section of the suction tube nozzle 130-1 can be defined as having an uppermost generator UG and a lowermost generator BG. The cross section of the suction tube nozzle 130-1 is a plane formed by cutting the suction tube nozzle 130-1 perpendicular to the central axis AO of the suction tube nozzle 130-1 (see FIG. 2B ).

[0135] <In the case of a vertical state> When the suction tube nozzle 130-1 is in a vertical state, the culture medium rises in the region formed between the plurality of cell aggregates settling within the suction tube nozzle 130-1. When the suction tube nozzle 130-1 is in a vertical state, the culture medium is sucked through the suction opening 134-1 and then sucked vertically upward within the suction tube nozzle 130-1. This is the direction opposite to the direction of gravity, and the direction of movement of the cell aggregates is determined by the magnitude relationship between the suction force and gravity. In other words, the movement direction of the cell aggregates is either up or down in the vertical direction.

[0136] <In the case of tilted state> On the other hand, as shown in Figures 1 and 2, tilting the suction tube nozzle 130-1 causes cell aggregates attempting to settle to move and gather in the area closer to the bottom bus line BG than in the area closer to the top bus line UG due to the action of gravity. As the gathered cell aggregates move toward the suction opening 134-1, the area closer to the bottom bus line BG becomes more likely to be occupied by cell aggregates. Due to the uneven distribution of cell aggregates along the cross section of the suction tube nozzle 130-1 (see Figure 2B), the culture medium moves and flows in the area closer to the top bus line UG, which is not occupied by cell aggregates, rather than in the area closer to the bottom bus line BG, which is occupied by cell aggregates. Tilting the suction tube nozzle 130-1 causes such movement of the cell aggregates and the culture medium, and two main flow regions, a first flow region FF and a second flow region SF, are formed in the enclosed region SR. In the cross-sectional view shown in Fig. 2B, the dashed, approximately circular shape is an imaginary line showing the boundary BD between the first flow region FF and the second flow region SF. In the cross-sectional view shown in Fig. 2A, the boundary BD is shown as a dashed straight line located between the uppermost bus line UG and the lowermost bus line BG along the longitudinal direction of the suction tube nozzle 130-1. The region outside the boundary BD is the first flow region FF, and the region inside the boundary BD is the second flow region SF.

[0137] <First flow region FF> The first flow region FF is a region where cell aggregates gather and then flow (settle) together with the culture medium toward the suction opening 134-1. That is, the first flow region FF is a region where a flow is formed in which the cell aggregates settle together with the culture medium. In particular, in the first flow region FF, cell aggregates are more likely to settle in the region close to the bottom bus line BG. The cell aggregates settle faster in the region close to the bottom bus line BG and settle more slowly as they move away from the bottom bus line BG.

[0138] The culture medium flows in succession from the suction opening 134-1 together with the cell aggregates. Therefore, some of the cell aggregates that have settled near the suction opening 134-1 flow out from the suction opening 134-1, while others move to the second flow region SF and rise again. Whether they flow out from the suction opening 134-1 or rise again is determined appropriately depending on the amount and size of the cell aggregates, the flow of the culture medium near the suction opening 134-1, and other factors.

[0139] <Second flow region SF> The second flow region SF is a region where, as the culture medium rises toward the discharge opening 136-1, the cell aggregates also flow toward the discharge opening 136-1. That is, the second flow region SF is a region where a flow of rising cell aggregates is formed. In particular, in the second flow region SF, cell aggregates are more likely to rise in regions close to the uppermost generatrix UG. In other words, the cell aggregates rise faster in regions close to the uppermost generatrix UG and rise more slowly as they move away from the uppermost generatrix UG.

[0140] <Top position UP> The cell aggregates that have risen toward the discharge opening 136-1 rise to a predetermined position below the discharge opening 136-1, for example, to a position halfway between the suction opening 134-1 and the discharge opening 136-1 (top position UP), and then move to the first flow region FF and settle again. By forming the top position UP, the cell aggregates are not discharged from the discharge opening 136-1, and only the culture medium can be discharged from the discharge opening 136-1, allowing only the culture medium to be circulated.

[0141] The highest position UP at which the cell aggregate can rise within the suction tube nozzle 130-1 is not limited to the midpoint between the suction opening 134-1 and the discharge opening 136-1. The highest position UP may be any position at which the possibility of the cell aggregate being discharged from the discharge opening 136-1 is sufficiently low. The highest position UP at which the cell aggregate can rise within the suction tube nozzle 130-1 can be determined by the inclination angle θ of the suction tube nozzle 130-1 and the circulation flow rate generated by driving the pump 150a.

[0142] <<Circulating Cell Aggregates>> With the formation of the first flow region FF, the cell aggregates settle toward the suction opening 134-1 and then move to the second flow region SF. With the formation of the second flow region SF, the cell aggregates that have moved to the second flow region SF rise to the uppermost position UP toward the discharge opening 136-1, and then move again to the first flow region FF. In this way, cell aggregates of a predetermined size or larger flow alternately between the first flow region FF and the second flow region SF and circulate within the suction tube nozzle 130-1. By circulating the cell aggregates within the suction tube nozzle 130-1, the cell aggregates can be retained within the suction tube nozzle 130-1, and only the culture medium can be discharged from the discharge opening 136-1 of the suction tube nozzle 130-1.

[0143] <Distribution of cell aggregates in the suction tube nozzle 130-1> Figure 2B is a cross-sectional view showing the distribution of cell aggregates in the suction tube nozzle 130-1 at the II section shown in Figure 2A. In Figure 2B, multiple white circular regions also represent cell aggregates.

[0144] As described above, the area close to the bottom generating line BG is occupied by the settling cell aggregates, and as a result, the cross section of the first flow region FF (the area along the direction perpendicular to the longitudinal direction of the suction tube nozzle 130-1) is enlarged by the settling cell aggregates.

[0145] On the other hand, since the area close to the bottom generating line BG is occupied by cell aggregates, the area close to the top generating line UG where the culture medium can flow is reduced. That is, the cross section of the second flow region SF (the area along the direction perpendicular to the longitudinal direction of the suction tube nozzle 130-1) is reduced by the cell aggregates in the first flow region FF.

[0146] The circulation flow rate of the culture medium discharged from the discharge opening 136-1 of the suction tube nozzle 130-1 is determined by the operation of the pump 150a. To maintain this circulation flow rate, the flow velocity of the culture medium flowing in the second flow region SF, where the cross section is reduced, becomes faster.

[0147] By forming the uppermost position UP, the cell aggregates that have moved to the second flow region SF rise to the uppermost position UP toward the discharge opening 136-1, and then move again to the first flow region FF. In this way, the cell aggregates remain within the suction tube nozzle 130-1, and only the culture medium is discharged from the discharge opening 136-1, thereby increasing the circulating flow rate of the culture medium.

[0148] In addition, as the circulation flow rate of the culture medium increases, the movement speed of the cell aggregates in the aspirating tube nozzle 130-1 also increases, and therefore the flow rate of the cell suspension attempting to rise in the second flow region SF also increases.

[0149] 3 is a graph showing the relationship between the tilt angle of the suction tube nozzle 130-1 and the circulation flow rate when the cell aggregate rises to a position halfway between the suction opening 134-1 and the discharge opening 136-1. The position halfway between the suction opening 134-1 and the discharge opening 136-1 is an example of the uppermost position UP.

[0150] The circulation flow rates shown in FIG. 3 were F1<F2<F3<F4. That is, as the tilt angle θ increases, the circulation flow rate must be increased to raise the cell aggregates to the intermediate position. In other words, as the tilt angle θ increases, the cell aggregates become less likely to be discharged from the suction opening 134-1 of the suction tube nozzle 130-1, allowing the circulation flow rate of the culture medium to be increased. The circulation flow rate of the culture medium can be increased until the cell aggregates reach the intermediate position so that they are not discharged from the suction opening 134-1 of the suction tube nozzle 130-1. In this way, the circulation flow rate of the culture medium can be increased by increasing the tilt angle θ. Increasing the circulation flow rate of the culture medium increases the amount of culture medium that comes into contact with the culture medium regeneration membrane 179 of the culture medium regeneration module 170, thereby improving the solute removal capacity.

[0151] Furthermore, even if the circulation flow rate of the culture medium is increased, the cell aggregates are not led out of the discharge opening 136-1 of the suction tube nozzle 130-1 into the culture medium circulation circuit (tubes 160a, 160b, 160c, etc.), and therefore the cell aggregates are not damaged. In this way, by increasing the inclination angle θ, the circulation flow rate of the culture medium can be increased, allowing the cell aggregates to grow efficiently.

[0152] Because the circulating flow rate of the culture medium can be increased without the cell aggregates being drawn into the culture medium circulation circuit, the amount of culture medium that can come into contact with the culture medium regeneration membrane 179 can be increased without damaging the cell aggregates, improving solute removal performance. Material exchange via the culture medium regeneration module 170 allows necessary components such as growth factors to remain on the culture side of the cell aggregates while waste products such as lactic acid can be removed, making it possible to reduce the amount of culture medium used compared to normal culture medium exchange, thereby reducing costs.

[0153] <<<<<Second Embodiment>>>>> Figure 4 is a schematic diagram showing the configuration of a culture medium regeneration system 200 according to a second embodiment. In Figure 4, the same components as those in the first embodiment are denoted by the same reference numerals. In Figure 4, pumps 150a, 150b, 150c, pipes 160a, 160b, 160c, 160d, 160e, 160f, 160g, culture medium regeneration module 170, and regeneration culture medium tank 180 are the same as those in the first embodiment and are therefore omitted. As shown in Figure 4, the culture vessel 110-1 of the culture medium regeneration system 200 is fixedly positioned vertically, unlike the culture medium regeneration system 100. The following mainly describes the differences from the culture medium regeneration system 100.

[0154] <Culture vessel 110-1> The structure and materials of the culture vessel 110-1 are basically the same as those of the culture medium regeneration system 100. The arrangement of the culture vessel 110-1 differs from that of the culture medium regeneration system 100. The direction of the central axis CO of the culture vessel 110-1 is vertical, which differs from the arrangement of the culture vessel 110-1 in the culture medium regeneration system 100. Therefore, the bottom 114 of the culture vessel 110-1 extends horizontally.

[0155] <Agitator 120> The configuration of the agitator 120 is basically the same as that of the culture medium regeneration system 100. The arrangement of the agitator 120 differs from that of the culture medium regeneration system 100. The direction of the rotation axis RO of the agitator 124 of the agitator 120 is vertical, which differs from the arrangement of the agitator 124 in the culture medium regeneration system 100. In other words, the central axis CO of the culture vessel 110-1 and the rotation axis RO of the agitator 124 are vertical, which differs from that of the culture medium regeneration system 100.

[0156] <Suction Tube Nozzle 130-2> The configuration and material of the suction tube nozzle 130-2 are basically the same as those of the culture medium regeneration system 100.

[0157] Similar to the suction tube nozzle 130-1, the suction tube nozzle 130-2 has an extension portion 132-1, an intake opening 134-1, and an exhaust opening 136-1.

[0158] The extension portion 132-1 has a similar configuration to the suction tube nozzle 130-1. The extension portion 132-1 extends in an inclined direction. The extension portion 132-1 is inclined at an inclination angle θ with respect to the vertical direction. Therefore, the suction tube nozzle 130-2 extends in an inclined direction. In other words, the central axis AO of the suction tube nozzle 130-2 extends in an inclined direction.

[0159] In the first embodiment, the central axis CO of the culture vessel 110-1, the rotation axis RO of the agitator blades 124a, and the central axis AO of the aspirating tube nozzle 130-1 are arranged along the inclined direction. In contrast, in the second embodiment, the central axis CO of the culture vessel 110-1 and the rotation axis RO of the agitator blades 124a extend vertically, and only the aspirating tube nozzle 130-2 extends in the inclined direction. The aspirating tube nozzle 130-2 is fixedly provided by a holding member or the like (not shown) so that it extends in a fixed inclined direction.

[0160] The suction tube nozzle 130-2 is positioned so that the suction opening 134-1 faces the side wall 112 of the culture vessel 110-1. By tilting the suction tube nozzle 130-2 so that the suction opening 134-1 faces the side wall 112 of the culture vessel 110-1, the suction tube nozzle 130-2 is less likely to interfere with the stirring blades 124a of the stirring bar 124, allowing the culture medium and cell aggregates to be sufficiently stirred. Furthermore, by tilting the suction tube nozzle 130-2, the degree of freedom in the size and shape of the stirring blades 124a of the stirring bar 124 can be increased.

[0161] The culture medium and cell aggregates flow in the suction tube nozzle 130-2 in the same manner as in the suction tube nozzle 130-1 of the first embodiment. Two flow regions, a first flow region FF and a second flow region SF, are formed in the suction tube nozzle 130-2. The cell aggregates remain within the suction tube nozzle 130-2, and the culture medium is discharged from the discharge opening 136-1 of the suction tube nozzle 130-2.

[0162] In this way, by using the inclined suction tube nozzle 130-2, the circulating flow rate of the culture medium can be increased without the cell aggregates being drawn into the culture medium circulation circuit, thereby increasing the amount of culture medium that can come into contact with the culture medium regeneration membrane without damaging the cell aggregates, and improving solute removal performance. Material exchange via the culture medium regeneration module 170 allows necessary components such as growth factors to remain on the culture side of the cell aggregates while removing the waste product lactic acid, thereby reducing the amount of culture medium used compared to normal culture medium exchange and reducing costs.

[0163] <<<<<Third Embodiment>>>>> Figure 5 is a schematic diagram showing the configuration of a culture medium regeneration system 300 according to a third embodiment. In Figure 5, the same components as those in the first and second embodiments are denoted by the same reference numerals. In Figure 5, pumps 150a, 150b, 150c, pipes 160a, 160b, 160c, 160d, 160e, 160f, 160g, culture medium regeneration module 170, and regeneration culture medium tank 180 are the same as those in the first embodiment and are therefore omitted. As shown in Figure 5, the culture vessel 110-3 of the culture medium regeneration system 300 is fixedly positioned vertically, as in the second embodiment. The following mainly describes the differences from the culture medium regeneration system 100 and the culture medium regeneration system 200.

[0164] <Culture Vessel 110-3> The structure and materials of the culture vessel 110-3 are basically the same as those of the culture medium regeneration system 100. In the culture medium regeneration system 100 and the culture medium regeneration system 200, the culture vessel 110-1 and the suction tube nozzle 130-1 and 130-2 are configured separately from each other. Therefore, the position and orientation of the suction tube nozzle 130-1 and 130-2 can be adjusted relative to the culture vessel 110-1. In contrast, the culture vessel 110-3 is formed integrally with the suction tube nozzle 130-3.

[0165] The suction tube nozzle 130-3 has an extension 132-3, an intake opening 134-3, and an exhaust opening 136-3.

[0166] The culture vessel 110-3 has a through-hole formed near the bottom 114. The suction opening 134-3 of the suction tube nozzle 130-3 is fixed so as to communicate with the through-hole formed near the bottom 114 of the culture vessel 110-3. The suction tube nozzle 130-3 forms an inclination angle θ with respect to the central axis CO of the culture vessel 110-3 and extends so as to move away from the culture vessel 110-3 as it extends upward. The central axis AO of the suction tube nozzle 130-3 extends in the inclined direction.

[0167] By forming the aspirating tube nozzle 130-3 integrally with the culture vessel 110-3, a holding member for holding the aspirating tube nozzle 130-3 is not required. Furthermore, since the tilt angle θ of the aspirating tube nozzle 130-3 can be kept constant, adjustment of the tilt angle θ is also not required. Furthermore, since the aspirating tube nozzle 130-3 is disposed outside the culture vessel 110-3, the aspirating tube nozzle 130-3 does not interfere with the stirring blades 124a of the stirring bar 124. This configuration allows for sufficient stirring of the culture medium and cell aggregates, and allows for greater freedom in the size and shape of the stirring blades 124a of the stirring bar 124.

[0168] 5 shows a configuration in which the suction opening 134-3 of the suction tube nozzle 130-3 is formed near the bottom 114 of the culture vessel 110-3, but the position of the suction opening 134-3 of the suction tube nozzle 130-3 is not limited to this. The position of the suction opening 134-3 can be determined appropriately depending on the type and amount of culture medium, the type and distribution of cell aggregates within the culture vessel 110-3, the rotation speed of the stirrer 124, etc. It is sufficient that the suction tube nozzle 130-3 is formed integrally with the culture vessel 110-3.

[0169] The culture medium and cell aggregates flow in the suction tube nozzle 130-3 in the same manner as in the suction tube nozzle 130-1 of the first embodiment. Two flow regions, a first flow region FF and a second flow region SF, are formed in the suction tube nozzle 130-3. The cell aggregates remain within the suction tube nozzle 130-3, and the culture medium is discharged from the discharge opening 136-3 of the suction tube nozzle 130-3.

[0170] In this way, by using the inclined suction tube nozzle 130-3, the circulating flow rate of the culture medium can be increased without the cell aggregates being drawn into the culture medium circulation circuit, thereby increasing the amount of culture medium that can come into contact with the culture medium regeneration membrane without damaging the cell aggregates, and improving solute removal performance. Material exchange via the culture medium regeneration module 170 allows necessary components such as growth factors to remain on the culture side of the cell aggregates while removing the waste product lactic acid, thereby reducing the amount of culture medium used compared to normal culture medium exchange and reducing costs.

[0171] <Other Configurations of Culture Vessel 110-3> In the example shown in Fig. 5, the aspirating tube nozzle 130-3 is configured to be integral with the culture vessel 110-3. The configuration of the culture vessel 110-3 is not limited to this. The aspirating tube nozzle 130-3 may be configured to be detachable and separate from the culture vessel 110-3.

[0172] For example, a generally cylindrical protruding portion (not shown) for attaching the aspirate tube nozzle 130-3 is provided on the side of the culture vessel 110-3. The aspirate tube nozzle 130-3 can be detachably attached to the culture vessel 110-3 by inserting the end of the aspirate tube nozzle 130-3, which is formed separately, into the protruding portion. By constructing the aspirate tube nozzle 130-3 separately from the culture vessel 110-3, it is possible to use aspirate tube nozzle 130-3 made of different materials from the culture vessel 110-3, or to use an aspirate tube nozzle 130-3 having a desired length or shape.

[0173] Furthermore, the position of the through-hole and protrusion for attaching the aspirating tube nozzle 130-3, the angle relative to the vertical direction, etc. can be set as desired, thereby increasing the degree of freedom in selecting the aspirating tube nozzle 130-3 and the culture vessel 110-3.

[0174] <<<<<Fourth Embodiment>>>>> Figure 6 is a schematic diagram showing the configuration of a culture medium regeneration system 400 according to a fourth embodiment. In Figure 6, the same components as those in the first embodiment are denoted by the same reference numerals. In Figure 6, pumps 150a, 150b, 150c, pipes 160a, 160b, 160c, 160d, 160e, 160f, 160g, culture medium regeneration module 170, and regeneration culture medium tank 180 are the same as those in the first embodiment and are therefore omitted. As shown in Figure 6, the culture vessel 110-1 of the culture medium regeneration system 400 is fixedly positioned vertically rather than inclined, unlike the culture medium regeneration system 100. The following mainly describes the differences from the culture medium regeneration system 100.

[0175] <Culture vessel 110-1> The structure and materials of the culture vessel 110-1 are basically the same as those of the culture medium regeneration system 100. The arrangement of the culture vessel 110-1 differs from that of the culture medium regeneration system 100. The direction of the central axis CO of the culture vessel 110-1 is vertical, which differs from the arrangement of the culture vessel 110-1 in the culture medium regeneration system 100. Therefore, the bottom 114 of the culture vessel 110-1 extends horizontally.

[0176] <Suction Tube Nozzle 130-4> Unlike the culture medium regeneration system 100, the culture medium regeneration system 400 has a suction tube nozzle 130-4.

[0177] The suction tube nozzle 130-4 has an inclined portion 130-2a, a vertical portion 130-2b, and a bent portion 130-2c. The suction tube nozzle 130-4 has an elongated shape overall due to the inclined portion 130-2a, the vertical portion 130-2b, and the bent portion 130-2c. The cross-sectional area of ​​the suction tube nozzle 130-4 is the area of ​​the inner diameter when the inclined portion 130-2a and the vertical portion 130-2b are cut along a direction perpendicular to the longitudinal direction of the inclined portion 130-2a and the vertical portion 130-2b.

[0178] <Inclined portion 130-2a> The inclined portion 130-2a is inclined at an inclination angle θ with respect to the vertical direction. The inclined portion 130-2a extends in the inclined direction. The inclined portion 130-2a has an elongated shape. The inclined portion 130-2a extends linearly along the longitudinal direction. The inclined portion 130-2a has a tubular shape. The inclined portion 130-2a has an intake opening 134-1.

[0179] The aspirating tube nozzle 130-4 is positioned so that the suction opening 134-1 of the inclined portion 130-2a faces the sidewall 112 of the culture vessel 110-1. That is, the inclined portion 130-2a is positioned so that the central axis AO of the inclined portion 130-2a moves away from the central axis CO of the culture vessel 110-1 as it extends downward. In other words, the direction in which the inclined portion 130-2a extends is the inclination direction, and the direction in which the extension portion 124b of the stirrer 124 extends is the vertical direction, forming an inclination angle θ. By inclining the inclined portion 130-2a so that the suction opening 134-1 faces the sidewall 112 of the culture vessel 110-1, the inclined portion 130-2a is less likely to interfere with the stirring blades 124a of the stirrer 124, and the culture medium and cell aggregates can be sufficiently stirred. Furthermore, by inclining the inclined portion 130-2a, the degree of freedom in the size and shape of the stirring blade 124a of the stirring bar 124 can be increased.

[0180] <Vertical section 130-2b> The vertical section 130-2b extends in the vertical direction. The vertical section 130-2b has an elongated shape. The vertical section 130-2b extends linearly along the longitudinal direction. The vertical section 130-2b has a tubular shape. The vertical section 130-2b has a discharge opening 136-1.

[0181] <Bent portion 130-2c> The inclined portion 130-2a and the vertical portion 130-2b are connected via the bent portion 130-2c. The bent portion 130-2c has a short tubular shape. The bent portion 130-2c connects the inclined portion 130-2a extending in the inclined direction and the vertical portion 130-2b extending in the vertical direction at an inclination angle θ. The inclined portion 130-2a and the vertical portion 130-2b communicate with each other via the bent portion 130-2c. Instead of the bent portion 130-2c, a curved member may be used to connect the inclined portion 130-2a extending in the inclined direction and the vertical portion 130-2b extending in the vertical direction.

[0182] <Inclined portion 130-2a, vertical portion 130-2b, bent portion 130-2c> The inclined portion 130-2a, vertical portion 130-2b, and bent portion 130-2c have an outer periphery and an inner periphery (not shown), similar to the extension portion 132-1 of the suction tube nozzle 130-1 of the culture medium regeneration system 100. The outer periphery constitutes the outer surface of the inclined portion 130-2a, vertical portion 130-2b, and bent portion 130-2c. The inner periphery constitutes the inner surface of the inclined portion 130-2a, vertical portion 130-2b, and bent portion 130-2c. The outer periphery and inner periphery are positioned concentrically. A long surrounding region SR, surrounded by the inner periphery and extending along the longitudinal direction, functions as a hollow conduit. The culture medium and cell aggregates can flow through the surrounding region SR.

[0183] The culture medium and cell aggregates flow in the inclined portion 130-2a in the same manner as in the suction tube nozzle 130-1 of the first embodiment. Two flow regions, a first flow region FF and a second flow region SF, are formed in the inclined portion 130-2a. The cell aggregates remain within the inclined portion 130-2a, and the culture medium is discharged from the discharge opening 136-1 via the inclined portion 130-2a.

[0184] In this way, by using the inclined suction tube nozzle 130-4, the circulating flow rate of the culture medium can be increased without the cell aggregates being drawn into the culture medium circulation circuit, thereby increasing the amount of culture medium that can come into contact with the culture medium regeneration membrane without damaging the cell aggregates, and improving solute removal performance. Material exchange via the culture medium regeneration module 170 allows necessary components such as growth factors to remain on the culture side of the cell aggregates while removing waste products such as lactic acid, thereby reducing the amount of culture medium used compared to normal culture medium exchange and reducing costs.

[0185] <Other Shapes of Suction Opening 134-1> Although only an example in which the inclined portion 130-2a of the suction opening 134-1 is inclined linearly so as to face the side wall portion 112 of the culture vessel 110-1 has been shown, the present invention is not limited to this. By appropriately changing the shape of the suction tube nozzle 130-4 depending on the size and shape of the stirring blades 124a of the stirrer 124, such as by curving the suction tube nozzle 130-4 in a spiral shape, interference with the stirring blades 124a can be avoided.

[0186] <<<<<Fifth Embodiment>>>>> Figure 7 is a schematic diagram showing the configuration of a culture medium regeneration system 500 according to a fifth embodiment. In Figure 7, the same components as those in the first embodiment are denoted by the same reference numerals. In Figure 7, pump 150a, pump 150b, pump 150c, pipe 160a, pipe 160b, pipe 160c, pipe 160d, pipe 160e, pipe 160f, pipe 160g, culture medium regeneration module 170, and culture medium tank for regeneration 180 are the same as those in the first embodiment and are therefore omitted.

[0187] 7, the culture medium regeneration system 500 is fixedly arranged in an inclined state at a certain inclination angle, similar to the culture medium regeneration system 100. The following mainly describes the differences from the culture medium regeneration system 100.

[0188] The culture medium regeneration system 500 has a suction tube nozzle 130-5. The suction tube nozzle 130-5 is made of glass, resin, stainless steel, or the like, and has a certain shape.

[0189] The suction tube nozzle 130-5 includes the suction tube nozzle 130-1 and an expanded diameter portion 470. In other words, the suction tube nozzle 130-5 according to the fifth embodiment has a structure in which the expanded diameter portion 470 is provided at the discharge opening 136-1 of the suction tube nozzle 130-1 of the first embodiment.

[0190] <Expanded Diameter Portion 470> The expanded diameter portion 470 extends in the vertical direction. The expanded diameter portion 470 has an elongated shape. The expanded diameter portion 470 extends linearly along the longitudinal direction. The expanded diameter portion 470 has a tubular shape. The expanded diameter portion 470 has a discharge opening 472. The lower end of the expanded diameter portion 470 has an opening that communicates with the discharge opening 136-1. In the example shown in FIG. 7, the suction tube nozzle 130-5 has a stepped portion where the suction tube nozzle 130-1 and the expanded diameter portion 470 are connected by a bent boundary. Alternatively, the suction tube nozzle 130-1 and the expanded diameter portion 470 may be connected so as to gradually widen (taper).

[0191] <Suction tube nozzle 130-1, expanded diameter portion 470, and enclosed region SR> The suction tube nozzle 130-1 and expanded diameter portion 470 have an outer circumferential portion and an inner circumferential portion (not shown), similar to the extension portion 132-1 of the suction tube nozzle 130-1 of the culture medium regeneration system 100. The outer circumferential portion constitutes the outer surface of the suction tube nozzle 130-1 and expanded diameter portion 470. The inner circumferential portion constitutes the inner surface of the suction tube nozzle 130-1 and expanded diameter portion 470. The outer circumferential portion and inner circumferential portion are concentric. The long enclosed region SR, surrounded by the inner circumferential portion and extending along the longitudinal direction, functions as a hollow conduit. The expanded diameter portion 470 communicates with the suction tube nozzle 130-1. The culture medium and cell aggregates can flow through the enclosed region SR.

[0192] <Flow of Culture Medium and Cell Aggregates> The culture medium and cell aggregates flow in the same manner as in the suction tube nozzle 130-1 of the first embodiment. Two flow regions, a first flow region FF and a second flow region SF, are formed in the suction tube nozzle 130-1. The cell aggregates remain within the suction tube nozzle 130-1, and the culture medium flows out from the discharge opening 136-1 of the suction tube nozzle 130-1, flows through the expanded diameter portion 470, and is discharged from the discharge opening 472 of the expanded diameter portion 470.

[0193] The expanded diameter section 470 has an inner diameter larger than the inner diameter of the suction tube nozzle 130-1. That is, the expanded diameter section 470 has a cross-sectional area larger than the cross-sectional area of ​​the suction tube nozzle 130-1. By increasing the cross-sectional area of ​​the expanded diameter section 470, the flow rate in the expanded diameter section 470 can be reduced. The cross-sectional area of ​​the expanded diameter section 470 may be determined according to the desired flow rate in the expanded diameter section 470. The cross-sectional area of ​​the expanded diameter section 470 refers to the area of ​​the inner diameter portion when the expanded diameter section 470 is cut in a direction perpendicular to the vertical direction in which the expanded diameter section 470 extends (i.e., horizontally).

[0194] In this way, by using the inclined suction tube nozzle 130-1, the circulating flow rate of the culture medium can be increased without the cell aggregates being drawn into the culture medium circulation circuit, thereby increasing the amount of culture medium that can come into contact with the culture medium regeneration membrane without damaging the cell aggregates, and improving solute removal performance. Material exchange via the culture medium regeneration module 170 allows necessary components such as growth factors to remain on the culture side of the cell aggregates while removing the waste product lactic acid, thereby reducing the amount of culture medium used compared to normal culture medium exchange and reducing costs.

[0195] <<<<<Sixth Embodiment>>>> Figure 8 is a schematic diagram showing the configuration of a culture medium regeneration system 600 according to a sixth embodiment. In Figure 8, the same components as those in the first embodiment are denoted by the same reference numerals. In Figure 8, pump 150a, pump 150b, pump 150c, pipe 160a, pipe 160b, pipe 160c, pipe 160d, pipe 160e, pipe 160f, pipe 160g, culture medium regeneration module 170, and culture medium tank for regeneration 180 are the same as those in the first embodiment and are therefore omitted.

[0196] 8, the culture vessel 110-1 of the culture medium regeneration system 600 is fixedly arranged in a vertical state, similar to the culture medium regeneration system 200. Below, differences from the culture medium regeneration system 100 and the culture medium regeneration system 200 will be mainly described.

[0197] The culture medium regeneration system 600 has a suction tube nozzle 130-6. The suction tube nozzle 130-6 is made of glass, resin, stainless steel, or the like, and has a certain shape.

[0198] The suction tube nozzle 130-6 includes the suction tube nozzle 130-4 and an expanded diameter portion 470. In other words, the suction tube nozzle 130-6 according to the sixth embodiment has a structure in which the expanded diameter portion 470 of the fifth embodiment is provided in the discharge opening 136-1 of the suction tube nozzle 130-4 of the fourth embodiment.

[0199] As in the fourth embodiment, the suction tube nozzle 130-4 has an inclined portion 130-2a, a vertical portion 130-2b, and a bent portion 130-2c. The inclined portion 130-2a, the vertical portion 130-2b, and the bent portion 130-2c give the suction tube nozzle 130-4 an elongated shape overall. The cross-sectional area of ​​the suction tube nozzle 130-4 is the area of ​​the inner diameter portion when the inclined portion 130-2a and the vertical portion 130-2b are cut along a direction perpendicular to the longitudinal direction of the inclined portion 130-2a and the vertical portion 130-2b.

[0200] Similar to the fifth embodiment, the expanded diameter portion 470 has a discharge opening 472. The lower end of the expanded diameter portion 470 has an opening that communicates with the discharge opening 136-1. In the example shown in FIG. 8 , the suction tube nozzle 130-6 has a stepped portion formed by a bent boundary between the suction tube nozzle 130-4 and the expanded diameter portion 470. Alternatively, the suction tube nozzle 130-4 and the expanded diameter portion 470 may be connected in a gradually expanding (tapered) manner.

[0201] <Suction tube nozzle 130-4, expanded diameter portion 470, and enclosed region SR> The suction tube nozzle 130-4 and expanded diameter portion 470 have an outer circumferential portion and an inner circumferential portion (not shown), similar to the extension portion 132-1 of the suction tube nozzle 130-1 of the culture medium regeneration system 100. The outer circumferential portion constitutes the outer surface of the suction tube nozzle 130-4 (inclined portion 130-2a, vertical portion 130-2b, and bent portion 130-2c) and the expanded diameter portion 470. The inner circumferential portion constitutes the inner surface of the suction tube nozzle 130-4 (inclined portion 130-2a, vertical portion 130-2b, and bent portion 130-2c) and the expanded diameter portion 470. The outer circumferential portion and the inner circumferential portion are concentrically positioned. The long enclosed region SR, surrounded by the inner circumferential portion and extending along the longitudinal direction, functions as a hollow conduit. The expanded diameter portion 470 communicates with the suction tube nozzle 130-4. The culture medium and cell aggregates can flow through the enclosed region SR.

[0202] <Flow of Culture Medium and Cell Aggregates> The culture medium and cell aggregates flow in the same manner as in the suction tube nozzle 130-1 of the first embodiment. Two flow regions, a first flow region FF and a second flow region SF, are formed in the inclined portion 130-2a. The cell aggregates remain within the inclined portion 130-2a, and the culture medium flows out from the discharge opening 136-1 of the suction tube nozzle 130-4, flows through the expanded diameter portion 470, and is discharged from the discharge opening 472 of the expanded diameter portion 470.

[0203] In this way, by using the suction tube nozzle 130-4 having the inclined slope 130-2a, the circulating flow rate of the culture medium can be increased without the cell aggregates being drawn into the culture medium circulation circuit, thereby increasing the amount of culture medium that can come into contact with the culture medium regeneration membrane without damaging the cell aggregates, and improving solute removal performance. Material exchange via the culture medium regeneration module 170 allows necessary components such as growth factors to remain on the culture side of the cell aggregates while removing the waste product lactic acid, thereby reducing the amount of culture medium used compared to normal culture medium exchange and reducing costs.

[0204] <<<<<Seventh Embodiment>>>> The seventh embodiment relates to a culture medium regeneration system that removes waste products (metabolites) while circulating a culture medium. The seventh embodiment mainly shows the results of verifying the relationship between the circulation flow rate and the ability to remove waste products (metabolites). The culture medium regeneration system of the seventh embodiment is a system for verification.

[0205] In the seventh embodiment, the circulation flow rate was confirmed using a liquid similar to the culture medium for culturing cell aggregates and a waste product similar to the waste product (metabolite). Specifically, phosphate-buffered saline (PBS) was used as the culture medium, and lithium lactate was used as the waste product. In the seventh embodiment, the circulation flow rate of the culture medium and the removal of the waste product are focused on, and substances similar to the culture medium and the waste product can be used as substitutes. Hereinafter, when there is no need to distinguish between them, the substitute for the culture medium will simply be referred to as the culture medium, and the substitute for the waste product will simply be referred to as the waste product.

[0206] <<<Outline of Seventh Embodiment>>> Culture medium replacement is essential in cell culture, and involves removing waste products and replenishing nutrients. However, the following problems arise when replacing the culture medium. First, there are cases where excessive medium replacement is necessary to remove waste products, even though nutrients still remain. In addition, volume fluctuations caused by medium replacement may result in insufficient nutrients being provided to the cell aggregates. Furthermore, when the culture medium is replaced, the concentrations of the culture medium components fluctuate significantly. To solve these problems, medium regeneration using dialysis technology has attracted attention.

[0207] As shown in the seventh embodiment, increasing the circulation flow rate in the culture medium circulation path and the circulation flow rate in the nutrient component supply path can improve the metabolic product removal capacity. Furthermore, adjusting the ratio between the circulation flow rate in the culture medium circulation path and the circulation flow rate in the nutrient component supply path can improve the metabolic product removal capacity. Furthermore, optimizing the ratio of the circulation flow rates can improve the culture efficiency.

[0208] <<<Details of the Seventh Embodiment>>> Figure 9 is a schematic diagram showing the configuration of a culture medium regeneration system 700 according to the seventh embodiment. In Figure 9, the same components as those in the first to sixth embodiments are denoted by the same reference numerals. In the seventh embodiment, a substitute for a culture medium or a substitute for a waste product is used, but the culture medium regeneration system 700 can have the same configuration as those in the first to sixth embodiments.

[0209] <Agitation Device> The culture medium regeneration system 700 according to the seventh embodiment has the agitation device 120, similar to that of the first embodiment, but is omitted for simplicity. The culture medium contained in the culture vessel 110-1 is agitated by the agitation device 120. The culture medium contained in the regeneration culture medium tank 180 is also agitated by an agitation device (not shown). Note that the culture medium contained in the regeneration culture medium tank 180 does not need to be agitated by an agitation device (not shown). Whether or not to agitate the culture medium can be determined depending on the type and temperature of the culture medium, the volume of the regeneration culture medium tank 180, etc.

[0210] 9, the culture medium regeneration system 700 also has a culture vessel 110-1. However, unlike the culture medium regeneration system 100, the culture vessel 110-1 is fixedly disposed in a vertical state.

[0211] <First end 162a of tube 160a> The culture medium regeneration system 700 has a suction tube nozzle 130-1, similar to the first embodiment. However, unlike the first embodiment, the suction tube nozzle 130-1 is not inclined but extends vertically.

[0212] The aspirating tube nozzle 130-1 has an extension 132-1, an intake opening 134-1, and a discharge opening 136-1. The intake opening 134-1 is located below the liquid level of the culture medium stored in the culture vessel 110-1. The culture medium stored in the culture vessel 110-1 is aspirated through the intake opening 134-1.

[0213] <<Control Device 710>> The control device 710 mainly includes a processor (such as a CPU (Central Processing Unit)), a ROM (Read Only Memory), a RAM (Random Access Memory), an I / F (Interface Device), an auxiliary storage device (such as a HDD (Hard Disk Drive) or an SSD (Solid State Drive)), and an input operation device (such as a keyboard, a mouse, or a touch panel). For example, the control device 710 may be a personal computer, a tablet computer, a portable terminal device, or a similar device. The HDD or SSD stores programs for controlling pumps 150a, 150b, and 150c, and the programs are deployed to RAM and executed.

[0214] The control device 710 issues control signals to the pumps 150a, 150b, and 150c via the I / F. The control signals control the rotation speeds of the motors (not shown) of the pumps 150a, 150b, and 150c, thereby adjusting the flow rates of the culture medium generated by driving the pumps 150a, 150b, and 150c. The circulation flow rate in the culture medium circulation circuit is adjusted by the pumps 150a, and the circulation flow rate in the culture medium regeneration circuit is adjusted by the pumps 150b and 150c.

[0215] <Culture medium circulation circuit and culture medium regeneration circuit> The culture medium regeneration system 700 has a culture medium circulation circuit and a culture medium regeneration circuit, similar to the culture medium regeneration system 100. The culture medium circulation circuit and the culture medium regeneration circuit form a closed circuit.

[0216] <Culture medium circulation circuit (culture medium circulation path)> The culture medium circulation circuit is mainly composed of the culture vessel 110-1, pump 150a, culture medium regeneration module 170, pipe 160a, pipe 160b, pipe 160c, etc. When pump 150a is driven, the culture medium is discharged from the culture vessel 110-1 and circulates through the culture medium circulation circuit. The culture medium circulates in the following order: culture vessel 110-1, pipe 160a, pump 150a, pipe 160b, culture medium regeneration module 170, and pipe 160c.

[0217] <Driving pump 150a> Driving pump 150a discharges culture medium containing waste products (lithium lactate) from culture vessel 110-1. The flow rate of culture medium per unit time generated in the culture medium circulation circuit by driving pump 150a is referred to as the circulation flow rate. The circulation flow rate can be adjusted by controlling the rotation speed of the motor (not shown) of pump 150a using control device 710.

[0218] <Culture medium regeneration circuit (nutrient component supply path)> The culture medium regeneration circuit is mainly composed of pump 150b, pump 150c, culture medium regeneration module 170, regeneration culture medium tank 180, pipe 160d, pipe 160e, pipe 160f, pipe 160g, etc. By driving pump 150b and pump 150c, the regeneration culture medium stored in regeneration culture medium tank 180 circulates through the culture medium regeneration circuit. The regeneration culture medium circulates in the following order: regeneration culture medium tank 180, pipe 160f, pump 150c, pipe 160g, culture medium regeneration module 170, pipe 160d, pump 150b, and pipe 160e.

[0219] <Driving pumps 150b and 150c> Pump 150b discharges liquid containing waste products removed by medium regeneration module 170 from the medium circulating through the medium circulation circuit to regeneration medium tank 180. Pump 150c supplies the regeneration medium stored in regeneration medium tank 180 to medium regeneration module 170. The circulating flow rate of the regeneration medium can be adjusted by controlling the rotation speeds of the motors (not shown) of pumps 150b and 150c using control device 710.

[0220] <Culture medium regeneration module 170> The culture medium regeneration module 170 has a culture medium regeneration membrane 179 for regenerating the culture medium. The culture medium regeneration membrane 179 may be a hollow fiber type culture medium regeneration module or a flat membrane type culture medium regeneration module. The culture medium regeneration module 170 may be, for example, a module for dialysis.

[0221] The medium regeneration module 170 has a medium inlet 172 and a medium outlet 174. The medium inlet 172 is an opening for introducing the medium flowing through the first end 162a of the tube 160a into the medium regeneration module 170. The medium outlet 174 is an opening for discharging the medium introduced into the medium regeneration module 170.

[0222] The medium regeneration module 170 has a regeneration medium supply port 176 and a regeneration medium discharge port 178. The regeneration medium supply port 176 is an opening for supplying the regeneration medium from the regeneration medium tank 180 to the medium regeneration module 170. The regeneration medium discharge port 178 is an opening for discharging the regeneration medium supplied from the regeneration medium supply port 176 to the medium regeneration module 170.

[0223] <Removal of waste product> Pipes 160d and 160e are used to guide waste product (lithium lactate) from the medium regeneration module 170 to the regeneration medium tank 180. This allows the waste product (lithium lactate) to be removed from the medium circulating through the medium circulation circuit. Pump 150b can adjust the flow rate of the medium being guided from the medium regeneration module 170 to the regeneration medium tank 180.

[0224] <Supply of Nutrients> Nutrients for the cell aggregates are delivered from the regeneration medium tank 180 to the medium regeneration module 170 via pipes 160f and 160g. This allows the nutrients to be supplied to the medium circulation circuit. The flow rate delivered from the regeneration medium tank 180 to the medium regeneration module 170 can be adjusted using pump 150c. Note that the seventh embodiment is intended to verify the relationship between the circulation flow rate and the capacity to remove waste products (metabolites), and it is not necessary to add nutrients to the regeneration medium tank 180. However, if nutrients are added to the regeneration medium tank 180, the relationship between the circulation flow rate and the capacity to add nutrients can also be verified.

[0225] <Operation of medium regeneration module 170> Nutrients for the cell aggregates are supplied from the regeneration medium tank 180 to the regeneration medium supply port 176 of the medium regeneration module 170 via pipes 160f and 160g. The supplied nutrients permeate the medium regeneration membrane 179 and move to the medium circulation circuit. As a result, the nutrients are supplied to the medium in the medium circulation circuit.

[0226] On the other hand, the waste product (lithium lactate) permeates the medium regeneration membrane 179 from the medium circulation circuit and is discharged from the regeneration medium outlet 178 of the medium regeneration module 170 through the pipes 160d and 160e to the regeneration medium tank 180. In this way, the waste product (lithium lactate) is removed from the medium in the medium circulation circuit.

[0227] <<Experiment to verify solute removal ability depending on flow rate>> In a closed circuit having a culture medium circulation circuit and a culture medium regeneration circuit, the circulation flow rate of the culture medium circulation circuit and the circulation flow rate of the culture medium regeneration circuit were adjusted to verify the solute removal ability depending on the circulation flow rate (the ability to remove waste product (lithium lactate)). Specifically, the ability to remove waste product (lithium lactate) depending on the circulation flow rate was verified. Figure 11 is a graph showing the relationship between the pump operation time and the concentration of waste product (lithium lactate) in the culture vessel 110-1, which changes over time.

[0228] <Conditions for Verification> As shown in FIG. 10A, the volume VL1 of the culture vessel 110-1 was 500 mL, and the volume VL2 of the regeneration medium tank 180 was 1500 mL. The ratio of the volume VL1 of the culture vessel 110-1 to the volume VL2 of the regeneration medium tank 180 was VL1:VL2 = 1:3. Also, as shown in FIG. 10A, 20 mM lithium lactate was pre-charged into the culture vessel 110-1 as a waste product substitute. Meanwhile, the regeneration medium tank 180 did not contain lithium lactate (0 mM) as a waste product substitute. Therefore, prior to the start of the medium regeneration experiment, the ratio of the concentration LC1 of lithium lactate contained in the culture vessel 110-1 to the concentration LC2 of lithium lactate contained in the regeneration medium tank 180 was LC1:LC2 = 20:0.

[0229] The change in concentration was confirmed under two circulation flow rate conditions. As shown in FIG. 10B, under the first circulation flow rate condition, the circulation flow rate CF1(1) of the culture medium circulation circuit was set to 5 mL / min, and the circulation flow rate CF2(1) of the culture medium regeneration circuit was set to 10 mL / min. The ratio of the circulation flow rate CF1(1) of the culture medium circulation circuit to the circulation flow rate CF2(1) of the culture medium regeneration circuit was CF1(1):CF2(1) = 1:2. Also, as shown in FIG. 10B, under the second circulation flow rate condition, the circulation flow rate CF1(2) of the culture medium circulation circuit was set to 20 mL / min, and the circulation flow rate CF2(2) of the culture medium regeneration circuit was set to 40 mL / min. The ratio of the circulation flow rate CF1(2) of the culture medium circulation circuit to the circulation flow rate CF2(2) of the culture medium regeneration circuit was CF1(2) = CF1(1) x 4, and CF2(2) = CF2(1) x 4, so that CF1(2):CF2(2) = 1:2.

[0230] <Verification Results> The control device 710 controlled pump 150a to generate a circulation flow rate in the culture medium circulation circuit, and pumps 150b and 150c to generate a circulation flow rate in the culture medium regeneration circuit. Every time a predetermined time elapsed after the start of operation of pumps 150a, 150b, and 150c, the concentrations of lithium lactate contained in the culture vessel 110-1 and the regeneration medium tank 180 were measured. The concentration of lithium lactate contained in the culture vessel 110-1 was measured by extracting the culture medium from the culture vessel 110-1. The concentration of lithium lactate contained in the regeneration medium tank 180 was measured by extracting the regeneration medium from the regeneration medium tank 180.

[0231] 11 is a graph showing the results of measurements of the concentration of lithium lactate contained in the culture vessel 110-1 and the concentration of lithium lactate contained in the regeneration medium tank 180. Note that because the concentration measurements were performed at predetermined time intervals, the lactic acid concentration is shown as a collection of discrete points, but to clearly show the change in concentration, the change in lactic acid concentration is shown as a continuous solid line. From top to bottom, FIG. 11 shows the change in concentration at a circulation flow rate CF1(1) in the culture medium circulation circuit, the change in concentration at a circulation flow rate CF1(2) in the culture medium circulation circuit, the change in concentration at a circulation flow rate CF2(2) in the culture medium regeneration circuit, and the change in concentration at a circulation flow rate CF2(1) in the culture medium regeneration circuit.

[0232] <Rate of change in concentration> As shown in Figure 11, the initial slope of the concentration change at the start of the experiment when the circulation flow rate in the culture medium circulation circuit was CF1(1) was designated S1(1). Similarly, the initial slope of the concentration change at the start of the experiment when the circulation flow rate in the culture medium regeneration circuit was CF2(1) was designated S2(1). The initial slope of the concentration change at the start of the experiment when the circulation flow rate in the culture medium circulation circuit was CF1(2) was designated S1(2). The initial slope of the concentration change at the start of the experiment when the circulation flow rate in the culture medium regeneration circuit was CF2(2) was designated S2(2).

[0233] As shown in FIG. 11 , |S1(1)|<|S1(2)|. The symbol "|x|" indicates the absolute value of x. Furthermore, S2(1)<S2(2). That is, when the circulation flow rate of the culture medium circulation circuit and the circulation flow rate of the culture medium regeneration circuit were increased, the concentration of lithium lactate contained in the culture vessel 110-1 rapidly decreased. From this, it can be seen that by increasing the circulation flow rate of the culture medium circulation circuit and the circulation flow rate of the culture medium regeneration circuit, the rate at which lithium lactate can be removed from the culture medium in the culture vessel 110-1 can be increased. In other words, the time required to remove lithium lactate from the culture medium in the culture vessel 110-1 can be shortened.

[0234] As time passes, the concentration of lithium lactate contained in the culture medium in the culture vessel 110-1 and the concentration of lithium lactate contained in the regeneration medium in the regeneration medium tank 180 both approach a common concentration C0. The culture medium regeneration system 700 shown in Figure 9 is a closed circuit having only a culture medium circulation circuit and a culture medium regeneration circuit. Therefore, after a sufficient amount of time has passed, lithium lactate gradually accumulates in the regeneration medium tank 180, and the capacity to remove lithium lactate gradually becomes saturated. As a result, the concentration of lactic acid contained in the culture medium in the culture vessel 110-1 and the concentration of lactic acid contained in the regeneration medium in the regeneration medium tank 180 approach each other.

[0235] By increasing the circulation flow rate of the regeneration medium flowing through the nutrient component supply path and the circulation flow rate of the medium circulating through the medium circulation path, the rate at which the concentration of the metabolic product (lithium lactate) in the culture vessel 110-1 decreases can be increased. Meanwhile, the nutrient components permeate the semipermeable membrane of the regeneration module 170 and move from the nutrient component supply path to the medium circulation path. Therefore, in contrast to the concentration of the metabolic product (lithium lactate), the rate at which the concentration of the nutrient components added to the medium in the culture vessel 110-1 increases can be increased.

[0236] <Variation 1 (Regeneration Medium Addition Tank)> In the seventh embodiment, the only tank storing new medium was the regeneration medium tank 180. However, an addition tank (not shown) that can add new medium to the regeneration medium tank 180 may be provided. Furthermore, by providing a pump (not shown), new medium can be supplied from the addition tank to the regeneration medium tank 180. By supplying new medium to the regeneration medium tank 180, it becomes easier to maintain the ability to remove lithium lactate, and the concentration C0 can be lowered. In this way, the medium can be continuously regenerated over a long period of time.

[0237] <Modification 2 (Formation of a Non-Circulating Path)> In the seventh embodiment, a closed system configuration was shown in which lithium lactate discharged from the culture medium regeneration module 170 was returned to the regeneration medium tank 180 via pipes 160d and 160e. That is, a circulation path was formed by pipes 160d, 160e, 160f, and 160g. Because lithium lactate is returned to the regeneration medium tank 180, lithium lactate gradually accumulates, and the capacity to remove lithium lactate gradually becomes saturated. As a result, the concentration of lactic acid contained in the culture medium in the culture vessel 110-1 and the concentration of lactic acid contained in the regeneration medium in the regeneration medium tank 180 become closer. From this perspective, a non-circulating path may be configured. For example, metabolic products discharged from the culture medium regeneration module 170 may be returned to a storage tank (not shown) other than the regeneration medium tank 180. This allows the concentration of lithium lactate in the regeneration medium tank 180 to be maintained, making it easier to maintain the capacity to remove lithium lactate and lowering the concentration C0.

[0238] <Modification 3 (Removal of Lithium Lactate Using an Adsorbent)> In the seventh embodiment, a closed-system configuration was shown in which lithium lactate discharged from the culture medium regeneration module 170 was returned to the regeneration medium tank 180 via pipes 160d and 160e. That is, a circulation path was formed by pipes 160d, 160e, 160f, and 160g. Because lithium lactate is returned to the regeneration medium tank 180, lithium lactate gradually accumulates, gradually saturating the capacity to remove lithium lactate. As a result, the concentration of lactic acid contained in the culture medium in the culture vessel 110-1 and the concentration of lactic acid contained in the regeneration medium in the regeneration medium tank 180 become closer. From this perspective, an adsorbent or the like may be used to selectively remove lithium lactate from cell aggregates. Furthermore, the configuration for removing lithium lactate using an adsorbent described in the first embodiment may be appropriately adopted. Specifically, the configurations of <Aspect 1 of Removal Using an Adsorbent> to <Aspect 4 of Removal Using an Adsorbent> of the first embodiment can be applied to the culture medium regeneration system 700 of the seventh embodiment. By doing so, the concentration of lithium lactate in the regeneration medium tank 180 can be maintained, making it easier to maintain the ability to remove lithium lactate, and lowering the concentration C0.

[0239] <Modification 4 (Circulation Flow Rate Control 1)> In the seventh embodiment, the circulation flow rate of the culture medium circulation circuit and the circulation flow rate of the culture medium regeneration circuit are set to constant values ​​independent of time. This is not limiting, and the control device 710 may control the pumps 150a, 150b, and 150c to change the circulation flow rates according to the treatment time. Treatment can be performed at an appropriate circulation flow rate or an appropriate ratio of circulation flow rates according to changes in the concentration of lithium lactate.

[0240] <Modification 5 (Circulation Flow Rate Control 2)> A sensor capable of measuring the concentration of lithium lactate in the culture vessel 110-1 in real time may be provided, and the circulation flow rates of the culture medium circulation circuit and the culture medium regeneration circuit may be determined based on the measurement results, and pumps 150a, 150b, and 150c may be controlled by the control device 710. Treatment can be performed at an appropriate circulation flow rate or an appropriate circulation flow rate ratio depending on changes in the concentration of lithium lactate.

[0241] <<<<<Eighth Embodiment>>>>> The eighth embodiment relates to a culture medium regeneration system that increases the flow rate of circulating culture medium while circulating the medium and preventing cell aggregates from being discharged into the circulation circuit. The eighth embodiment mainly illustrates the results of a verification of the relationship between the inclination angle of the suction tube nozzle 130-2 and the circulating flow rate of the medium. The culture medium regeneration system of the eighth embodiment is a system for verification. In the eighth embodiment, a liquid similar to the culture medium for culturing cell aggregates is used, and resin simulant particles are used as a substitute for the cell aggregates. Specifically, phosphate-buffered saline (PBS) is used as the culture medium, and resin beads with a particle size of 800 μm and a specific gravity of 1.025 g / cc are used as the cell aggregates. Hereinafter, unless otherwise necessary, the resin beads will be referred to as cell aggregates, and phosphate-buffered saline (PBS) will be referred to as the culture medium.

[0242] Circulation of the culture medium is essential in cell culture processes. However, if cell aggregates are discharged into the circulation circuit, fewer cell aggregates are available for culture. On the other hand, suppressing the discharge of cell aggregates limits the circulation flow rate, which reduces the time rate of change in the concentration of metabolic products in the culture section and the time rate of change in the concentration of nutrients.

[0243] In the eighth embodiment, a culture medium regeneration system is provided that uses inclined suction to increase the circulation flow rate of the culture medium in the circulation circuit while suppressing the discharge of cell aggregates, thereby maintaining the number of cell aggregates to be cultured and increasing the time rate of change of the metabolic product concentration in the culture section as well as the time rate of change of the nutrient concentration.

[0244] 12A and 12B are schematic diagrams showing the configuration of a culture medium regeneration system 800 according to the eighth embodiment. In FIG. 12A, the same components as those in the first and second embodiments are denoted by the same reference numerals. In FIG. 12A, the agitator 120, pump 150a, culture medium regeneration module 170, pipes 160a, 160b, pumps 150b, 150c, regeneration culture medium tank 180, pipes 160d, 160e, 160f, and 160g are the same as those in the first embodiment and are therefore omitted. The culture medium is aspirated from the culture vessel 110-1 by pump 150a, circulated, and returned to the culture vessel 110-1.

[0245] As shown in FIG. 12A, the culture vessel 110-1 of the culture medium regeneration system 800 is fixedly arranged in a vertical state, similar to the culture medium regeneration system 200.

[0246] <Agitation Device> The culture medium regeneration system 800 according to the eighth embodiment also has an agitation device similar to that of the first embodiment, but for simplicity, it is omitted. The culture medium contained in the culture vessel 110-1 is appropriately agitated by the agitation device.

[0247] <Suction tube nozzle 130-2> The culture medium regeneration system 800 has a suction tube nozzle 130-2. The configuration and material of the suction tube nozzle 130-2 are the same as those of the suction tube nozzle 130-1 of the culture medium regeneration system 100.

[0248] Similar to the suction tube nozzle 130-1, the suction tube nozzle 130-2 has an extension portion 132-1, an intake opening 134-1, and an exhaust opening 136-1.

[0249] <Tilt angle θ> The extension portion 132-1 has a similar configuration to the suction tube nozzle 130-1. As shown in Figures 12A and 12B, the extension portion 132-1 extends in an inclined direction. The extension portion 132-1 is inclined at an inclination angle θ with respect to the vertical direction. Therefore, the entire suction tube nozzle 130-2 extends in an inclined direction. In other words, the central axis AO of the suction tube nozzle 130-2 extends in the inclined direction.

[0250] In the eighth embodiment, the central axis CO of the culture vessel 110-1 extends vertically, and the aspirating tube nozzle 130-2 extends in an inclined direction. The aspirating tube nozzle 130-2 is disposed so that the suction opening 134-1 faces the side wall 112 of the culture vessel 110-1.

[0251] In the eighth embodiment, the central axis AO of the aspirating tube nozzle 130-2 is inclined with respect to the central axis CO of the culture vessel 110-1. In the eighth embodiment, the inclination angle θ of the aspirating tube nozzle 130-2 is selected from a plurality of angles. For example, three types of interchangeable aspirating tube nozzles 130-2 with inclination angles θ of 10 degrees, 15 degrees, and 20 degrees are selectively used. Each of the three types of aspirating tube nozzles 130-2 is stably held by a holding member (not shown) at its respective inclination angle θ. Note that, for clarity, FIG. 12A shows only the aspirating tube nozzle 130-2 with an inclination angle θ of 20 degrees in solid lines. FIG. 12B shows three types of aspirating tube nozzles 130-2 with inclination angles θ of 10 degrees, 15 degrees, and 20 degrees. Note that the inclination angle θ is not limited to these three and can be set to any desired angle within the range of 0<θ<90°.

[0252] <Flow of culture medium and cell aggregates within suction tube nozzle 130-2> The culture medium and cell aggregates flow within suction tube nozzle 130-2 in the same manner as in suction tube nozzle 130-1 of the first embodiment (see FIG. 2). Two flow regions, a first flow region FF and a second flow region SF, are also formed in suction tube nozzle 130-2. The cell aggregates remain within suction tube nozzle 130-2, and the culture medium is discharged from discharge opening 136-1 of suction tube nozzle 130-2.

[0253] In this way, by using the inclined suction pipe nozzle 130-2, the circulation flow rate of the culture medium can be increased without discharging the cell aggregates into the culture medium circulation circuit. Therefore, when a culture medium regeneration module (not shown) is used, the amount of culture medium that can come into contact with the culture medium regeneration membrane (not shown) can be increased without damaging the cell aggregates, thereby improving solute removal performance.

[0254] <<Verification Experiment>> Using simulated particles as a substitute for cell aggregates, conditions were determined under which the simulated particles would not be sucked into the suction tube nozzle 130-2.

[0255] <Conditions> The simulated particles were made of resin, had a particle size of 800 μm, and a specific gravity of 1.025 g / cc. A solution consisting of 0.02% surfactant and phosphate-buffered saline (PBS) was used as the culture medium. Air bubbles around the resin beads prevented them from settling, so a surfactant was used as a countermeasure. The tilt angle θ was 10 degrees, 15 degrees, and 20 degrees.

[0256] The circulation flow rate of the medium was determined by the following preliminary experiment. First, the flow rate of the medium relative to the rotation speed of the motor of the pump 150a was measured in advance. The flow rate of the medium can be determined from the weight difference in the amount of liquid per minute. For example, the flow rate of the medium was determined to be 40 (mL / min) for a motor rotation speed of 100 (rpm) of the pump 150a.

[0257] From this, the flow rate per rotation is calculated from the measurement results of the culture medium flow rate. For example, the flow rate of the culture medium is calculated as 40 (mL / min) / 100 (rpm) = 0.4 (mL / rotation). The calculated flow rate per rotation is stored in the RAM of the control device.

[0258] Using the results of the preliminary experiment, the flow rate is determined as follows: The desired flow rate can be determined by setting it on the operation panel of the control device. For example, the desired flow rate is set to 10 (mL / min) on the operation panel.

[0259] The CPU of the control device calculates the number of rotations corresponding to the desired flow rate from the desired flow rate set on the operation panel and the stored flow rate per rotation. For example, it calculates 10 (mL / min) / 0.4 (mL / rotation) = 25 rotations / min and controls the motor of pump 150a to achieve 25 rotations / min. In this way, the flow rate set on the operation panel was used as the actual medium flow rate, and the maximum flow rate at which flow-mimicking particles were not sucked into the suction tube nozzle 130-2 was measured.

[0260] <Experimental Method> Simulated particles were introduced into the culture vessel 110-1. The flow rate in the suction tube nozzle 130-2 was gradually increased to confirm the state in which the simulative particles were not sucked in. The tilt angle θ was changed to similarly confirm the conditions in which the simulative particles were not sucked in. While changing the flow rate on the operation panel of the control device, the flow rate at which the simulative particles were not sucked in was determined for each tilt angle θ.

[0261] <Experimental Results> FIG. 13 is a graph showing the relationship between the inclination angle θ of the suction pipe nozzle 130-2 and the maximum flow rate at which the simulative particles are not sucked into the suction pipe nozzle 130-2.

[0262] When the inclination angle θ of the suction tube nozzle 130-2 was 10 degrees, the flow rate at which the simulative particles were not sucked into the suction tube nozzle 130-2 was minimum. When the inclination angle θ of the suction tube nozzle 130-2 was 20 degrees, the flow rate at which the simulative particles were not sucked into the suction tube nozzle 130-2 was maximum. By increasing the suction angle of the suction tube nozzle 130-2, the effect of suppressing suction into the suction tube nozzle 130-2 is improved, allowing the culture medium to flow at a greater flow rate.

[0263] By tilting the suction tube nozzle 130-2, the circulating flow rate of the culture medium can be increased without the cell aggregates being sucked into the suction tube nozzle 130-2, thereby suppressing cell loss and increasing the time rate of change in the concentration of metabolic products in the culture section, as well as the time rate of change in the concentration of nutrients.

[0264] 13 , there are tilt angles and circulation flow rates at which increasing the suction angle of the suction tube nozzle 130-2 can increase the circulation flow rate of the culture medium without the cell aggregates being sucked into the suction tube nozzle 130-2. Although the relationship between the specific tilt angle and circulation flow rate may vary depending on the type of cell aggregate, the type of culture medium, and the diameter and length of the suction tube nozzle 130-2, similar verification experiments can be used to determine whether there are tilt angles and circulation flow rates at which increasing the suction angle of the suction tube nozzle 130-2 can increase the circulation flow rate of the culture medium. Furthermore, similar verification experiments can be used to determine the optimal range of tilt angle and circulation flow rate depending on the type of cell aggregate, the type of culture medium, and the diameter and length of the suction tube nozzle 130-2.

[0265] <Modification 1> In the eighth embodiment, an example has been shown in which the culture medium is aspirated using only one aspirating tube nozzle 130-2, but a plurality of aspirating tube nozzles 130-2 may also be used.

[0266] <<<<<Scope of the Embodiments>>>>> As described above, the first to eighth embodiments have been described. However, the descriptions and drawings that form part of this disclosure should not be understood as limiting. Various embodiments not described here are also included. Therefore, the first to eighth embodiments are not mutually independent configurations, and also include embodiments in which the configurations of the first to eighth embodiments are appropriately selected.

[0267] According to a verification experiment using the culture medium regeneration system 700 of the seventh embodiment, increasing the circulation flow rate of the culture medium circulation path and the circulation flow rate of the nutrient supply path can improve at least one of the metabolic product removal capacity and the nutrient supply capacity. For example, regarding the nutrient supply capacity, a graph of nutrient concentration change can be obtained by swapping the circulation flow rates CF1(1) and CF1(2) of the culture medium circulation circuit with the circulation flow rates CF2(1) and CF2(2) of the culture medium regeneration circuit in the graph shown in Figure 11 (equivalent to an inverted graph). It is believed that the concentration change of nutrient components also shows a similar trend. It is expected that the rate of change of nutrient components will also increase as the flow rate increases. From the above, the rate of change of nutrient components can also be explained from the data on lithium lactate.

[0268] It is clear that the verification results obtained in the seventh embodiment can be applied to a culture medium regeneration system 100 having a configuration similar to that of the culture medium regeneration system 700. Specifically, the verification results obtained in the seventh embodiment can be applied to any system that has a culture medium circulation circuit and a culture medium regeneration circuit and is capable of adjusting the circulation flow rate of the culture medium in the culture medium circulation circuit and the circulation flow rate of the regeneration culture medium in the culture medium regeneration circuit. Therefore, the verification results obtained in the seventh embodiment can be applied not only to the first embodiment but also to the second to sixth embodiments.

[0269] Furthermore, verification experiments using the culture medium regeneration system 800 of the eighth embodiment showed that the circulating flow rate of the culture medium in the circulation circuit can be increased while suppressing the discharge of cell aggregates using inclined suction. Therefore, it is clear that the verification results obtained in the eighth embodiment can be applied to a culture medium regeneration system 100 having a similar configuration to the culture medium regeneration system 800. Specifically, the verification results obtained in the eighth embodiment can be applied to any system that has an inclined suction tube nozzle 130-2 and can adjust the flow rate of the culture medium discharged from the suction tube nozzle 130-2. Therefore, the verification results obtained in the eighth embodiment can be applied not only to the first embodiment, but also to the second to sixth embodiments.

[0270] A medium regeneration system is provided that can efficiently culture cell aggregates by appropriately adjusting the components during the culture process of the cell aggregates. CROSS-REFERENCE TO RELATED APPLICATIONS

[0271] This application claims priority based on Japanese Patent Application No. 2024-018124, filed with the Japan Patent Office on February 8, 2024, the entire disclosure of which is incorporated herein by reference in its entirety.

[0272] 100, 200, 300, 400, 500, 600, 700, 800 Culture medium regeneration system 110-1, 110-3 Culture vessel 130-1, 130-2, 130-3 Suction tube nozzle 170 Culture medium regeneration module 172 Culture medium regeneration membrane

Claims

1. A medium regeneration system comprising: a medium circulation path through which a medium contained in a culture section for culturing cell aggregates circulates; a nutrient component supply path through which a regeneration medium containing nutritional components for the cell aggregates flows; and a medium regeneration module connected to the medium circulation path and the nutrient component supply path and containing a semipermeable membrane that is permeable to at least one of the nutritional components and metabolic products of the cell aggregates, wherein at least one of the opportunities for the nutritional components to permeate the semipermeable membrane and come into contact with the cell aggregates and the opportunities for the metabolic products to permeate the semipermeable membrane and come into contact with the regeneration medium in the nutrient component supply path is regulated.

2. A culture medium regeneration system according to claim 1, which adjusts the concentration of either a nutrient component supplied to the cell aggregate via the semipermeable membrane or a metabolic product of the cell aggregate.

3. The culture medium regeneration system according to claim 2, further comprising a nutrient component storage tank in which the nutrient components are stored, and the flow path between the nutrient component storage tank and the semipermeable membrane is a non-circulating path that has the nutrient component supply path and does not have a flow path from the semipermeable membrane to the nutrient component storage tank.

4. The culture medium regeneration system according to claim 2, further comprising a nutrient component storage tank in which the nutrient components are stored, and a nutrient component adding device that adds new nutrient components to the nutrient component storage tank.

5. The culture medium regeneration system according to claim 1, wherein the flow rate of the culture medium flowing through the culture medium circulation path is adjusted.

6. The culture medium regeneration system according to claim 5, further comprising an outlet nozzle for discharging the culture medium from the culture section toward the semipermeable membrane, the outlet nozzle being disposed at an angle to the vertical direction.

7. A medium regeneration system according to any one of claims 1 to 6, wherein the regeneration medium contains an adsorbent that adsorbs the metabolic products.

8. The culture medium regeneration system according to claim 7, wherein the target of the adsorbent is at least one of waste products selected from the group consisting of lactic acid, ammonia, glutamic acid, isovaleric acid, butyric acid, and citric acid.

9. A culture medium regeneration system comprising: a culture medium circulation path through which the culture medium and metabolic products contained in a culture unit are circulated; a nutrient component supply path through which a regeneration culture medium containing nutritional components is circulated; a culture medium regeneration module connected to the culture medium circulation path and the nutrient component supply path and containing a semipermeable membrane that is permeable to the nutritional components and metabolic products; and a control unit that adjusts the circulation flow rate of the culture medium circulating through the culture medium circulation path and the circulation flow rate of the regeneration culture medium circulating through the nutrient component supply path, wherein the rate of change of at least one of the concentration of the metabolic products and the concentration of the nutritional components in the culture unit is changed by the circulation flow rate of the culture medium circulating through the culture medium circulation path and the circulation flow rate of the regeneration culture medium circulating through the nutrient component supply path.

10. A culture medium regeneration system as described in claim 9, which increases at least one of the rate of decrease in the concentration of the metabolic product in the culture section and the rate of increase in the concentration of the nutrient component in the culture section by increasing the circulation flow rate of the regeneration culture medium flowing through the nutrient component supply path and the circulation flow rate of the culture medium circulating in the culture medium circulation path.

11. A culture medium regeneration system comprising: a culture medium circulation path through which the culture medium contained in the culture section circulates together with cell aggregates; and an outlet nozzle that discharges the culture medium from the culture section, wherein the outlet nozzle is arranged at an angle to the vertical direction, and the circulation flow rate is increased while preventing cell aggregates from being discharged from the culture section into the culture medium circulation path according to the inclination angle of the outlet nozzle.

12. A culture medium regeneration system that increases at least one of the rate at which the concentration of the metabolic product in the culture section decreases and the rate at which the concentration of the nutrient component in the culture section increases by increasing the circulation flow rate through the culture medium circulation path.

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