Medium regeneration system

Abstract

A culture medium regeneration system is provided that allows for appropriate adjustment of the composition during the culture of cell aggregates. The culture medium regeneration system comprises: a culture medium circulation path for circulating culture medium housed in a culture section for culturing cell aggregates; a nutrient supply path for supplying regeneration culture medium containing nutrients for the cell aggregates; and a culture medium regeneration module connected to the culture medium circulation path and the nutrient supply path, and housing a semi-permeable membrane capable of allowing at least one of the nutrients and metabolites produced by the cell aggregates to pass through. The culture medium regeneration system adjusts the opportunity for the nutrients to contact the cell aggregates through the semi-permeable membrane and the opportunity for the metabolites to contact the regeneration culture medium in the nutrient supply path through the semi-permeable membrane.

Description

Technical Field

[0001] This invention relates to a culture medium regeneration system for regenerating culture media. Background Technology

[0002] Typically, during the culture (proliferation) process, cell aggregates (cells) consume nutrients (such as glucose) and produce metabolic waste (such as lactic acid). Therefore, it is necessary to replenish nutrients and remove metabolic waste. As a method, the culture medium is usually replaced at prescribed intervals (e.g., Patent Documents 1, 2, 3, etc.).

[0003] Existing technical documents

[0004] Patent documents

[0005] Patent Document 1: Japanese Patent Application Publication No. 2002-85049

[0006] Patent Document 2: Japanese Patent No. 6422221

[0007] Patent Document 3: Japanese Patent No. 2003-521877 Summary of the Invention

[0008] The problem that the invention aims to solve

[0009] The aforementioned conventional techniques involve regenerating the culture medium through dialysis, which uses a separation membrane within a portion of the culture loop to adjust the composition of multiple tanks. However, the performance (dialysis capacity) of the separation membrane cannot keep up with the concentration changes of components that occur during culture, making proper composition adjustment impossible.

[0010] The present invention was made in view of the above-mentioned points. Its object is to provide a culture medium regeneration system that can appropriately adjust the composition during the culture of cell aggregates.

[0011] Solution for solving the problem

[0012] The culture medium regeneration system based on the present invention is characterized by having:

[0013] A culture medium circulation circuit that circulates the culture medium contained in the culture section used for culturing cell aggregates;

[0014] A nutrient supply path for supplying a regeneration culture medium containing nutrients for the cell aggregates; and

[0015] A culture medium regeneration module, connected to the culture medium circulation path and the nutrient supply path, and housing a semi-permeable membrane capable of allowing at least one of the nutrients and the metabolites produced by the cell aggregate to pass through.

[0016] The culture medium regeneration system regulates the chance of at least one of the following: the chance of the nutrients coming into contact with the cell aggregate through the semipermeable membrane and the chance of the metabolites coming into contact with the regeneration culture medium of the nutrient supply path through the semipermeable membrane.

[0017] Invention Effects

[0018] The composition can be appropriately adjusted during the culture of cell aggregates. Attached Figure Description

[0019] Figure 1 This is a schematic diagram showing the structure of the culture medium regeneration system 100 according to the first embodiment.

[0020] Figure 2 This is a cross-sectional view showing the general movement of the cell aggregate within the suction nozzle 130-1. Figure 2 A) and shown Figure 2 A cross-sectional view of the distribution of cell aggregates within the suction nozzle 130-1 in section II shown in Figure A. Figure 2 B).

[0021] Figure 3 This is a graph showing the relationship between the tilt angle of the suction nozzle 130-1 and the circulation flow rate when the cell aggregate rises to the midpoint between the suction opening 134-1 and the discharge opening 136-1.

[0022] Figure 4 This is a schematic diagram showing the structure of the culture medium regeneration system 200 according to the second embodiment.

[0023] Figure 5 This is a schematic diagram showing the structure of the culture medium regeneration system 300 according to the third embodiment.

[0024] Figure 6 This is a schematic diagram showing the structure of the culture medium regeneration system 400 according to the fourth embodiment.

[0025] Figure 7 This is a schematic diagram showing the structure of the culture medium regeneration system 500 according to the fifth embodiment.

[0026] Figure 8 This is a schematic diagram showing the structure of the culture medium regeneration system 600 according to the sixth embodiment.

[0027] Figure 9 This is a schematic diagram showing the structure of the culture medium regeneration system 700 according to the seventh embodiment.

[0028] Figure 10A This is a table showing the conditions for the verification experiment of the culture medium regeneration system 700 according to the seventh embodiment.

[0029] Figure 10B This is a table showing the conditions for the verification experiment of the culture medium regeneration system 700 according to the seventh embodiment.

[0030] Figure 11 This is a graph showing the change in lactic acid concentration in the culture medium regeneration system 700 of the seventh embodiment as a result of a verification experiment.

[0031] Figure 12A This is a schematic diagram showing the structure of the culture medium regeneration system 800 according to the eighth embodiment.

[0032] Figure 12B This is a schematic diagram showing the structure of the culture medium regeneration system 800 according to the eighth embodiment.

[0033] Figure 13 It is a graph showing the flow rate of cell aggregates that are not attracted by the use of tilted attraction. Detailed Implementation

[0034] <<<<Summary of Implementation Methods>>>>

[0035] To culture cell aggregates (cells (hereinafter, mainly referred to as cell aggregates)), nutrients need to be supplemented and metabolic waste needs to be removed. As a method, culture medium replacement is usually performed.

[0036] However, in methods based on culture medium replacement, the following three issues are envisioned.

[0037] (1) Replacement that matches the removal of metabolic waste

[0038] Even if nutrients remain in the culture medium, it is necessary to replace the medium to remove metabolic waste. In cases where it is necessary to replace the medium more than once, it may be considered an overuse of the medium.

[0039] (2) Volume change during culture medium replacement

[0040] When the culture medium is changed, the volume of the culture medium in the culture vessel is temporarily reduced when the culture medium is removed, and the density of cell aggregates increases, which may lead to an increase in the shear stress of the cell aggregates.

[0041] (3) Changes in the concentration of the culture medium

[0042] Imagine a situation where the concentration of the culture medium components changes drastically before and after the culture medium is replaced due to the addition of new culture medium to the culture vessel.

[0043] The culture medium regeneration system of this embodiment was developed to address the aforementioned problems.

[0044] <<First Feature>>

[0045] According to the first feature, a culture medium regeneration system is provided, wherein...

[0046] The culture medium regeneration system comprises:

[0047] A culture medium circulation circuit (such as a culture medium circulation loop described later) supplies culture medium to the culture section housed for culturing cell aggregates.

[0048] Nutrient supply pathways (e.g., culture medium regeneration circuits described later) supply a regeneration culture medium containing nutrients for the cell aggregates; and

[0049] The culture medium regeneration module is connected to the culture medium circulation path and the nutrient supply path, and contains a semi-permeable membrane (e.g., the culture medium regeneration membrane 179 described later) that allows at least one of the nutrients and the metabolites produced by the cell aggregate to pass through.

[0050] The culture medium regeneration system regulates the chance of at least one of the following: the chance of the nutrients coming into contact with the cell aggregate through the semipermeable membrane and the chance of the metabolites coming into contact with the regeneration culture medium of the nutrient supply path through the semipermeable membrane.

[0051] The culture medium regeneration system comprises a culture medium circulation path, a nutrient supply path, and a culture medium regeneration module. The culture medium regeneration system includes a culture system for culturing cell aggregates in a culture section. The culture medium regeneration system is used to regenerate the culture medium used in the culture system for culturing cell aggregates.

[0052] <Culture Media Circulation Path>

[0053] The culture medium circulation path is a flow path for circulating culture medium. The culture medium is contained within a culture section used to culture cell aggregates. Cell aggregates also flow into a portion of the culture medium circulation path (e.g., suction nozzles 130-1, 130-2, 130-3, etc., described later), but the cell aggregates are not circulated throughout the entire culture medium circulation path. In contrast, the culture medium circulates throughout the entire culture medium circulation path.

[0054] <Nutritional Component Supply Path>

[0055] The nutrient supply path provides a fluid containing nutrients for cell aggregates, such as a regeneration culture medium.

[0056] <Culture Media Regeneration Module>

[0057] The culture medium regeneration module is connected to both the culture medium circulation path and the nutrient supply path. The culture medium regeneration module has a semi-permeable membrane. This membrane allows at least one of the nutrients and metabolic products generated from the cell aggregates to pass through. Nutrients move from the nutrient supply path to the culture medium circulation path through the semi-permeable membrane. Conversely, metabolic products move from the culture medium circulation path to the nutrient supply path through the semi-permeable membrane. Through this exchange of nutrients and metabolic products via the semi-permeable membrane, cell aggregates can be cultured.

[0058] <Regulating the Opportunities for Contact>

[0059] Furthermore, it regulates the opportunities for nutrients to pass through the semipermeable membrane and come into contact with cell aggregates, and the opportunities for metabolic products produced by cell aggregates to pass through the semipermeable membrane and come into contact with the fluid in the nutrient supply path.

[0060] Because of the ability to regulate contact opportunities, it is possible to supply necessary nutrients to the culture medium circulation loop and remove unwanted metabolites from the culture medium circulation loop, and to properly adjust the composition during the culture of cell aggregates.

[0061] <<Second Feature>>

[0062] The second feature is based on the first feature.

[0063] The concentration of at least one of the nutrients supplied to the cell aggregate via the semipermeable membrane and the metabolites of the cell aggregate is adjusted.

[0064] By regulating the concentration of at least one of the nutrients and the metabolites of the cell aggregates, an environment appropriate for the growth of the cell aggregates can be provided.

[0065] <<Third Feature>>

[0066] The third feature is based on the first or second feature.

[0067] It also includes a nutrient storage tank (such as the regeneration culture medium tank 180 described later) for storing the nutrients.

[0068] The flow path between the nutrient storage tank and the semi-permeable membrane is a non-circulating path that has the nutrient supply path but does not have a flow path from the semi-permeable membrane to the nutrient storage tank.

[0069] By employing a non-circulating pathway, the effects of metabolic byproducts can be prevented, ensuring that nutrients are at the desired concentration. For example, nutrient concentrations can be maintained at a constant level. An environment appropriate for the growth of cell aggregates can always be provided.

[0070] <<Fourth Feature>>

[0071] The fourth feature is based on the first to third features.

[0072] It also includes a nutrient storage tank for storing the aforementioned nutrients.

[0073] It also includes a nutrient addition device for adding new nutrients to the nutrient storage tank.

[0074] By adding new nutrients to the nutrient storage tank, the nutrient concentration can be adjusted to a desired level. For example, the nutrient concentration can be maintained at a constant level. By adjusting the nutrient concentration, a balance can be achieved between the supply of nutrients and the removal of unwanted metabolic products.

[0075] <<Fifth Feature>>

[0076] The fifth feature is based on the first through fourth features.

[0077] Adjust the flow rate of the culture medium flowing in the culture medium circulation path.

[0078] It can supply the necessary nutrients according to the growth of cell aggregates and provide an environment that corresponds to the growth of cell aggregates.

[0079] <<Sixth Feature>>

[0080] The sixth feature is based on the first through fifth features.

[0081] It also has an outlet nozzle (e.g., suction nozzles 130-1, 130-2, 130-3, etc., described later) for discharging culture medium from the culture section toward the semipermeable membrane.

[0082] The outlet nozzle is set at an angle relative to the vertical direction.

[0083] By tilting the outlet nozzle relative to the vertical direction, the flow rate of the culture medium flowing through the outlet nozzle can be adjusted, thereby regulating the amount of nutrients, metabolites, and contact with the semipermeable membrane. This allows for a balance between the amount of nutrients supplied and the amount of metabolites removed.

[0084] <<Seventh Feature>>

[0085] The seventh feature is based on the first through sixth features.

[0086] The regeneration culture medium contains an adsorbent that adsorbs the metabolites.

[0087] It can maintain a low concentration of metabolites present in the regeneration medium that pass through the semipermeable membrane.

[0088] <<Eighth Feature>>

[0089] The eighth feature is based on features one through seven.

[0090] The adsorbent targets at least one of the following as metabolic waste products: lactic acid, ammonia, glutamic acid, isovaleric acid, butyric acid, and citric acid.

[0091] It can maintain a low concentration of metabolites present in the regeneration medium that pass through the semipermeable membrane.

[0092] <<Ninth Feature>>

[0093] According to the ninth feature, a culture medium regeneration system is provided, wherein...

[0094] The culture medium regeneration system comprises:

[0095] Culture medium circulation circuit (such as the culture medium circulation loop described later) is used to circulate the culture medium and metabolites contained in the culture section;

[0096] Nutrient supply pathways (such as the culture medium regeneration loop described later) circulate regeneration culture medium containing nutrients.

[0097] A culture medium regeneration module (e.g., culture medium regeneration module 170 described later) is connected to the culture medium circulation path and the nutrient supply path, and houses a semi-permeable membrane (e.g., culture medium regeneration membrane 179 described later) that allows the nutrients and metabolites to pass through; and

[0098] The control unit adjusts the circulation flow rate of the culture medium circulating in the culture medium circulation path and the circulation flow rate of the regeneration culture medium circulating in the nutrient supply path.

[0099] The culture medium regeneration system alters the rate of change of at least one of the concentrations of the metabolites and the concentrations of the nutrients in the culture section by varying the circulation flow rate of the culture medium circulating in the culture medium circulation path and the circulation flow rate of the regeneration culture medium circulating in the nutrient supply path (e.g., as described later). Figure 11 ).

[0100] <<Tenth Feature>>

[0101] The tenth feature is based on the ninth feature.

[0102] By increasing the circulation flow rate of the regeneration medium flowing in the nutrient supply path and the circulation flow rate of the medium circulating in the medium circulation path, at least one of the rate at which the concentration of the metabolites in the culture section decreases and the rate at which the concentration of the nutrients in the culture section increases is improved.

[0103] <<Eleventh Feature>>

[0104] have:

[0105] A culture medium circulation circuit that circulates the culture medium contained in the culture section along with the cell aggregates; and

[0106] A discharge nozzle that discharges the culture medium from the culture section.

[0107] The outlet nozzle is set at an angle relative to the vertical direction.

[0108] The flow rate is increased by inhibiting cell aggregates from being discharged from the culture section into the culture medium circulation path, depending on the tilt angle of the outlet nozzle.

[0109] <<Twelfth Feature>>

[0110] The twelfth feature is based on features nine through eleven.

[0111] By increasing the circulation flow rate in the culture medium circulation path, at least one of the rate at which the concentration of the metabolites in the culture section decreases and the rate at which the concentration of the nutrients in the culture section increases is improved.

[0112] <<<<Details of this implementation method>>>>

[0113] The embodiments will now be described with reference to the accompanying drawings. It should be noted that these embodiments refer to the first to eighth embodiments described below.

[0114] <<<Cell>>>

[0115] Cells are objects cultivated through a culture system. Cells are increased by dividing during cultivation. Cells then form aggregates (cell clumps) through contact with each other. A culture system is a system that allows cells to grow and form these aggregates.

[0116] The cells used are any cells that form cell aggregates in the culture medium; there are no particular limitations. Cells are preferably derived from mammals, particularly primates such as humans and apes, or species commonly used in research, such as mice. Examples of cells include those used in regenerative medicine research and those used as cell preparations. Specifically, examples include pluripotent stem cells such as ES cells and iPS cells, various precursor cells such as nephron precursor cells, ureteral bud cells, and matrix precursor cells, as well as various stem cells such as mesenchymal stem cells, neural stem cells, and adipose stem cells. Cells can be induced from pluripotent stem cells, immortalized cells, or lineage cells, or primary cultured cells isolated from tissues. Furthermore, depending on the purpose, cells can be normal cells or cells with disease, gene abnormalities, or introduced genes.

[0117] In addition, cell aggregates also include substances substituted by non-growing resins, etc. Substitutes such as resins do not require nutrients and do not produce metabolic waste (metabolic products). Substitutes such as resins only need to be similar in size, shape, specific gravity, and flowability in the culture medium to grown cell aggregates. In this specification, there are instances where substances substituted by resins are also referred to as cell aggregates.

[0118] <<<Culture Medium>>>

[0119] The culture media used in the culture medium regeneration system include fresh culture medium and recycled culture medium. Fresh culture medium is used in culture vessel 110-1 (described later). Figure 1 , Figure 4 , Figure 6 , Figure 7 , Figure 8 ), 110-3 (described later) Figure 5 (reactor) and other newly supplied culture media. Returned culture media are culture media that have been supplied to culture vessels 110-1, 110-3, etc. and used in culture, and are returned due to recycling or disposal.

[0120] Regarding the culture medium, there are no particular limitations on the selection and use of an appropriate medium based on the cells. Furthermore, conventionally known materials and additives beneficial to cell culture can be used appropriately. 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. Preferred additives include amino acids, vitamins, inorganic salts, proteins (growth factors), glucose, antibiotics, signal transduction inhibitors, reducing agents, and buffers. Serum (preferably derived from mammals, such as fetal bovine serum, human serum, etc.) can also be added. Supplements can also be used as alternatives to serum.

[0121] It should be noted that in this instruction manual, even liquids that do not contain nutrients, with similar pH, osmotic pressure, viscosity, etc., are referred to as culture media from a fluidity perspective. For example, phosphate-buffered saline (PBS) can be used as a substitute for culture media.

[0122] <<<Cell suspension>>>

[0123] Cell suspensions refer to systems in which cells or cell aggregates are dispersed in a culture medium. Culture containers 110-1 and 110-3 store cells, cell aggregates, and culture medium as cell suspensions.

[0124] <<<Metabolic waste (metabolites)>>>

[0125] Metabolic waste products are generated along with the growth of cell aggregates. Common metabolic waste products include lactic acid, ammonia, glutamic acid, isovaleric acid, butyric acid, and citric acid. It should be noted that in this specification, not only metabolic waste products generated along with the growth of cell aggregates, but also substances similar to these metabolic waste products are referred to as metabolic waste. For example, lithium lactate, which is similar to lactic acid as an example of metabolic waste, is also referred to as metabolic waste.

[0126] <<<Stirring>>>

[0127] Stirring refers to the displacement of cells, cell aggregates, and culture medium in a cell suspension. Through stirring, the cells, cell aggregates, and culture medium move together. Primarily, the cells and cell aggregates move along with the culture medium (convection). It should be noted that any action that causes cell displacement in a cell suspension is acceptable; this includes not only stirring but also actions such as shaking.

[0128] <<<Direction, state, etc.>>>

[0129] <Horizontal direction>

[0130] The horizontal direction refers to the direction that intersects the Earth's gravity at a right angle.

[0131] <Vertical direction>

[0132] The vertical direction refers to the direction of gravity. It is the direction indicated by the line suspending the object. It is the direction perpendicular to the horizontal direction. Specifically, it refers to the central axis CO of culture containers 110-1 and 110-3, the rotation axis RO of the stirring blade 124a, and the central axis AO of the suction nozzles 130-1, 130-2, and 130-3. Figure 1 , Figure 2 A, Figure 4 , Figure 5 , Figure 6 , Figure 7 , Figure 8 ( ) becomes the direction of the vertical direction.

[0133] <Direction of tilt>

[0134] The tilt direction refers to a direction that is tilted from the vertical and horizontal directions, but is neither vertical nor horizontal. Specifically, it refers to the direction in which the central axis CO of the culture containers 110-1 and 110-3, the rotation axis RO of the stirring blade 124a, and the central axis AO of the suction nozzles 130-1, 130-2, and 130-3 are tilted at a specified angle relative to the vertical and horizontal directions.

[0135] For simplicity, the angle between the vertical direction and the inclined direction may also be referred to as the tilt angle. The tilt angle is the angle between the vertical direction and the vertical direction. The tilt angle is preferably an acute angle. For example, a tilt angle preferably in the range of 5 to 30 degrees is preferred. More preferably, a tilt angle in the range of 10 to 20 degrees is also preferred. The tilt angle is not limited to the above range and can be appropriately determined based on factors such as the circulation flow rate of the culture medium, the type and size of the cell aggregates, and the desired treatment time.

[0136] <Axial>

[0137] The axial direction refers to the direction along the central axis CO of the culture containers 110-1 and 110-3, the rotation axis RO of the stirring blade 124a, and the central axis AO of the suction nozzles 130-1, 130-2, and 130-3. The axial direction can be defined primarily for elongated or cylindrical shapes. It is not limited to a straight line; it can be along the central axis CO, rotation axis RO, or central axis AO.

[0138] <Plumb State>

[0139] The vertical orientation refers to the state where the direction of the central axis CO of culture containers 110-1 and 110-3, the direction of the rotation axis RO of the stirring blade 124a, and the direction of the central axis AO of suction nozzles 130-1, 130-2, and 130-3 are parallel to the vertical direction. In other words, the vertical orientation means that the central axis CO of culture containers 110-1 and 110-3, the rotation axis RO of the stirring blade 124a, and the central axis AO of suction nozzles 130-1, 130-2, and 130-3 extend along the vertical direction.

[0140] <Tilted State>

[0141] The tilted state refers to the state in which the direction of the central axis CO of culture containers 110-1 and 110-3, the rotation axis RO of the stirring blade 124a, and the central axis AO of the suction nozzles 130-1, 130-2, and 130-3 are tilted relative to the vertical direction.

[0142] <Size of cell aggregates>

[0143] The size of a cell aggregate is simply an indicator of the degree of its growth and is not limited to its diameter. Besides the radius, it can refer to the density distribution, occupied area, or other indicators representing the approximate extent of the cell aggregate's growth process. It should be noted that in this specification, both the term "size of the cell aggregate" and the term "diameter of the cell aggregate" are used.

[0144] <<<<First Implementation>>>>

[0145] Figure 1 This is a schematic diagram showing the structure of the culture medium regeneration system 100 according to the first embodiment.

[0146] <<<Main Structure of Culture Medium Regeneration System 100>>>

[0147] The culture medium regeneration system 100 mainly includes a culture container 110-1, a stirring device 120, a suction nozzle 130-1, a pump 150a, a pump 150b, a pump 150c, a culture medium regeneration module 170, and a culture medium tank 180 for regeneration.

[0148] <<<Culture media circulation loop and culture media regeneration loop>>>

[0149] The culture medium regeneration system 100 has a culture medium circulation loop and a culture medium regeneration loop.

[0150] <<Culture Media Circulation Loop (Culture Media Circulation Circuit)>>

[0151] The culture medium circulation loop mainly consists of a culture container 110-1, a suction nozzle 130-1, a pump 150a, a culture medium regeneration module 170, and tubes 160a, 160b, and 160c. Driven by pump 150a, the culture medium circulates within the loop. The culture medium circulates in the following order: culture container 110-1, suction nozzle 130-1, tube 160a, pump 150a, tube 160b, culture medium regeneration module 170, and tube 160c.

[0152] <<Culture Media Regeneration Circuit (Nutrient Supply Circuit)>>

[0153] The culture medium regeneration loop mainly consists of pump 150b, pump 150c, culture medium regeneration module 170, regeneration culture medium tank 180, tubes 160d, 160e, 160f, and 160g. Driven by pumps 150b and 150c, the culture medium stored in the regeneration culture medium tank 180 circulates within the culture medium regeneration loop. The regeneration culture medium circulates in the following order: regeneration culture medium tank 180, tube 160f, pump 150c, tube 160g, culture medium regeneration module 170, tube 160d, pump 150b, and tube 160e.

[0154] <<<Cultivation container 110-1, stirring device 120, suction nozzle 130-1>>>

[0155] like Figure 1 As shown, the culture container 110-1, the stirring device 120, and the suction nozzle 130-1 are fixedly configured in a tilted state with a constant tilt angle θ.

[0156] <<Cultivation Container 110-1>>

[0157] The culture medium regeneration system 100 has a culture container 110-1. During cell culture, the culture medium and cells (cell suspension) are contained in the culture container 110-1. Within the culture container 110-1, the cells are cultured and grow into cell aggregates.

[0158] The culture container 110-1 has a generally cylindrical shape. The culture container 110-1 has a generally cylindrical side wall portion 112 and a generally circular bottom 114. The shape of the culture container 110-1 is not limited to a cylindrical shape, as long as it has a shape that allows the stirring blade 124a (described later) to rotate and stir the cell suspension so as to smoothly culture the cells or to smoothly circulate the culture medium.

[0159] <Central Axis CO>

[0160] The culture vessel 110-1 has a central axis CO extending along the center of the sidewall portion 112.

[0161] <<Stirring Device 120>>

[0162] <Structure of the stirring device 120>

[0163] The stirring device 120 stirs the cell suspension stored in the culture container 110-1. The stirring device 120 includes a drive unit 122, a stirring element 124, and a holding member 126. The drive unit 122 can be, for example, a magnetic stirrer. The drive unit 122 has a motor (not shown). By rotating the motor, the drive unit 122 uses magnetic force to rotate the stirring element 124, which is separated from the drive unit 122. The rotation speed of the motor can be appropriately determined according to the amount of cell suspension, the number and size of cell aggregates, etc.

[0164] The motor's rotational speed and direction are controlled by a control device (not shown). This control device includes a processor (CPU (Central Processing Unit), etc.), ROM (Read-Only Memory), RAM (Random Access Memory), and input / output interfaces. The ROM and RAM store programs used to control the motor's rotational speed and direction. The processor reads these programs from the ROM and RAM and executes them.

[0165] It should be noted that a magnetic stirrer can also be omitted, and the stirring piece 124 can be directly connected to the shaft of the motor (not shown) and made to rotate.

[0166] <Stirring based on stirring device 120>

[0167] The cell suspension is stirred by rotating the agitator 124. By stirring the cell suspension, it is possible to prevent cell aggregates from contacting each other or with the side wall 112 or the generally circular bottom 114 of the culture container 110-1 during culture and recovery.

[0168] <Agitator 124>

[0169] The stirring element 124 has stirring blades 124a and extensions 124b. The stirring blades 124a have a thin, plate-like shape that is approximately an isosceles triangle. The stirring blades 124a are located inside the culture container 110-1 and are separated from the drive unit 122. The bottom of the stirring blades 124a is opposite to the bottom 114 of the culture container 110-1.

[0170] It should be noted that the shape of the stirring blade 124a is not limited to an approximate isosceles triangle shape; any shape capable of stirring the cell suspension and rotating around the central axis CO is acceptable. The shape of the stirring blade 124a should be able to create the desired flow of the cell suspension.

[0171] The extension 124b has a generally rod-shaped form. The extension 124b extends from the top of the stirring blade 124a (the top opposite the bottom of the stirring blade 124a) in a direction away from the stirring blade 124a. The direction in which the extension 124b extends is the direction in which the rotation axis RO of the stirring element 124 extends. Preferably, the stirring element 124 is configured such that the rotation axis RO is substantially aligned with the central axis CO of the culture vessel 110-1. With this configuration, the cell suspension can be stirred throughout the culture vessel 110-1. It should be noted that the extension direction of the rotation axis RO only needs to be parallel to the extension direction of the central axis CO.

[0172] <Retaining Component 126>

[0173] The retaining member 126 holds the agitator 124 in a rotatable position. Even if the rotation axis RO of the agitator 124 is tilted, the retaining member 126 will still hold the agitator 124 in a rotatable position. By using the retaining member 126 to hold the agitator 124, the rotation axis RO can be made approximately aligned with the central axis CO.

[0174] Although an example is shown in which the rotation axis RO is roughly aligned with the central axis CO, the rotation axis RO can be appropriately determined based on factors such as the tilt angle, the amount of cell suspension, the number and size of cell aggregates, and the offset of cell aggregates within the culture vessel 110-1.

[0175] <<Suction Nozzle 130-1>>

[0176] The suction nozzle 130-1 is used to separate the cell aggregates remaining in the suction nozzle 130-1 from the circulating culture medium discharged from the discharge opening 136-1 of the suction nozzle 130-1. The cell aggregates cultured in the culture vessel 110-1 come in various sizes. The suction nozzle 130-1 is used to ensure that the cultured cell aggregates remain in the suction nozzle 130-1, and that only the culture medium is discharged from the discharge opening 136-1. The operation of the cell aggregates and culture medium within the suction nozzle 130-1 is described later. The suction nozzle 130-1 is formed of glass, resin, stainless steel, etc., and has a constant shape. The suction nozzle 130-1 is preferably made of a sterilizable material.

[0177] <Extension 132-1, Intake opening 134-1, Exhaust opening 136-1>

[0178] Suction nozzle 130-1 has:

[0179] Extension 132-1;

[0180] Inhalation opening 134-1; and

[0181] Discharge opening 136-1.

[0182] <Extension 132-1>

[0183] The extension 132-1 has an elongated shape. That is, the suction nozzle 130-1 has an elongated shape. The extension 132-1 extends in a straight line along the length direction. The extension 132-1 extends in an inclined direction. That is, the suction nozzle 130-1 extends in an inclined direction. In other words, the central axis AO of the suction nozzle 130-1 extends in an inclined direction.

[0184] like Figure 2 As shown, the extension 132-1 has a tubular shape. The extension 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 arranged concentrically. The elongated surrounding region SR, surrounded by the inner peripheral portion 132I-1 and extending along its length, functions as a hollow conduit. Culture medium and cell aggregates can flow through the surrounding region SR. It should be noted that... Figure 2 In A, the area surrounding SR is shown as the region enclosed by dashed lines along the four corners of the inner perimeter 132I-1. Additionally, in Figure 2 In section B, the area surrounding region SR is shown as the region enclosed by a circular dashed line along the inner periphery 132I-1. The extension 132-1 has a constant cross-sectional area A1. The cross-sectional area A1 is the area of ​​the inner diameter portion when the suction nozzle 130-1 is cut off in the vertical direction relative to the central axis AO of the suction nozzle 130-1.

[0185] <Inhalation opening 134-1 and discharge opening 136-1>

[0186] The suction nozzle 130-1 has two ends in the longitudinal direction. The suction nozzle 130-1 has a suction opening 134-1 at one end and a discharge opening 136-1 at the other end. The extension 132-1 extends in such a way that it is sandwiched between the suction opening 134-1 and the discharge opening 136-1.

[0187] The aspiration opening 134-1 has a generally circular opening. The discharge opening 136-1 has a generally circular opening. The aspiration opening 134-1, the discharge opening 136-1, and the surrounding region SR are connected. The culture medium and cell aggregates stored in the culture vessel 110-1 are aspirated through the aspiration opening 134-1 and guided towards the surrounding region SR. The cell aggregates guided towards the surrounding region SR remain in the surrounding region SR while flowing there.

[0188] It should be noted that the cell aggregates guided to the area surrounding the SR also exist as cell aggregates that, after flowing around the area surrounding the SR, are discharged from the aspiration opening 134-1 and return to the culture vessel 110-1. The culture medium guided to the area surrounding the SR flows around the area surrounding the SR and is discharged from the discharge opening 136-1.

[0189] The suction opening 134-1 and the discharge opening 136-1 have a constant opening area A1, which is the same as the cross-sectional area A1 of the extension 132-1. The suction nozzle 130-1 has a cross-sectional area A1.

[0190] <<<Pump 150a, Pipe 160a, Pipe 160b, Pipe 160c>>>

[0191] <Pump 150a>

[0192] Pump 150a can adjust the flow rate of the culture medium. Pump 150a can be, for example, a tubular pump, a reciprocating pump, a centrifugal pump, or a propeller pump. Pump 150a has a motor (not shown). The flow rate of pump 150a can be adjusted by the rotational speed of the motor, the frequency of the drive motor, etc. For simplicity, the term "drive of the motor of pump 150a" may be used below.

[0193] The motor's rotational speed and the timing of its drive are controlled by a control device (not shown). This control device includes a processor (CPU (Central Processing Unit), etc.), ROM (Read-Only Memory), RAM (Random Access Memory), and input / output interfaces. The ROM and RAM store programs used to control the motor's rotational speed and drive timing. The processor reads these programs from the ROM and RAM and executes them.

[0194] The control device that controls the motor of pump 150a may be the same as or different from the control device that controls the motor of drive unit 122 of stirring device 120.

[0195] <Driver of Pump 150a>

[0196] Driven by pump 150a, cell aggregates along with culture medium can be discharged from culture vessel 110-1. The flow rate of culture medium generated per unit time by pump 150a is called the circulation flow rate.

[0197] <<<Pipe 160a, Pipe 160b, Pipe 160c>>>

[0198] Tubes 160a, 160b, and 160c are made of flexible resin, etc. It should be noted that... Figure 1For simplicity, tubes 160a, 160b, and 160c are shown in a straight line shape.

[0199] Tubes 160a, 160b, and 160c are elongated. Tube 160a has a first end 162a and a second end 164a along its length. Tube 160b has a first end 162b and a second end 164b along its length. Tube 160c has a first end 162c and a second end 164c along its length.

[0200] <Pipe 160a>

[0201] The first end 162a of the tube 160a is connected to the discharge opening 136-1 of the suction nozzle 130-1. The second end 164a of the tube 160a is connected to the first end 152a of the pump 150a. The suction nozzle 130-1, the tube 160a, and the pump 150a are connected.

[0202] <Pipe 160b>

[0203] The first end 162b of tube 160b is connected to the second end 154a of pump 150a. The second end 164b of tube 160b is connected to the culture medium inlet 172 of culture medium regeneration module 170. Pump 150a, tube 160b and culture medium regeneration module 170 are connected in series.

[0204] <Pipe 160c>

[0205] The first end 162c of tube 160c is connected to the culture medium outlet 174 of culture medium regeneration module 170. The second end 164c of tube 160c is fixed in a predetermined position inside culture container 110-1. Tube 160c is in communication with culture medium regeneration module 170.

[0206] <<<Culture Media Regeneration Circuit>>>

[0207] The culture medium regeneration loop mainly consists of pump 150b, pump 150c, culture medium regeneration module 170, regeneration culture medium tank 180, tubes 160d, 160e, 160f, and 160g. The regeneration culture medium stored in the regeneration culture medium tank 180 circulates in the culture medium regeneration loop.

[0208] <Pump 150b, Pump 150c>

[0209] Pump 150b discharges the liquid, from which metabolic waste has been removed by the culture medium regeneration module 170, to the regeneration culture medium tank 180. Pump 150c supplies the regeneration culture medium stored in the regeneration culture medium tank 180 to the culture medium regeneration module 170. Thus, the components required for cell growth can be supplied to the culture medium.

[0210] Pumps 150b and 150c can adjust the flow rate of the regeneration culture medium. Pumps 150b and 150c can be, for example, tubular pumps, reciprocating pumps, centrifugal pumps, propeller pumps, etc. Pumps 150b and 150c have motors (not shown). The flow rate of pumps 150b and 150c can be adjusted using the rotational speed of the motor, the frequency of the drive motor, etc. For simplicity, the drive of the motors of pumps 150b and 150c may be referred to simply as the drive of pumps 150b and 150c. Pump 150b has a first end 152b on the inflow side and a second end 154b on the outflow side. Pump 150c has a first end 152c on the inflow side and a second end 154c on the outflow side.

[0211] The motor's rotational speed and the timing of its drive are controlled by a control device (not shown). This control device includes a processor (CPU (Central Processing Unit), etc.), ROM (Read-Only Memory), RAM (Random Access Memory), and input / output interfaces. The ROM and RAM store programs used to control the motor's rotational speed and drive timing. The processor reads these programs from the ROM and RAM and executes them.

[0212] The control device for controlling the motors of pumps 150b and 150c may be the same as or different from the control device for controlling the motor of the drive unit 122 of the stirring device 120.

[0213] <<<Tube 160d, Tube 160e, Tube 160f, Tube 160g>>>

[0214] Tubes 160d, 160e, 160f, and 160g are made of flexible resin, etc. It should be noted that... Figure 1 For simplicity, tubes 160d, 160e, 160f, and 160g are shown in a straight line shape. Tubes 160d, 160e, 160f, and 160g have an elongated shape.

[0215] Pipe 160d has a first end 162d and a second end 164d in the length direction. Pipe 160e has a first end 162e and a second end 164e in the length direction. Pipe 160f has a first end 162f and a second end 164f in the length direction. Pipe 160g has a first end 162g and a second end 164g in the length direction.

[0216] <Culture Media Regeneration Module 170>

[0217] The culture medium regeneration module 170 has a culture medium regeneration membrane 179 for regenerating the culture medium. The culture medium regeneration membrane 179 can be a hollow fiber type culture medium regeneration module or a flat membrane type culture medium regeneration module.

[0218] The culture medium regeneration module 170 has a culture medium inlet 172 and a culture medium outlet 174. The culture medium inlet 172 is an opening for introducing culture medium flowing from the suction nozzle 130-1 into the culture medium regeneration module 170. The culture medium outlet 174 is an opening for discharging the culture medium introduced into the culture medium regeneration module 170.

[0219] The culture medium regeneration module 170 has a culture medium supply port 176 and a culture medium discharge port 178. The culture medium supply port 176 is an opening for supplying culture medium from the culture medium tank 180 to the culture medium regeneration module 170. The culture medium discharge port 178 is an opening for discharging the culture medium supplied to the culture medium regeneration module 170 from the culture medium supply port 176.

[0220] <160d tube>

[0221] The first end 162d of tube 160d is connected to the regeneration medium outlet 178 of the medium regeneration module 170. The second end 164d of tube 160d is connected to the first end 152b of pump 150b.

[0222] <pipe 160e>

[0223] The first end 162e of tube 160e is connected to the second end 154b of pump 150b. The second end 164e of tube 160e is fixed in a predetermined position inside the regeneration culture medium tank 180.

[0224] <Removal of metabolic waste>

[0225] Using tubes 160d and 160e, metabolic products (such as lactic acid, ammonia, glutamic acid, isovaleric acid, butyric acid, and citric acid, which are metabolic waste products) generated by cell aggregates are discharged from the culture medium regeneration module 170 to the regeneration culture medium tank 180. This removes metabolic products from the culture medium circulation loop. Pump 150b allows for the adjustment of the flow rate discharged from the culture medium regeneration module 170 to the regeneration culture medium tank 180.

[0226] <pipe 160f>

[0227] The first end 162f of tube 160f is fixed in a predetermined position inside the regeneration culture medium tank 180. The second end 164f of tube 160f is connected to the first end 152c of pump 150c.

[0228] <160g tube>

[0229] The first end 162g of tube 160g is connected to the second end 154c of pump 150c. The second end 164g of tube 160g is connected to the regeneration culture medium supply port 176 of culture medium regeneration module 170.

[0230] <Nutritional Supply>

[0231] Nutrients for cell aggregation are supplied from the regeneration culture tank 180 to the culture medium regeneration module 170 via tubes 160f and 160g. This allows for the supply of nutrients to the culture medium circulation loop. The flow rate supplied from the regeneration culture tank 180 to the culture medium regeneration module 170 can be adjusted using pump 150c.

[0232] <Operation of Culture Medium Regeneration Module 170>

[0233] Nutrients for cell aggregates are supplied from the regeneration culture medium tank 180 to the regeneration culture medium supply port 176 of the culture medium regeneration module 170 via tubes 160f and 160g. The supplied nutrients move into the culture medium circulation loop through the culture medium regeneration membrane 179. Thus, nutrients are supplied to the culture medium circulation loop.

[0234] On the other hand, metabolic products generated by cell aggregates (such as lactic acid, ammonia, glutamic acid, isovaleric acid, butyric acid, citric acid, etc., which are metabolic wastes) pass through the culture medium regeneration membrane 179 from the culture medium circulation loop and are discharged from the regeneration culture medium outlet 178 of the culture medium regeneration module 170 via pipes 160d and 160e to the regeneration culture medium tank 180. Thus, metabolic products are removed from the culture medium circulation loop.

[0235] In this way, the culture medium regeneration module 170 regenerates the culture medium by bringing the culture medium supplied from the culture medium supply port 176 into contact with the culture medium introduced from the culture medium inlet port 172 via the culture medium regeneration membrane 179. The regenerated culture medium is discharged from the culture medium outlet port 174 and guided to the culture container 110-1 via the tube 160c. The culture medium that has come into contact with the culture medium via the culture medium regeneration membrane 179 is discharged from the culture medium discharge port 178 and returned to the culture medium tank 180.

[0236] The culture medium regeneration module 170 removes metabolic waste and other substances discharged during the growth of cell aggregates in the culture container 110-1 by filtration, and supplies the culture medium with the components required for the growth of cell aggregates (such as glucose, amino acids, vitamins, inorganic salts, etc.).

[0237] <Regeneration Culture Medium Box 180>

[0238] Regeneration medium is stored in regeneration culture tank 180. This regeneration medium fully contains glucose, amino acids, vitamins, and inorganic salts. For example, regeneration media include the aforementioned basal media 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, etc. Furthermore, amino acids, vitamins, inorganic salts, proteins (growth factors), glucose-containing sugars, antibiotics, signal transduction inhibitors, reducing agents, and buffers can also be added as additives.

[0239] <Formation of Non-Cyclic Pathways>

[0240] In the aforementioned example, a structure is shown that uses tubes 160d and 160e to return metabolic products discharged from the culture medium regeneration module 170 to the regeneration culture medium tank 180. That is, a circulation path is formed by tubes 160d, 160e, 160f, and 160g. Because metabolic products are returned to the regeneration culture medium tank 180, the concentration of nutrients in the regeneration culture medium tank 180 gradually changes with the increase of metabolic products. Therefore, it is conceivable that an appropriate amount of nutrients cannot be supplied to the culture vessel 110-1.

[0241] From this perspective, a non-cyclic approach can also be adopted. For example, metabolic products discharged from the culture medium regeneration module 170 can be returned to a storage tank (not shown) different from the regeneration culture medium tank 180. In this way, the concentration of nutrients in the regeneration culture medium tank 180 can be maintained, and the appropriate amount of nutrients can always be supplied to the culture vessel 110-1.

[0242] Furthermore, nutrients can be appropriately supplied from the nutrient storage tank to the regeneration culture medium tank 180 by setting up a nutrient storage tank containing nutrients and a supply pump (not shown) and driving the supply pump. The concentration of nutrients in the regeneration culture medium tank 180 can be maintained at the desired concentration, and an appropriate amount of nutrients can always be supplied to the culture container 110-1.

[0243] <Capacity and quantity of regeneration culture medium tank 180>

[0244] The capacity of the regeneration culture medium tank 180 can be set to a desired size. By increasing the capacity of the regeneration culture medium tank 180, the concentration of the regeneration culture medium stored in the regeneration culture medium tank 180 can be less affected by the regeneration culture medium returned from the culture medium regeneration module 170. The capacity of the regeneration culture medium tank 180 can be appropriately determined based on factors such as the flow rate of the regeneration culture medium returned from the culture medium regeneration module 170 per unit time.

[0245] Furthermore, the number of regeneration culture medium tanks 180 is not limited to one. Multiple regeneration culture medium tanks 180 can also be used.

[0246] For example, it is also possible to supply regeneration culture medium from the first regeneration culture medium tank 180a to the culture medium regeneration module 170, and return the regeneration culture medium from the culture medium regeneration module 170 to a second regeneration culture medium tank 180b that is different from the first regeneration culture medium tank 180a, etc.

[0247] Alternatively, a tank for storing the prepared regeneration culture medium can be provided to maintain the state of the regeneration culture medium tank 180. When using multiple regeneration culture medium tanks 180, the flow can be controlled by connecting them with interconnecting pipes or by using pumps, valves, etc. When multiple regeneration culture medium tanks 180 are connected, at least one culture medium regeneration module other than the culture medium regeneration module 170 can be provided between the regeneration culture medium tanks 180. This is as long as the concentration of the regeneration culture medium supplied up to the culture medium regeneration module 170 remains relatively stable.

[0248] <Removal based on adsorbent>

[0249] In the aforementioned example, a structure is shown in which metabolic products discharged from the culture medium regeneration module 170 are returned to the regeneration culture medium tank 180 using tubes 160d and 160e. That is, a circulation path is formed by tubes 160d, 160e, 160f, and 160g. With this structure, metabolic products flow into the regeneration culture medium tank 180 along with the regeneration culture medium, thus leading to a gradual accumulation of metabolic waste in the regeneration culture medium tank 180 as the amount of metabolic products increases. Therefore, it is anticipated that the concentration difference of metabolic waste components between the culture vessel 110-1 and the regeneration culture medium tank 180 will decrease, resulting in a reduction in solute removal capacity.

[0250] From this perspective, adsorbents that selectively remove metabolic products (such as lactic acid, ammonia, glutamic acid, isovaleric acid, butyric acid, citric acid, etc.) generated by cell aggregates can also be used.

[0251] <Solution 1 based on adsorbent>

[0252] like Figure 1 As shown, an adsorbent column 190, filled with adsorbent 192 for adsorbing metabolic products as metabolic waste, can be positioned midway through the tube 160g. This allows the metabolic products contained in the regenerated culture medium to be adsorbed and removed during the flow of the regenerated culture medium within the adsorbent column 190, as it flows from the regenerated culture medium tank 180 to the culture medium regeneration module 170. In this configuration, the regenerated culture medium, now free of metabolic products, can be exported to the culture medium regeneration module 170.

[0253] The adsorbent 192, which is filled in a single adsorbent column 190, can also be mixed with multiple types of adsorbents for adsorbing substances such as lactic acid and ammonia.

[0254] exist Figure 1 The example shown illustrates a structure in which the adsorbent column 190 is positioned midway through tube 160g, but the adsorbent column 190 can also be positioned midway through tubes 160d, 160e, and 160f. The adsorbent column 190 can be positioned at any location in at least one of tubes 160d, 160e, 160f, and 160g.

[0255] Moreover, in Figure 1 The example shown illustrates a single adsorbent column 190, but multiple adsorbent columns 190 can also be connected in series or in parallel with at least one of tubes 160d, 160e, 160f, and 160g.

[0256] Alternatively, an adsorbent column can be provided in a manner that allows communication between the outlet pipe 160d or 160e from the culture medium regeneration module 170 to the regeneration culture medium tank 180 and the nutrient supply pipe 160f or 160g from the regeneration culture medium tank 180 to the culture medium regeneration module 170. This newly formed flow path of the adsorbent column, parallel to the flow path between the culture medium regeneration module 170 and the regeneration module 170, allows for the adsorption of metabolic products contained in the regeneration culture medium while achieving flow balance with the culture medium regeneration module 170.

[0257] In this case, a separate solenoid valve can be installed to switch the connection state. Furthermore, a separate pump can be installed to adjust the flow rate of the regeneration medium. Additionally, a concentration sensor can be installed. This sensor can detect the concentration of metabolites contained in the regeneration medium and control the solenoid valve and pump based on the detection results.

[0258] Furthermore, instead of tubes 160d, 160e, 160f, and 160g, the metabolic products can be adsorbed in the regeneration culture medium tank 180. Specifically, the regeneration culture medium tank 180 can also be configured to contain an adsorbent.

[0259] By placing the adsorbent into the regeneration culture medium tank 180, the metabolic products accumulated in the regeneration culture medium tank 180 are adsorbed, and the concentration of metabolic waste in the regeneration culture medium tank 180 can be maintained at a low level. As a result, the concentration difference of metabolic waste components between the culture container 110-1 and the regeneration culture medium tank 180 can be maintained, and the solute removal capacity can be fully utilized.

[0260] <Solution 2 based on adsorbent>

[0261] For example, by maintaining the adsorbent along the wall of the regeneration culture medium tank 180, metabolites close to the wall of the regeneration culture medium tank 180 can be adsorbed.

[0262] <Solution 3 based on adsorbent>

[0263] Alternatively, a regeneration culture medium tank 180 can be used, which is divided into an upper region and a lower region by a septum (not shown) such as a porous membrane. The regeneration culture medium and metabolites are stored in the upper region. In the lower region, the regeneration culture medium and metabolites are stored across the septum, and an adsorbent is also contained. The regeneration culture medium and metabolites can move between the upper and lower regions through the septum. In contrast, the adsorbent cannot pass through the septum and remains only in the lower region. With this configuration, the metabolites are adsorbed onto the adsorbent as they move from the upper region to the lower region through the septum.

[0264] <Solution 4 based on adsorbent>

[0265] Furthermore, a structure can be adopted in which the particulate adsorbent is dispersed, settled, or floated in the regeneration culture medium within the regeneration culture tank 180. With this configuration, metabolites can be adsorbed onto the adsorbent within the regeneration culture tank 180.

[0266] Furthermore, in the case of a circuit that constitutes multiple regeneration culture medium tanks, an adsorbent can be added to each regeneration culture medium tank.

[0267] Furthermore, in addition to placing the adsorbent in the regeneration culture medium tank, it can also be combined with the method of adding new regeneration culture medium from the tank where the prepared regeneration culture medium is stored.

[0268] It should be noted that pumps 150b and 150c can also be activated by monitoring glucose concentration, lactate concentration, and pH. For example, pumps 150b and 150c can be activated based on the flow rate of pump 150a, glucose concentration, lactate concentration, and pH. In this way, glucose, amino acids, vitamins, inorganic salts, etc., can be added to the culture medium according to the flow rate of the culture medium.

[0269] <<<Motion of the cell aggregate within the suction nozzle 130-1>>>

[0270] Figure 2 A is a cross-sectional view showing the outline of the action of the cell aggregate within the suction nozzle 130-1. Figure 2 In A, multiple white ring-shaped regions indicate cell aggregates.

[0271] <Upper busbar UG and lower busbar BG>

[0272] The suction 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 generatrices along its length. A generatrice refers to a straight line at each position when a curved surface (tubular (cylindrical) surface) is drawn (formed) by moving a straight line. By setting the suction nozzle 130-1 to an inclined state, it is possible to draw the uppermost generatrice UG located at the uppermost side of the suction nozzle 130-1 and the lowermost generatrice BG located at the lowermost side of the suction nozzle 130-1 in its cross-section. It should be noted that the cross-section of the suction nozzle 130-1 refers to the surface formed by cutting the suction nozzle 130-1 vertically relative to its central axis AO (see reference). Figure 2 B).

[0273] <Status of plumb>

[0274] With the suction nozzle 130-1 in a vertical position, the culture medium rises in the region between multiple cell aggregates that settle within the suction nozzle 130-1. With the suction nozzle 130-1 in a vertical position, after being drawn in from the suction opening 134-1, the culture medium is drawn upwards in the vertical direction within the suction nozzle 130-1. This is the opposite direction to gravity, and the direction of movement of the cell aggregates is determined by the relationship between the magnitude of the suction force and gravity. That is, the direction of movement of the cell aggregates can be either upwards or downwards in the vertical direction.

[0275] <Situation of tilting>

[0276] On the other hand, such as Figure 1 as well as Figure 2As shown, by tilting the suction nozzle 130-1, the cell aggregates to be settled are more easily moved and concentrated towards the area near the lowermost generatrix BG due to gravity, compared to the area near the uppermost generatrix UG. As the concentrated cell aggregates move towards the suction opening 134-1, the area near the lowermost generatrix BG is further more easily occupied by cell aggregates. This creates an uneven distribution of cell aggregates along the cross-section of the suction nozzle 130-1 (see reference). Figure 2 The culture medium flows from the region near the lowermost generatrix BG, which is occupied by cell aggregates, to the region near the uppermost generatrix UG, which is not occupied by cell aggregates. By tilting the suction nozzle 130-1, the movement of the cell aggregates and the movement of the culture medium create two main flow regions around region SR: a first flow region FF and a second flow region SF. It should be noted that... Figure 2 In the cross-sectional view shown in B, the approximately circular dashed line is an imaginary line representing the boundary BD between the first flow region FF and the second flow region SF. Figure 2 In the cross-sectional view shown in Figure A, the boundary BD is indicated by a straight line, which is a dashed line arranged along the length of the suction nozzle 130-1 between the uppermost generatrix UG and the lowermost generatrix BG. 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.

[0277] <First Flow Region FF>

[0278] The first flow zone FF is the area where cell aggregates are concentrated and flow (sediment) together with the culture medium toward the aspiration opening 134-1. In other words, the first flow zone FF is the region where cell aggregates settle together with the culture medium. Particularly within the first flow zone FF, cell aggregates settle readily near the lowermost generatrix (BG). Cell aggregates settle rapidly near the lowermost generatrix (BG) and then slowly as they move away from it.

[0279] It should be noted that the culture medium and cell aggregates flow in sequentially from the aspiration opening 134-1. Therefore, the cell aggregates that settle near the aspiration opening 134-1 include those that flow out of the aspiration opening 134-1 and those that move towards the second flow zone SF and rise again. Whether they flow out of the aspiration opening 134-1 or rise again depends on the amount and size of the cell aggregates, the flow of the culture medium near the aspiration opening 134-1, and other factors.

[0280] <Second Flow Area SF>

[0281] The second flow zone SF is the region where the culture medium rises towards the discharge opening 136-1, and the cell aggregates also flow towards the discharge opening 136-1. That is, the second flow zone SF is the region where cell aggregates rise. Specifically, in the second flow zone SF, cell aggregates tend to rise more easily in the region near the uppermost generatrix UG. In other words, cell aggregates rise rapidly near the uppermost generatrix UG and rise slowly as they move away from it.

[0282] <Top Position Up>

[0283] The cell aggregates rising toward the discharge opening 136-1, after reaching a predetermined position below the discharge opening 136-1, such as the position midway between the aspiration opening 134-1 and the discharge opening 136-1 (the uppermost position UP), move toward the first flow zone FF and settle again. By forming the uppermost position UP, the cell aggregates are prevented from being discharged from the discharge opening 136-1, and only the culture medium is discharged from the discharge opening 136-1, thus enabling the culture medium to circulate.

[0284] It should be noted that the highest position UP where the cell aggregate can rise to its maximum extent within the suction 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 should be any position where the likelihood of the cell aggregate being discharged from the discharge opening 136-1 is sufficiently low. The highest position UP where the cell aggregate can rise within the suction nozzle 130-1 can be determined based on the tilt angle θ of the suction nozzle 130-1 and the circulating flow rate generated by the pump 150a.

[0285] <<Circulating Cell Aggregates>>

[0286] Through the formation of the first flow zone FF, the cell aggregates settle towards the suction opening 134-1 and then move towards the second flow zone SF. Through the formation of the second flow zone SF, the cell aggregates that have moved to the second flow zone SF rise towards the discharge opening 136-1 to the uppermost position UP, and then move back towards the first flow zone FF. In this way, cell aggregates of a specified size and above flow alternately in the first flow zone FF and the second flow zone SF, circulating within the suction nozzle 130-1. By circulating the cell aggregates within the suction nozzle 130-1, the cell aggregates can be retained within the suction nozzle 130-1, allowing only the culture medium to be discharged from the discharge opening 136-1 of the suction nozzle 130-1.

[0287] <Distribution of cell aggregates within suction nozzle 130-1>

[0288] Figure 2B is shown Figure 2 A cross-sectional view showing the distribution of cell aggregates within the suction nozzle 130-1 in section II, as shown in section A. Figure 2 In B, multiple white ring-shaped regions also indicate cell aggregates.

[0289] As mentioned above, the region near the lowest busbar BG is occupied by the settled cell aggregates. As a result, the cross-section of the first flow region FF (the area of ​​the suction nozzle 130-1 along the direction perpendicular to the length direction) is enlarged by the settled cell aggregates.

[0290] On the other hand, because the area near the lowermost generatrix BG is occupied by cell aggregates, the area where the culture medium can flow becomes smaller in the area near the uppermost generatrix UG. That is, the cross-section of the second flow region SF (the area of ​​the suction nozzle 130-1 along the direction perpendicular to the length direction) is reduced due to the cell aggregates in the first flow region FF.

[0291] The circulation flow rate of the culture medium discharged from the discharge opening 136-1 of the suction nozzle 130-1 is determined by the drive of the pump 150a. In order to maintain this circulation flow rate, the flow rate of the culture medium flowing in the second flow zone SF, where the cross-section becomes smaller, is increased.

[0292] By forming the uppermost position UP, the cell aggregate that has moved to the second flow zone SF rises towards the discharge opening 136-1 to the uppermost position UP, and then moves again towards the first flow zone FF. In this way, the cell aggregate remains in the suction nozzle 130-1, and only the culture medium is discharged from the discharge opening 136-1, thus increasing the circulation flow rate of the culture medium.

[0293] It should be noted that by increasing the circulation flow rate of the culture medium, the movement speed of the cell aggregates within the suction nozzle 130-1 also increases. Therefore, the flow rate of the cell suspension rising in the second flow zone also accelerates.

[0294] <<<Relationship between the inclination of suction nozzle 130-1 and the circulation flow rate>>>

[0295] Figure 3 This is a graph showing the relationship between the tilt angle of the suction nozzle 130-1 and the circulation flow rate when the cell aggregate rises to the midpoint between the suction opening 134-1 and the discharge opening 136-1. The midpoint between the suction opening 134-1 and the discharge opening 136-1 is an example of the highest position UP.

[0296] Figure 3The circulation flow rates shown are F1 < F2 < F3 < F4. That is, when the tilt angle θ is increased, the circulation flow rate needs to be increased in order for the cell aggregate to rise to the middle position. In other words, as the tilt angle θ increases, it becomes difficult for the cell aggregate to be discharged from the suction opening 134-1 of the suction nozzle 130-1, thus increasing the circulation flow rate of the culture medium. The circulation flow rate of the culture medium can be increased until the cell aggregate reaches the middle position, preventing the cell aggregate from being discharged from the suction opening 134-1 of the suction nozzle 130-1. In this way, by increasing the tilt angle θ, the circulation flow rate of the culture medium can be increased. By increasing the circulation flow rate of the culture medium, the amount of contact between the culture medium and the culture medium regeneration membrane 179 of the culture medium regeneration module 170 can be increased, thereby improving the ability to remove solutes.

[0297] Furthermore, even with increased culture medium circulation flow, the cell aggregates are not discharged from the discharge opening 136-1 of the suction nozzle 130-1 into the culture medium circulation loop (tubes 160a, 160b, 160c, etc.), thus preventing damage to the cell aggregates. In this way, by increasing the culture medium circulation flow by increasing the tilt angle θ, the cell aggregates can grow efficiently.

[0298] The circulation flow rate of the culture medium can be increased without leading the cell aggregates into the culture medium circulation loop. Therefore, 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, thus improving solute removal performance. Through the exchange of substances via the culture medium regeneration module 170, essential components such as growth factors can be retained on the culture side of the cell aggregates while metabolic waste such as lactic acid can be removed. Therefore, compared with conventional culture medium replacement, the amount of culture medium used can be reduced, thereby reducing costs.

[0299] <<<Second Implementation>>>>

[0300] Figure 4 This is a schematic diagram showing the structure of the culture medium regeneration system 200 according to the second embodiment. Figure 4 In this drawing, structures identical to those in the first embodiment are shown using the same reference numerals. Figure 4 In this embodiment, pumps 150a, 150b, 150c, pipes 160a, 160b, 160c, 160d, 160e, 160f, 160g, culture medium regeneration module 170, and culture medium regeneration tank 180 are the same as in the first embodiment, but are omitted. Figure 4 As shown, the culture container 110-1 of the culture medium regeneration system 200 differs from that of the culture medium regeneration system 100 and is fixedly configured in a vertical position. Hereinafter, the differences from the culture medium regeneration system 100 will be mainly explained.

[0301] <Cultivation Container 110-1>

[0302] The structure and material of culture container 110-1 are basically the same as those of culture medium regeneration system 100. However, the configuration of culture container 110-1 differs from that of culture medium regeneration system 100. The central axis CO of culture container 110-1 is vertical, unlike the configuration of culture container 110-1 in culture medium regeneration system 100. Therefore, the bottom 114 of culture container 110-1 extends horizontally.

[0303] <Stirring device 120>

[0304] The structure of the stirring device 120 is basically the same as that of the culture medium regeneration system 100. However, the configuration of the stirring device 120 differs from that of the culture medium regeneration system 100. Specifically, the rotation axis RO of the stirring element 124 in the stirring device 120 is vertical, unlike the configuration of the stirring element 124 in the culture medium regeneration system 100. That is, both the central axis CO of the culture vessel 110-1 and the rotation axis RO of the stirring element 124 are vertical, unlike the culture medium regeneration system 100.

[0305] <Suction nozzle 130-2>

[0306] The structure and material of the suction nozzle 130-2 are basically the same as those of the culture medium regeneration system 100.

[0307] Like suction nozzle 130-1, suction nozzle 130-2 has the following characteristics:

[0308] Extension 132-1;

[0309] Inhalation opening 134-1; and

[0310] Discharge opening 136-1.

[0311] The extension 132-1 also has the same structure as the suction nozzle 130-1. The extension 132-1 extends in an inclined direction. The extension 132-1 is inclined at an angle θ relative to the vertical direction. Therefore, the suction nozzle 130-2 extends in an inclined direction. In other words, the central axis AO of the suction nozzle 130-2 extends in an inclined direction.

[0312] In the first embodiment, the central axis CO of the culture vessel 110-1, the rotation axis RO of the stirring blade 124a, and the central axis AO of the suction nozzle 130-1 are arranged along an 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 stirring blade 124a extend in a vertical direction, while only the suction nozzle 130-2 extends in an inclined direction. The suction nozzle 130-2 is fixedly provided using a retaining member (not shown) to extend in a constant inclined direction.

[0313] The suction nozzle 130-2 is configured such that the suction opening 134-1 faces the side wall 112 of the culture container 110-1. By tilting the suction nozzle 130-2 so that the suction opening 134-1 faces the side wall 112 of the culture container 110-1, the suction nozzle 130-2 is less likely to interfere with the stirring blades 124a of the stirrer 124, and the culture medium and cell aggregates can be thoroughly stirred. Moreover, by tilting the suction nozzle 130-2, the degree of freedom in the size and shape of the stirring blades 124a of the stirrer 124 can be increased.

[0314] The culture medium and cell aggregates flow into the suction nozzle 130-2 in the same manner as in the suction 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 nozzle 130-2. The cell aggregates remain inside the suction nozzle 130-2, while the culture medium is discharged from the discharge opening 136-1 of the suction nozzle 130-2.

[0315] In this way, by using the inclined suction nozzle 130-2, the circulation flow rate of the culture medium can be increased without leading the cell aggregates into the culture medium circulation loop. Therefore, the amount of culture medium that can come into contact with the culture medium regeneration membrane can be increased without damaging the cell aggregates, thus improving solute removal performance. Through the material exchange via the culture medium regeneration module 170, essential components such as growth factors can be retained on the culture side of the cell aggregates while lactic acid, a metabolic waste product, can be removed. Therefore, compared with conventional culture medium replacement, the amount of culture medium used can be reduced, thereby reducing costs.

[0316] <<<<Third Implementation Method>>>>

[0317] Figure 5 This is a schematic diagram showing the structure of the culture medium regeneration system 300 according to the third embodiment. Figure 5 In this drawing, structures identical to those in the first and second embodiments are shown using the same reference numerals. Figure 5 In this embodiment, pumps 150a, 150b, 150c, pipes 160a, 160b, 160c, 160d, 160e, 160f, 160g, culture medium regeneration module 170, and culture medium regeneration tank 180 are the same as in the first embodiment and are omitted. Figure 5 As shown, the culture container 110-3 of the culture medium regeneration system 300 is fixedly configured in a vertical position, similar to that in the second embodiment. Hereinafter, the differences from the culture medium regeneration system 100 and the culture medium regeneration system 200 will be mainly described.

[0318] <Cultivation Container 110-3>

[0319] The structure and material of the culture container 110-3 are basically the same as those of the culture medium regeneration system 100. It should be noted that in both the culture medium regeneration systems 100 and 200, the culture container 110-1 and the suction nozzles 130-1 and 130-2 are separately constructed. Therefore, the suction nozzles 130-1 and 130-2 can be adjusted in position and orientation relative to the culture container 110-1. In contrast, the culture container 110-3 and the suction nozzles 130-3 are integrally formed.

[0320] Suction nozzle 130-3 has:

[0321] Extension 132-3;

[0322] Inhalation opening 134-3; and

[0323] Discharge opening 136-3.

[0324] A through hole is formed near the bottom 114 of the culture container 110-3. The suction opening 134-3 of the suction nozzle 130-3 is fixedly disposed in communication with the through hole formed near the bottom 114 of the culture container 110-3. The suction nozzle 130-3 is inclined at an angle θ relative to the central axis CO of the culture container 110-3 and extends from the culture container 110-3 in a manner that tends upwards. The central axis AO of the suction nozzle 130-3 extends in an inclined direction.

[0325] By integrally forming the suction nozzle 130-3 with the culture container 110-3, a holding member for retaining the suction nozzle 130-3 is eliminated. Furthermore, the tilt angle θ of the suction nozzle 130-3 can be kept constant, thus eliminating the need for tilt angle θ adjustment. Moreover, since the suction nozzle 130-3 is positioned on the outside of the culture container 110-3, it will not interfere with the stirring blades 124a of the stirrer 124. This structure allows for thorough mixing of the culture medium and cell aggregates, or increases the freedom of choice in the size and shape of the stirring blades 124a of the stirrer 124.

[0326] exist Figure 5 In the example shown, the suction opening 134-3 of the suction nozzle 130-3 is formed near the bottom 114 of the culture container 110-3, but the position of the suction opening 134-3 of the suction nozzle 130-3 is not limited thereto. The position of the suction opening 134-3 can be appropriately determined according to the type and amount of culture medium, the type of cell aggregates, their distribution within the culture container 110-3, the rotation speed of the stirrer 124, etc. The suction nozzle 130-3 can be integrally formed with the culture container 110-3.

[0327] The culture medium and cell aggregates flow in the suction nozzle 130-3 in the same manner as in the suction 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 nozzle 130-3. The cell aggregates remain within the suction nozzle 130-3, while the culture medium is discharged from the discharge opening 136-3 of the suction nozzle 130-3.

[0328] In this way, by using the inclined suction nozzle 130-3, the circulation flow rate of the culture medium can be increased without leading the cell aggregates into the culture medium circulation loop. Therefore, the amount of culture medium that can come into contact with the culture medium regeneration membrane can be increased without damaging the cell aggregates, thus improving solute removal performance. Through the material exchange via the culture medium regeneration module 170, essential components such as growth factors can be retained on the culture side of the cell aggregates while lactic acid, a metabolic waste product, can be removed. Therefore, compared with conventional culture medium replacement, the amount of culture medium used can be reduced, thereby reducing costs.

[0329] <Other Structures of Culture Container 110-3>

[0330] exist Figure 5 In the example shown, the suction nozzle 130-3 is integrally formed with the culture container 110-3. The structure of the culture container 110-3 is not limited to this. The suction nozzle 130-3 may also be configured to be detachable from the culture container 110-3.

[0331] For example, a generally cylindrical protrusion (not shown) for mounting a suction nozzle 130-3 is provided on the side of the culture container 110-3. By inserting the end of the separately formed suction nozzle 130-3 into the protrusion for assembly, the suction nozzle 130-3 can be detachably mounted to the culture container 110-3. By separately constructing the suction nozzle 130-3 and the culture container 110-3, the materials of the suction nozzle 130-3 and the culture container 110-3 can be different, or a suction nozzle 130-3 with a desired length and shape can be used.

[0332] Furthermore, the position and angle of the through hole and protrusion used to install the suction nozzle 130-3 can be customized to the desired position and angle. This increases the flexibility in selecting the suction nozzle 130-3 and the culture container 110-3.

[0333] <<<<Fourth Implementation Method>>>>

[0334] Figure 6 This is a schematic diagram showing the structure of the culture medium regeneration system 400 according to the fourth embodiment. Figure 6 In this drawing, structures identical to those in the first embodiment are shown using the same reference numerals. Figure 6 In this embodiment, pumps 150a, 150b, 150c, pipes 160a, 160b, 160c, 160d, 160e, 160f, 160g, culture medium regeneration module 170, and culture medium regeneration tank 180 are the same as in the first embodiment and are omitted. Figure 6 As shown, the culture container 110-1 of the culture medium regeneration system 400 differs from that of the culture medium regeneration system 100; it is not tilted but fixed in a vertical position. The following mainly describes the differences from the culture medium regeneration system 100.

[0335] <Cultivation Container 110-1>

[0336] The structure and material of culture container 110-1 are basically the same as those of culture medium regeneration system 100. However, the configuration of culture container 110-1 differs from that of culture medium regeneration system 100. The central axis CO of culture container 110-1 is vertical, unlike the configuration of culture container 110-1 in culture medium regeneration system 100. Therefore, the bottom 114 of culture container 110-1 extends horizontally.

[0337] <Suction nozzle 130-4>

[0338] The culture medium regeneration system 400 differs from the culture medium regeneration system 100 in that it has a suction nozzle 130-4.

[0339] Suction nozzle 130-4 has:

[0340] Inclined part 130-2a;

[0341] Vertical part 130-2b; and

[0342] Bending portion 130-2c. The suction nozzle 130-4 is elongated in shape as a whole by means of the inclined portion 130-2a, the vertical portion 130-2b, and the bending portion 130-2c. It should be noted that the cross-sectional area of ​​the suction 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 vertically in the direction relative to the length direction of the inclined portion 130-2a and the vertical portion 130-2b.

[0343] <Inclined section 130-2a>

[0344] The inclined portion 130-2a is inclined at an angle θ relative to the vertical direction. The inclined portion 130-2a extends along the inclined direction. The inclined portion 130-2a has an elongated shape. The inclined portion 130-2a extends in a straight line along its length. The inclined portion 130-2a has a tubular shape. The inclined portion 130-2a has a suction opening 134-1.

[0345] The suction nozzle 130-4 is configured such that the suction opening 134-1 of the inclined portion 130-2a faces the side wall 112 of the culture container 110-1. That is, the inclined portion 130-2a is configured such that its central axis AO moves away from the central axis CO of the culture container 110-1 as it tends downwards. In other words, the direction in which the inclined portion 130-2a extends is the inclined direction, and the direction in which the extension 124b of the stirrer 124 extends is the vertical direction, forming an inclined angle θ. By tilting the inclined portion 130-2a so that the suction opening 134-1 faces the side wall 112 of the culture container 110-1, the inclined portion 130-2a is less likely to interfere with the stirring blades 124a of the stirrer 124, allowing for thorough stirring of the culture medium and cell aggregates. Furthermore, by tilting the inclined portion 130-2a, the degree of freedom in the size and shape of the stirring blades 124a of the stirrer 124 can be increased.

[0346] <Vertical section 130-2b>

[0347] The vertical portion 130-2b extends in the vertical direction. The vertical portion 130-2b has an elongated shape. The vertical portion 130-2b extends in a straight line along its length. The vertical portion 130-2b has a tubular shape. The vertical portion 130-2b has a discharge opening 136-1.

[0348] <Bending section 130-2c>

[0349] The inclined portion 130-2a and the vertical portion 130-2b are connected via a bent portion 130-2c. The bent portion 130-2c has a short tubular shape. Through the bent portion 130-2c, the inclined portion 130-2a extending in the inclined direction and the vertical portion 130-2b extending in the vertical direction are connected at an angle θ. The inclined portion 130-2a and the vertical portion 130-2b are connected via the bent portion 130-2c. Alternatively, instead of the bent portion 130-2c, the inclined portion 130-2a extending in the inclined direction and the vertical portion 130-2b extending in the vertical direction can be connected by a bent member.

[0350] <Inclined section 130-2a, Vertical section 130-2b, Bending section 130-2c>

[0351] The inclined portion 130-2a, vertical portion 130-2b, and bent portion 130-2c, like the extension 132-1 of the suction nozzle 130-1 of the culture medium regeneration system 100, have an outer peripheral portion and an inner peripheral portion (not shown). The outer peripheral portion forms the outer surface of the inclined portion 130-2a, vertical portion 130-2b, and bent portion 130-2c. The inner peripheral portion forms the inner surface of the inclined portion 130-2a, vertical portion 130-2b, and bent portion 130-2c. The outer peripheral portion and the inner peripheral portion are arranged concentrically. The long strip surrounding region SR, which is surrounded by the inner peripheral portion and extends along its length, functions as a hollow conduit. Culture medium and cell aggregates can flow in the surrounding region SR.

[0352] The culture medium and cell aggregates flow in the inclined section 130-2a in the same manner as the suction nozzle 130-1 in the first embodiment. Two flow regions, a first flow region FF and a second flow region SF, are formed in the inclined section 130-2a. The cell aggregates remain within the inclined section 130-2a, while the culture medium is discharged through the inclined section 130-2a from the discharge opening 136-1.

[0353] In this way, by using the inclined suction nozzle 130-4, the circulation flow rate of the culture medium can be increased without leading the cell aggregates into the culture medium circulation loop. Therefore, the amount of culture medium that can contact the culture medium regeneration membrane can be increased without damaging the cell aggregates, thus improving solute removal performance. Through the material exchange via the culture medium regeneration module 170, essential components such as growth factors can be retained on the culture side of the cell aggregates while metabolic waste such as lactic acid can be removed. Therefore, compared with conventional culture medium replacement, the amount of culture medium used can be reduced, thereby reducing costs.

[0354] <Other shapes of the suction opening 134-1>

[0355] Only one example is shown where the inclined portion 130-2a is inclined in a straight line with the suction opening 134-1 facing the side wall portion 112 of the culture container 110-1, but it is not limited to this. By making the suction nozzle 130-4 spirally bent or appropriately changing the shape of the suction nozzle 130-4 according to the size and shape of the stirring blade 124a of the stirring member 124, interference with the stirring blade 124a can be avoided.

[0356] <<<<Fifth Implementation Method>>>>

[0357] Figure 7 This is a schematic diagram showing the structure of the culture medium regeneration system 500 according to the fifth embodiment. Figure 7 In this drawing, structures identical to those in the first embodiment are shown using the same reference numerals. Figure 7In this embodiment, pumps 150a, 150b, 150c, tubes 160a, 160b, 160c, 160d, 160e, 160f, 160g, culture medium regeneration module 170, and culture medium box 180 for regeneration are the same as in the first embodiment and are omitted.

[0358] like Figure 7 As shown, the culture medium regeneration system 500, like the culture medium regeneration system 100, is fixedly configured in a tilted state with a constant tilt angle. The following mainly describes the differences between the 500 and the 100 culture medium regeneration systems.

[0359] The culture medium regeneration system 500 has a suction nozzle 130-5. The suction nozzle 130-5 is made of glass, resin, stainless steel, etc., and has a constant shape.

[0360] Suction nozzle 130-5 has:

[0361] Suction nozzle 130-1; and

[0362] Enlarged diameter portion 470. In other words, the suction nozzle 130-5 of the fifth embodiment has a structure in which the discharge opening 136-1 of the suction nozzle 130-1 of the first embodiment is provided with an enlarged diameter portion 470.

[0363] <Expanded Diameter Section 470>

[0364] The enlarged diameter section 470 extends vertically. The enlarged diameter section 470 has an elongated shape. The enlarged diameter section 470 extends linearly along its length. The enlarged diameter section 470 has a tubular shape. The enlarged diameter section 470 has a discharge opening 472. Furthermore, the lower end of the enlarged diameter section 470 has an opening and communicates with the discharge opening 136-1. It should be noted that... Figure 7 In the example shown, the suction nozzle 130-5 is connected to the enlarged diameter portion 470 by bending at the boundary, thus having a stepped portion. In contrast, it can also be configured to connect the suction nozzle 130-1 and the enlarged diameter portion 470 in a gradually expanding manner (in a tapered shape).

[0365] <Suction nozzle 130-1, enlarged section 470, surrounding area SR>

[0366] Like the extension 132-1 of the suction nozzle 130-1 in the culture medium regeneration system 100, the suction nozzle 130-1 and the expansion section 470 have an outer peripheral portion and an inner peripheral portion (not shown). The outer peripheral portion forms the outer surface of the suction nozzle 130-1 and the expansion section 470. The inner peripheral portion forms the inner surface of the suction nozzle 130-1 and the expansion section 470. The outer peripheral portion and the inner peripheral portion are arranged concentrically. The long strip surrounding region SR, which is surrounded by the inner peripheral portion and extends along the length direction, functions as a hollow conduit. The expansion section 470 communicates with the suction nozzle 130-1. Culture medium and cell aggregates can flow in the surrounding region SR.

[0367] <Flow of culture medium and cell aggregates>

[0368] The culture medium and cell aggregates flow in the same manner as in the suction 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 nozzle 130-1. The cell aggregates remain inside the suction nozzle 130-1, while the culture medium flows out from the discharge opening 136-1 of the suction nozzle 130-1, flows in the expansion section 470, and is discharged from the discharge opening 472 of the expansion section 470.

[0369] The enlarged diameter section 470 has an inner diameter larger than that of the suction nozzle 130-1. That is, the enlarged diameter section 470 has a cross-sectional area larger than that of the suction nozzle 130-1. By increasing the cross-sectional area of ​​the enlarged diameter section 470, the flow velocity within the enlarged diameter section 470 can be reduced. The cross-sectional area of ​​the enlarged diameter section 470 can be determined based on the desired flow velocity within the enlarged diameter section 470. It should be noted that the cross-sectional area of ​​the enlarged diameter section 470 is the area of ​​the portion of the inner diameter when the enlarged diameter section 470 is cut off along the vertical direction (i.e., the horizontal direction) relative to the vertical direction in which the enlarged diameter section 470 extends.

[0370] In this way, by using the inclined suction nozzle 130-1, the circulation flow rate of the culture medium can be increased without leading the cell aggregates into the culture medium circulation loop. Therefore, the amount of culture medium that can come into contact with the culture medium regeneration membrane can be increased without damaging the cell aggregates, thus improving solute removal performance. Through the material exchange via the culture medium regeneration module 170, essential components such as growth factors can be retained on the culture side of the cell aggregates while lactic acid, a metabolic waste product, can be removed. Therefore, compared with conventional culture medium replacement, the amount of culture medium used can be reduced, thereby reducing costs.

[0371] <<<<Sixth Implementation Method>>>>

[0372] Figure 8 This is a schematic diagram showing the structure of the culture medium regeneration system 600 according to the sixth embodiment. Figure 8In this drawing, structures identical to those in the first embodiment are shown using the same reference numerals. Figure 8 In this embodiment, pumps 150a, 150b, 150c, tubes 160a, 160b, 160c, 160d, 160e, 160f, 160g, culture medium regeneration module 170, and culture medium box 180 for regeneration are the same as in the first embodiment and are omitted.

[0373] like Figure 8 As shown, the culture container 110-1 of the culture medium regeneration system 600 is fixedly configured in a vertical position, just like the culture medium regeneration system 200. The following mainly describes the differences between this system and the culture medium regeneration system 100 and 200.

[0374] The culture medium regeneration system 600 has a suction nozzle 130-6. The suction nozzle 130-6 is formed of glass, resin, stainless steel, etc., and has a constant shape.

[0375] Suction nozzle 130-6 has:

[0376] Suction nozzle 130-4; and

[0377] Enlarged diameter portion 470. In other words, the suction nozzle 130-6 of the sixth embodiment has a structure in which the discharge opening 136-1 of the suction nozzle 130-4 of the fourth embodiment is provided with the enlarged diameter portion 470 of the fifth embodiment.

[0378] The suction nozzle 130-4, like in the fourth embodiment, has:

[0379] Inclined part 130-2a;

[0380] Vertical part 130-2b; and

[0381] Bending portion 130-2c. The suction nozzle 130-4 is elongated in shape as a whole by means of the inclined portion 130-2a, the vertical portion 130-2b, and the bending portion 130-2c. It should be noted that the cross-sectional area of ​​the suction 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 vertically in the direction relative to the length direction of the inclined portion 130-2a and the vertical portion 130-2b.

[0382] Similar to the fifth embodiment, the enlarged diameter portion 470 also has a discharge opening 472. Furthermore, the lower end of the enlarged diameter portion 470 has an opening that communicates with the discharge opening 136-1. It should be noted that... Figure 8In the example shown, the suction nozzle 130-6 is connected to the enlarged diameter portion 470 by bending at the boundary through the suction nozzle 130-4, thus having a stepped portion. In contrast, the suction nozzle 130-4 and the enlarged diameter portion 470 can also be connected in a gradually expanding manner (in a tapered shape).

[0383] <Suction nozzle 130-4, enlarged section 470, surrounding area SR>

[0384] Like the extension 132-1 of the suction nozzle 130-1 in the culture medium regeneration system 100, the suction nozzle 130-4 and the expansion section 470 have an outer peripheral portion and an inner peripheral portion (not shown). The outer peripheral portion forms the outer surface of the suction nozzle 130-4 (sloping portion 130-2a, vertical portion 130-2b, and bent portion 130-2c) and the expansion section 470. The inner peripheral portion forms the inner surface of the suction nozzle 130-4 (sloping portion 130-2a, vertical portion 130-2b, and bent portion 130-2c) and the expansion section 470. The outer peripheral portion and the inner peripheral portion are arranged concentrically. The long strip surrounding region SR, which is surrounded by the inner peripheral portion and extends along its length, functions as a hollow conduit. The expansion section 470 communicates with the suction nozzle 130-4. Culture medium and cell aggregates can flow in the surrounding region SR.

[0385] <Flow of culture medium and cell aggregates>

[0386] The culture medium and cell aggregates flow in the same manner as the suction nozzle 130-1 in the first embodiment. Two flow regions, a first flow region FF and a second flow region SF, are formed in the inclined section 130-2a. The cell aggregates remain within the inclined section 130-2a, while the culture medium flows out from the discharge opening 136-1 of the suction nozzle 130-4, flows in the expansion section 470, and is discharged from the discharge opening 472 of the expansion section 470.

[0387] In this way, by using the suction nozzle 130-4 with the inclined section 130-2a, the circulation flow rate of the culture medium can be increased without leading the cell aggregates into the culture medium circulation loop. Therefore, the amount of culture medium that can contact the culture medium regeneration membrane can be increased without damaging the cell aggregates, thus improving solute removal performance. Through the material exchange via the culture medium regeneration module 170, essential components such as growth factors can be retained on the culture side of the cell aggregates while lactic acid, a metabolic waste product, is removed. Therefore, compared to conventional culture medium replacement, the amount of culture medium used can be reduced, thus lowering costs.

[0388] <<<<Seventh Implementation Method>>>>

[0389] The seventh embodiment relates to a culture medium regeneration system that removes metabolic waste (metabolites) while circulating the culture medium. The seventh embodiment primarily demonstrates verification results regarding the relationship between circulation flow rate and the ability to remove metabolic waste (metabolites). The culture medium regeneration system of the seventh embodiment is a system used for verification.

[0390] In the seventh embodiment, a liquid similar to the culture medium used to culture cell aggregates was used, and metabolic waste similar to metabolic waste (metabolites) was used, and the circulation flow rate was confirmed. Specifically, phosphate-buffered saline (PBS) was used as the culture medium, and lithium lactate was used as the metabolic waste. In the seventh embodiment, considering the circulation flow rate of the culture medium and the removal of metabolic waste, substances similar to the culture medium and metabolic waste could be substituted. Hereinafter, without distinction, the substitute for the culture medium will be simply referred to as culture medium, and the substitute for the metabolic waste will be simply referred to as metabolic waste.

[0391] <<<Summary of the Seventh Embodiment>>>

[0392] In cell culture, media replacement is essential for removing metabolic waste and replenishing nutrients. However, media replacement presents several problems. First, even if some nutrients remain, excessive media replacement may be necessary to remove metabolic waste. Second, the volume changes caused by media replacement may prevent sufficient nutrient distribution to cell aggregates. Furthermore, the concentration fluctuations of media components become drastic during media replacement. To address these issues, attention has been paid to media regeneration using dialysis techniques.

[0393] As shown in the seventh embodiment, by increasing the circulation flow rate of the culture medium circulation path and the circulation flow rate of the nutrient supply path, the ability to remove metabolic products can be improved. Furthermore, by adjusting the ratio of the circulation flow rate of the culture medium circulation path to the circulation flow rate of the nutrient supply path, the ability to remove metabolic products can be improved. Moreover, by optimizing the ratio of circulation flow rates, the culture efficiency can be improved.

[0394] <<<Details of the Seventh Embodiment>>>

[0395] Figure 9 This is a schematic diagram showing the structure of the culture medium regeneration system 700 according to the seventh embodiment. Figure 9 In the seventh embodiment, structures identical to those in the first to sixth embodiments are shown using the same reference numerals. While a substitute for the culture medium and a substitute for metabolic waste are used in the seventh embodiment, the same structure as in the first to sixth embodiments can be used as the culture medium regeneration system 700.

[0396] <Stirring device>

[0397] In the culture medium regeneration system 700 of the seventh embodiment, the stirring device 120 is present in the same manner as in the first embodiment, but is omitted for simplicity. The culture medium contained in the culture container 110-1 is stirred by the stirring device 120. In addition, the culture medium contained in the regeneration culture medium tank 180 is also stirred by a stirring device (not shown). It should be noted that the culture medium contained in the regeneration culture medium tank 180 may also be stirred without using a stirring device (not shown). Whether to stir the culture medium depends on the type of culture medium, temperature, volume of the regeneration culture medium tank 180, etc.

[0398] <Cultivation Container 110-1>

[0399] like Figure 9 As shown, the culture medium regeneration system 700 also includes a culture container 110-1. However, unlike the culture medium regeneration system 100, the culture container 110-1 is fixedly configured in a vertical position.

[0400] <First end 162a of pipe 160a>

[0401] The culture medium regeneration system 700, like the first embodiment, has a suction nozzle 130-1. However, unlike the first embodiment, the suction nozzle 130-1 is not inclined, but extends in a vertical direction.

[0402] Suction nozzle 130-1 has:

[0403] Extension 132-1;

[0404] Inhalation opening 134-1; and

[0405] Discharge opening 136-1. Suction opening 134-1 is located below the liquid level of the culture medium stored in culture container 110-1. The culture medium stored in culture container 110-1 is drawn in through suction opening 134-1.

[0406] <<Control Device 710>>

[0407] The control device 710 mainly includes a processor (CPU (Central Processing Unit), ROM (Read-Only Memory), RAM (Random Access Memory), I / F (Interface Device), auxiliary storage devices (HDD (Hard Disk Drive), SSD (Solid State Drive), etc.), and input operation devices (keyboard, mouse, touch panel, etc.). For example, it can use a personal computer, tablet computer, portable terminal device, or other devices based on these standards. The programs used to control pumps 150a, 150b, and 150c are stored in the HDD or SSD, and the programs are expanded and executed in RAM.

[0408] The control device 710 sends control signals to pumps 150a, 150b, and 150c via I / F. These control signals control the rotational speed of the motors (not shown) of pumps 150a, 150b, and 150c, adjusting the flow rate of the culture medium generated by the drives of pumps 150a, 150b, and 150c. Pump 150a is used to regulate the circulation flow rate of the culture medium circulation loop, while pumps 150b and 150c are used to regulate the circulation flow rate of the culture medium regeneration loop.

[0409] <Culture media circulation loop and culture media regeneration loop>

[0410] Like the culture medium regeneration system 100, the culture medium regeneration system 700 has a culture medium circulation loop and a culture medium regeneration loop. The culture medium circulation loop and the culture medium regeneration loop form a closed system loop.

[0411] <Culture Media Circulation Loop (Culture Media Circulation Circuit)>

[0412] The culture medium circulation loop mainly consists of a culture container 110-1, a pump 150a, a culture medium regeneration module 170, and tubes 160a, 160b, and 160c. Driven by pump 150a, the culture medium is discharged from the culture container 110-1 and circulates in the culture medium circulation loop. The culture medium circulates in the following order: culture container 110-1, tube 160a, pump 150a, tube 160b, culture medium regeneration module 170, and tube 160c.

[0413] <Driver of Pump 150a>

[0414] Driven by pump 150a, culture medium containing metabolic waste (lithium lactate) is discharged from culture vessel 110-1. The flow rate of culture medium generated per unit time in the culture medium circulation loop driven by pump 150a is called the circulation flow rate. The circulation flow rate can be adjusted by controlling the rotational speed of the motor (not shown) of pump 150a using control device 710.

[0415] <Culture Media Regeneration Circuit (Nutrient Supply Circuit)>

[0416] The culture medium regeneration loop mainly consists of pump 150b, pump 150c, culture medium regeneration module 170, regeneration culture medium tank 180, tubes 160d, 160e, 160f, and 160g. Driven by pumps 150b and 150c, the culture medium stored in the regeneration culture medium tank 180 circulates within the culture medium regeneration loop. The regeneration culture medium circulates in the following sequence: regeneration culture medium tank 180, tube 160f, pump 150c, tube 160g, culture medium regeneration module 170, tube 160d, pump 150b, and tube 160e.

[0417] <Driver for Pump 150b and Pump 150c>

[0418] Pump 150b discharges liquid containing metabolic waste removed from the culture medium circulating in the culture medium circulation loop by the culture medium regeneration module 170 to the regeneration culture medium tank 180. Pump 150c supplies the regeneration culture medium stored in the regeneration culture medium tank 180 to the culture medium regeneration module 170. The circulation flow rate of the regeneration culture medium can be adjusted by controlling the rotational speed of the motors (not shown) of pumps 150b and 150c using the control device 710.

[0419] <Culture Media Regeneration Module 170>

[0420] The culture medium regeneration module 170 has a culture medium regeneration membrane 179 for regenerating the culture medium. The culture medium regeneration membrane 179 can be a hollow fiber type culture medium regeneration module or a flat membrane type culture medium regeneration module. The culture medium regeneration module 170 can, for example, be a dialysis module.

[0421] The culture medium regeneration module 170 has a culture medium inlet 172 and a culture medium outlet 174. The culture medium inlet 172 is an opening for introducing culture medium flowing from the first end 162a of the tube 160a into the culture medium regeneration module 170. The culture medium outlet 174 is an opening for discharging the culture medium introduced into the culture medium regeneration module 170.

[0422] The culture medium regeneration module 170 has a culture medium supply port 176 and a culture medium discharge port 178. The culture medium supply port 176 is an opening for supplying culture medium from the culture medium tank 180 to the culture medium regeneration module 170. The culture medium discharge port 178 is an opening for discharging the culture medium supplied to the culture medium regeneration module 170 from the culture medium supply port 176.

[0423] <Removal of metabolic waste>

[0424] Metabolic waste (lithium lactate) is discharged from the culture medium regeneration module 170 to the regeneration culture medium tank 180 using pipes 160d and 160e. This removes metabolic waste (lithium lactate) from the culture medium circulating in the culture medium circulation loop. The flow rate discharged from the culture medium regeneration module 170 to the regeneration culture medium tank 180 can be adjusted using pump 150b.

[0425] <Nutritional Supply>

[0426] Nutrients for cell aggregation are supplied from the regeneration culture tank 180 to the culture medium regeneration module 170 via tubes 160f and 160g. This allows nutrient supply to the culture medium circulation loop. The flow rate supplied from the regeneration culture tank 180 to the culture medium regeneration module 170 can be adjusted using pump 150c. It should be noted that the seventh embodiment is an embodiment for verifying the relationship between circulation flow rate and the removal capacity of metabolic waste (metabolites), and it is also possible not to introduce nutrients into the regeneration culture tank 180. However, even when nutrients are introduced into the regeneration culture tank 180, the relationship between circulation flow rate and the nutrient absorption capacity can still be verified.

[0427] <Operation of Culture Medium Regeneration Module 170>

[0428] Nutrients for cell aggregates are supplied from the regeneration culture medium tank 180 to the regeneration culture medium supply port 176 of the culture medium regeneration module 170 via tubes 160f and 160g. The supplied nutrients move into the culture medium circulation loop through the culture medium regeneration membrane 179. Thus, nutrients are supplied to the culture medium in the culture medium circulation loop.

[0429] On the other hand, metabolic waste (lithium lactate) is discharged from the culture medium circulation loop through the culture medium regeneration membrane 179 and from the regeneration culture medium outlet 178 of the culture medium regeneration module 170 through pipes 160d and 160e to the regeneration culture medium tank 180. Thus, metabolic waste (lithium lactate) is removed from the culture medium in the culture medium circulation loop.

[0430] <<Verification Experiment of Solute Removal Capacity Based on Flow Rate>>

[0431] In a closed system with a culture medium circulation loop and a culture medium regeneration loop, the circulation flow rates of the culture medium circulation loop and the culture medium regeneration loop were adjusted to verify the solute removal capability (removal capability of metabolic waste (lithium lactate)) based on circulation flow rate. Specifically, the ability to remove metabolic waste (lithium lactate) by means of circulation flow rate was verified. Figure 11 This is a graph showing the relationship between pump running time and the concentration of metabolic waste (lithium lactate) in culture vessel 110-1, which varies with time.

[0432] <Conditions for verification>

[0433] like Figure 10A As shown, the capacity VL1 of the culture container 110-1 is set to 500 mL, and the capacity VL2 of the regeneration culture medium tank 180 is set to 1500 mL. The ratio of the capacity VL1 of the culture container 110-1 to the capacity VL2 of the regeneration culture medium tank 180 is set to VL1:VL2 = 1:3. Furthermore, as... Figure 10A As shown, 20 mM lithium lactate was pre-added to culture container 110-1 as a substitute for metabolic waste. On the other hand, lithium lactate (0 mM) was not included in the regeneration culture tank 180 as a substitute for metabolic waste. Therefore, at the time point before the start of the culture medium regeneration experiment, the ratio of the concentration LC1 of lithium lactate in culture container 110-1 to the concentration LC2 of lithium lactate in regeneration culture tank 180 was set as LC1:LC2 = 20:0.

[0434] The concentration changes were confirmed for both circulation flow rates. Figure 10B As shown, as the first circulation flow rate condition, the circulation flow rate CF1(1) of the culture medium circulation loop is set to 5 mL / min, and the circulation flow rate CF2(1) of the culture medium regeneration loop is set to 10 mL / min. The ratio of the circulation flow rate CF1(1) of the culture medium circulation loop to the circulation flow rate CF2(1) of the culture medium regeneration loop is CF1(1):CF2(1) = 1:2. Furthermore, as... Figure 10B As shown, as a condition for the second circulation flow rate, the circulation flow rate CF1(2) of the culture medium circulation loop is set to 20 mL / min, and the circulation flow rate CF2(2) of the culture medium regeneration loop is set to 40 mL / min. The ratio of the circulation flow rate CF1(2) of the culture medium circulation loop to the circulation flow rate CF2(2) of the culture medium regeneration loop is set to CF1(2) = CF1(1) × 4 and CF2(2) = CF2(1) × 4, and CF1(2):CF2(2) = 1:2.

[0435] <Verification Results>

[0436] Using control device 710, pump 150a is controlled to generate a circulating flow in the culture medium circulation loop, and pumps 150b and 150c are controlled to generate a circulating flow in the culture medium regeneration loop. From the start of operation of pumps 150a, 150b, and 150c, the concentrations of lithium lactate contained in culture container 110-1 and regeneration culture medium tank 180 are measured at predetermined intervals. The concentration of lithium lactate in culture container 110-1 is measured by extracting culture medium from culture container 110-1. The concentration of lithium lactate in regeneration culture medium tank 180 is measured by extracting regeneration culture medium from regeneration culture medium tank 180.

[0437] Figure 11This is a graph showing the measured concentrations of lithium lactate contained in culture vessel 110-1 and the measured concentrations of lithium lactate contained in regeneration culture tank 180. It should be noted that the concentration measurements were taken at specified time intervals; therefore, the lactic acid concentration is shown as a discrete set of multiple points. However, to clearly illustrate the concentration changes, a continuous solid line is used to represent the variation in lactic acid concentration. Figure 11 The concentration changes of the culture medium circulation loop at the circulation flow rate CF1(1), the culture medium circulation loop at the circulation flow rate CF1(2), the culture medium regeneration loop at the circulation flow rate CF2(2), and the culture medium regeneration loop at the circulation flow rate CF2(1) are shown sequentially from top to bottom.

[0438] <Rate of change in concentration>

[0439] like Figure 11 As shown, the slope of the initial concentration change at the beginning of the experiment when the circulation flow rate of the culture medium circulation loop is CF1(1) is set as S1(1). Similarly, the slope of the initial concentration change at the beginning of the experiment when the circulation flow rate of the culture medium regeneration loop is CF2(1) is set as S2(1). The slope of the initial concentration change at the beginning of the experiment when the circulation flow rate of the culture medium circulation loop is CF1(2) is set as S1(2). The slope of the initial concentration change at the beginning of the experiment when the circulation flow rate of the culture medium regeneration loop is CF2(2) is set as S2(2).

[0440] like Figure 11 As shown, |S1(1)| < |S1(2)|. It should be noted that the notation "|x|" represents the absolute value of x. In addition, S2(1) < S2(2). That is, when the circulation flow rate of the culture medium circulation loop and the circulation flow rate of the culture medium regeneration loop are increased, the concentration of lithium lactate contained in culture vessel 110-1 decreases rapidly. Therefore, by increasing the circulation flow rate of the culture medium circulation loop and the circulation flow rate of the culture medium regeneration loop, the rate at which lithium lactate can be removed from the culture medium in culture vessel 110-1 can be increased. In other words, the time required to remove lithium lactate from the culture medium in culture vessel 110-1 can be shortened.

[0441] It should be noted that, over time, the concentration of lithium lactate in the culture medium in culture container 110-1 and the concentration of lithium lactate in the regeneration culture medium in regeneration culture tank 180 will approach a common concentration C0. Figure 9The culture medium regeneration system 700 shown is a closed system loop consisting only of a culture medium circulation loop and a culture medium regeneration loop. Therefore, as sufficient time passes, lithium lactate gradually accumulates in the regeneration culture medium tank 180, and the ability to remove lithium lactate gradually becomes saturated. As a result, the concentration of lactic acid in the culture medium of culture vessel 110-1 is close to the concentration of lactic acid in the regeneration culture medium of the regeneration culture medium tank 180.

[0442] By increasing the circulation flow rate of the regeneration medium flowing in the nutrient supply path and the circulation flow rate of the medium circulating in the medium circulation path, the rate at which the concentration of metabolites (lithium lactate) decreases within the culture vessel 110-1 can be increased. On the other hand, nutrients move from the nutrient supply path to the medium circulation path through the semi-permeable membrane of the regeneration module 170. Therefore, in contrast to the concentration of metabolites (lithium lactate), the rate at which the concentration of nutrients imparted to the medium within the culture vessel 110-1 increases can be increased.

[0443] <Modified Example 1 (Supplementation box for regeneration culture medium)>

[0444] In the seventh embodiment, the tank for storing new culture medium is simply the regeneration culture medium tank 180. In contrast, an additional tank (not shown) can also be provided to add new culture medium to the regeneration culture medium tank 180. Furthermore, by providing a pump (not shown), new culture medium can be supplied from the additional tank to the regeneration culture medium tank 180. By supplying new culture medium to the regeneration culture medium tank 180, the ability to remove lithium lactate is easily maintained, and the CO concentration can be reduced. In this way, the culture medium can be continuously regenerated over a long period of time.

[0445] <Variation Example 2 (Formation of a Non-Circular Path)>

[0446] In the seventh embodiment, a closed system is shown in which lithium lactate discharged from the culture medium regeneration module 170 is returned to the regeneration culture medium tank 180 using pipes 160d and 160e. That is, a circulation path is formed by pipes 160d, 160e, 160f, and 160g. Because lithium lactate is returned to the regeneration culture medium tank 180, lithium lactate gradually accumulates, and the ability to remove lithium lactate gradually becomes saturated. As a result, the concentration of lactic acid in the culture medium of culture container 110-1 is close to the concentration of lactic acid in the regeneration culture medium of the regeneration culture medium tank 180. From this viewpoint, a non-circulation path can also be constructed. For example, the metabolites discharged from the culture medium regeneration module 170 can be returned to a storage tank (not shown) different from the regeneration culture medium tank 180. In this way, the concentration of lithium lactate in the regeneration culture medium tank 180 can be maintained, the ability to remove lithium lactate can be easily maintained, and the concentration CO can be reduced.

[0447] <Modified Example 3 (Removal of Lithium Lactate Based on Adsorbent)>

[0448] In the seventh embodiment, a closed system is shown in which lithium lactate discharged from the culture medium regeneration module 170 is returned to the regeneration culture medium tank 180 using pipes 160d and 160e. That is, a circulation path is formed by pipes 160d, 160e, 160f, and 160g. Since lithium lactate is returned to the regeneration culture medium tank 180, lithium lactate gradually accumulates, and the ability to remove lithium lactate gradually becomes saturated. As a result, the concentration of lactic acid contained in the culture medium of culture container 110-1 is close to the concentration of lactic acid contained in the regeneration culture medium of regeneration tank 180. From this point of view, adsorbents or the like for selectively removing lithium lactate generated from cell aggregates can also be used. In addition, the structure for removing lithium lactate based on adsorbents described in the first embodiment can be appropriately introduced. Specifically, the structures of <Adsorbent-based removal scheme 1> to <Adsorbent-based removal scheme 4> of the first embodiment can be applied to the culture medium regeneration system 700 of the seventh embodiment. In this way, the concentration of lithium lactate in the regeneration culture medium tank 180 can be maintained, making it easy to maintain the ability to remove lithium lactate and reduce the concentration CO.

[0449] <Variation Example 4 (Control of Circulating Flow 1)>

[0450] In the seventh embodiment, the circulation flow rate of the culture medium circulation loop and the circulation flow rate of the culture medium regeneration loop are set to constant values ​​independent of time. Not limited to this, the control device 710 can also control pumps 150a, 150b, and 150c, and control them by changing the circulation flow rate according to the processing time. This allows for processing with appropriate circulation flow rates and appropriate circulation flow rate ratios based on changes in the lithium lactate concentration.

[0451] <Variation Example 5 (Control of Circulating Flow 2)>

[0452] Alternatively, a sensor capable of measuring the concentration of lithium lactate in the culture vessel 110-1 in real time can be installed. Based on the measurement results, the circulation flow rate of the culture medium circulation loop and the circulation flow rate of the culture medium regeneration loop can be determined, and pumps 150a, 150b, and 150c can be controlled using the control device 710. This allows for processing based on changes in the concentration of lithium lactate with appropriate circulation flow rates and appropriate circulation flow rate ratios.

[0453] <<<<Eighth Implementation Method>>>>

[0454] The eighth embodiment relates to a culture medium regeneration system that inhibits cell aggregates from being discharged into the circulation loop while circulating the culture medium, and increases the flow rate of the culture medium circulation. The eighth embodiment primarily demonstrates the verification results of the relationship between the tilt angle of the suction nozzle 130-2 and the circulation flow rate of the culture medium. The culture medium regeneration system of the eighth embodiment is a system used for verification. In the eighth embodiment, a liquid similar to the culture medium used to culture cell aggregates is used, and resin-made simulated particles are used instead of 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 cell aggregates. Hereinafter, unless otherwise specified, the resin beads will be referred to as cell aggregates, and the phosphate-buffered saline (PBS) will be referred to as the culture medium.

[0455] In cell culture processes, the circulation of the culture medium is indispensable. However, when cell aggregates are discharged into the circulation loop, fewer cell aggregates become available for culture. On the other hand, when the discharge of cell aggregates is inhibited, the circulation flow rate is limited, and the time-varying rate of change of the concentration of metabolites in the culture section decreases, as does the time-varying rate of change of the concentration of nutrients.

[0456] In the eighth embodiment, a culture medium regeneration system is provided that increases the circulation flow rate of the culture medium circulation loop while using tilting suction to suppress the discharge of cell aggregates. This allows for the maintenance of the number of cell aggregates in the culture medium, thereby increasing the time-varying rate of change in the concentration of metabolites in the culture section, and also increasing the time-varying rate of change in the concentration of nutrients.

[0457] Figure 12A as well as Figure 12B This is a schematic diagram showing the structure of the culture medium regeneration system 800 according to the eighth embodiment. Figure 12A In this drawing, structures identical to those in the first and second embodiments are shown using the same reference numerals. It should be noted that... Figure 12A In this embodiment, the stirring device 120, pump 150a, culture medium regeneration module 170, tubes 160a, 160b, pump 150b, pump 150c, culture medium tank 180 for regeneration, tubes 160d, 160e, 160f, and 160g are the same as in the first embodiment and are omitted. The culture medium is drawn from the culture container 110-1 by pump 150a and circulated back to the culture container 110-1.

[0458] like Figure 12A As shown, the culture container 110-1 of the culture medium regeneration system 800 is fixedly configured in a vertical position, just like the culture medium regeneration system 200.

[0459] <Stirring device>

[0460] The culture medium regeneration system 800 of the eighth embodiment also has the same stirring device as that of the first embodiment, but it is omitted for simplicity. The culture medium contained in the culture container 110-1 is appropriately stirred by the stirring device.

[0461] <Suction nozzle 130-2>

[0462] The culture medium regeneration system 800 has a suction nozzle 130-2. The structure and material of the suction nozzle 130-2 are the same as those of the suction nozzle 130-1 of the culture medium regeneration system 100.

[0463] Like suction nozzle 130-1, suction nozzle 130-2 has the following characteristics:

[0464] Extension 132-1;

[0465] Inhalation opening 134-1; and

[0466] Discharge opening 136-1.

[0467] <Inclination angle θ>

[0468] The extension 132-1 also has the same structure as the suction nozzle 130-1. For example... Figure 12A as well as Figure 12B As shown, the extension 132-1 extends in an inclined direction. The extension 132-1 is inclined at an angle θ relative to the vertical direction. Therefore, the entire suction nozzle 130-2 extends in an inclined direction. In other words, the central axis AO of the suction nozzle 130-2 extends in an inclined direction.

[0469] In the eighth embodiment, the central axis CO of the culture container 110-1 extends in a vertical direction, and the suction nozzle 130-2 extends in an inclined direction. The suction nozzle 130-2 is arranged such that the suction opening 134-1 faces the side wall 112 of the culture container 110-1.

[0470] In the eighth embodiment, the central axis AO of the suction nozzle 130-2 is inclined relative to the central axis CO of the culture container 110-1. In the eighth embodiment, the inclination angle θ of the suction nozzle 130-2 is selected from multiple angles. For example, selectively using the inclination angle θ allows for the replacement of three types of suction nozzles 130-2: 10 degrees, 15 degrees, and 20 degrees. The three types of suction nozzles 130-2 are stably held by a holding member (not shown) at their respective inclination angles θ. It should be noted that... Figure 12A For clarity, only the suction nozzle 130-2 with an inclination angle θ of 20 degrees is shown in solid line. Figure 12BThe diagram shows three types of suction nozzles 130-2 with tilt angles θ of 10 degrees, 15 degrees, and 20 degrees. It should be noted that the tilt angle θ is not limited to the above three types and can be appropriately set to the desired angle within the range of 0 < θ < 90 degrees.

[0471] <Flow of culture medium and cell aggregates within aspiration nozzle 130-2>

[0472] The culture medium and cell aggregates flow within the suction nozzle 130-2 in the same manner as in the suction nozzle 130-1 of the first embodiment (see reference). Figure 2 Two flow zones, a first flow zone FF and a second flow zone SF, are also formed in the suction nozzle 130-2. The cell aggregate remains inside the suction nozzle 130-2, and the culture medium is discharged from the discharge opening 136-1 of the suction nozzle 130-2.

[0473] In this way, by using the inclined suction nozzle 130-2, the circulation flow rate of the culture medium can be increased without discharging the cell aggregates into the culture medium circulation loop. Therefore, when using the culture medium regeneration module (not shown), 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.

[0474] <<Verification Experiment>>

[0475] As a substitute for cell aggregates, simulated particles were used to determine the conditions under which the simulated particles would not be attracted by the attracting nozzle 130-2.

[0476] <Conditions>

[0477] The simulated particles were made of resin, with 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. The beads did not settle when air bubbles adhered around them, so a surfactant was used to address this. The tilt angles θ were 10 degrees, 15 degrees, and 20 degrees.

[0478] The circulation flow rate of the culture medium was determined through the following preliminary experiments. First, the flow rate of the culture medium relative to the rotational speed of the motor of pump 150a was measured beforehand. The flow rate of the culture medium can be determined based on the weight difference of the liquid volume per minute. For example, with a motor rotational speed of 100 (rpm) relative to pump 150a, the flow rate of the culture medium was determined to be 40 (mL / min).

[0479] Therefore, based on the measured flow rate of the culture medium, the flow rate per revolution is calculated. For example, the calculated flow rate of the culture medium is 40 (mL / min) / rotation speed is 100 (rpm) = 0.4 (mL / revolution). The calculated flow rate per revolution is then stored in the RAM of the control device, etc.

[0480] Using the results of the preliminary experiment, determine the flow rate as follows. The desired flow rate can be determined by setting it using the control panel. For example, using the control panel, set the desired flow rate to 10 (mL / min).

[0481] The CPU of the control device calculates the rotational speed corresponding to the desired flow rate based on the desired flow rate set using the operation panel and the stored flow rate per revolution. For example, it calculates 10 (mL / min) / 0.4 (mL / rotation) = 25 revolutions / min, and controls the motor of pump 150a at 25 revolutions / min. In this way, by setting the flow rate using the operation panel as the actual flow rate of the culture medium, the maximum flow rate at which simulated particles are not attracted by the aspiration nozzle 130-2 is measured.

[0482] <Experimental Methods>

[0483] The simulated particles are introduced into the culture container 110-1. The flow rate in the suction nozzle 130-2 is gradually increased to confirm that the simulated particles are not being attracted. The tilt angle θ is changed, and the condition under which the simulated particles are not attracted is similarly confirmed. While changing the flow rate using the control panel, the flow rate at which the simulated particles are not attracted is determined for each tilt angle θ.

[0484] <Experimental Results>

[0485] Figure 13 This is a graph showing the relationship between the tilt angle θ of the suction nozzle 130-2 and the maximum flow rate at which simulated particles are not attracted by the suction nozzle 130-2.

[0486] With a tilt angle θ of 10 degrees for the suction nozzle 130-2, the flow rate of simulated particles not attracted by the nozzle 130-2 is minimized. With a tilt angle θ of 20 degrees for the suction nozzle 130-2, the flow rate not attracted by the nozzle 130-2 is maximized. By increasing the suction angle of the suction nozzle 130-2, the effect of inhibiting attraction to the nozzle 130-2 can be improved, allowing the culture medium to flow at a greater flow rate.

[0487] By tilting the suction nozzle 130-2, the circulation flow rate of the culture medium can be increased without attracting cell aggregates. This increases the rate of change of metabolite concentrations in the culture section, as well as the rate of change of nutrient concentrations, while simultaneously suppressing cell loss.

[0488] according to Figure 13 The results show that by increasing the suction angle of the suction nozzle 130-2, the circulation flow rate of the culture medium can be increased without the cell aggregates being attracted by the suction nozzle 130-2. While the specific relationship between the 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 nozzle 130-2, finding the tilt angle and circulation flow rate that increase the circulation flow rate of the culture medium by increasing the suction angle of the suction nozzle 130-2 can be determined through the same validation experiments. Furthermore, 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 nozzle 130-2, can also be determined through the same validation experiments.

[0489] <Variation Example 1>

[0490] In the eighth embodiment, an example is shown of using only one suction nozzle 130-2 to aspirate the culture medium, but multiple suction nozzles 130-2 may also be used.

[0491] <<<<Scope of Implementation>>>>

[0492] 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 construed as limiting. Various embodiments not described herein are included. Therefore, the first to eighth embodiments are not mutually independent structures, and also include embodiments obtained by appropriately selecting the structures of each of the first to eighth embodiments.

[0493] According to the verification experiment using the culture medium regeneration system 700 in the seventh embodiment, by increasing the circulation flow rate of the culture medium circulation path and the circulation flow rate of the nutrient supply path, at least one of the ability to remove metabolites and the ability to supply nutrients can be improved. For example, regarding the ability to supply nutrients, by adopting... Figure 11 The graph showing the change in lactic acid concentration, obtained by swapping the circulation flow rates CF1(1), CF1(2) of the culture medium circulation loop with the circulation flow rates CF2(1), CF2(2) of the culture medium regeneration loop (equivalent to an upside-down graph), yields a graph showing the change in nutrient concentration. It is assumed that the change in nutrient concentration also shows the same trend. It is anticipated that as the flow rate increases, the rate of change in nutrient concentration also increases. Based on the above, the change rate of nutrient concentration can be explained using data on lithium lactate.

[0494] The verification results obtained by the seventh embodiment can be clearly applied to culture medium regeneration systems such as the culture medium regeneration system 100, which have the same structure as the culture medium regeneration system 700. Specifically, the verification results obtained by the seventh embodiment can be applied to any system that has a culture medium circulation loop and a culture medium regeneration loop, and that can adjust the circulation flow rate of the culture medium in the culture medium circulation loop and the circulation flow rate of the culture medium for regeneration in the culture medium regeneration loop. Therefore, the verification results obtained by the seventh embodiment can be applied not only to the first embodiment, but also to the second to sixth embodiments.

[0495] Furthermore, according to the verification experiment using the culture medium regeneration system 800 according to the eighth embodiment, it is possible to increase the circulation flow rate of the culture medium circulation loop while suppressing the discharge of cell aggregates using tilted suction. Therefore, it is evident that the verification results obtained by the eighth embodiment can be applied to culture medium regeneration systems such as the culture medium regeneration system 100, which have the same structure as the culture medium regeneration system 800. Specifically, as long as the system has a tilted suction nozzle 130-2 and the flow rate of the culture medium discharged from the suction nozzle 130-2 can be adjusted, the verification results obtained by the eighth embodiment can be applied. Therefore, not only the first embodiment, but also the second to sixth embodiments can be applied.

[0496] Industrial applicability

[0497] A culture medium regeneration system is provided that allows for the efficient culture of cell aggregates by appropriately adjusting the composition during the culture process.

[0498] Cross-referencing of related applications

[0499] This application claims priority based on Japan 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.

[0500] Explanation of reference numerals in the attached figures

[0501] 100, 200, 300, 400, 500, 600, 700, 800 Culture Medium Regeneration Systems

[0502] 110-1 and 110-3 culture containers

[0503] 130-1, 130-2, 130-3 suction nozzles

[0504] 170 Culture Medium Regeneration Module

[0505] 172 Culture medium regeneration membrane.

Claims

1. A culture medium regeneration system, wherein, The culture medium regeneration system comprises: A culture medium circulation circuit that circulates the culture medium contained in the culture section used for culturing cell aggregates; A nutrient supply path for supplying a regeneration culture medium containing nutrients for the cell aggregates; and A culture medium regeneration module, connected to the culture medium circulation path and the nutrient supply path, and housing a semi-permeable membrane capable of allowing at least one of the nutrients and the metabolites produced by the cell aggregate to pass through. The culture medium regeneration system regulates the chance of at least one of the following: the chance of the nutrients coming into contact with the cell aggregate through the semipermeable membrane and the chance of the metabolites coming into contact with the regeneration culture medium of the nutrient supply path through the semipermeable membrane.

2. The culture medium regeneration system according to claim 1, wherein, The culture medium regeneration system regulates the concentration of either the nutrients supplied to the cell aggregate via the semipermeable membrane or the metabolites of the cell aggregate.

3. The culture medium regeneration system according to claim 2, wherein, The culture medium regeneration system also includes a nutrient storage tank for storing the nutrients. The flow path between the nutrient storage tank and the semi-permeable membrane is a non-circulating path that has the nutrient supply path but does not have a flow path from the semi-permeable membrane to the nutrient storage tank.

4. The culture medium regeneration system according to claim 2, wherein, The culture medium regeneration system also includes a nutrient storage tank for storing the nutrients. The culture medium regeneration system also includes a nutrient addition device for adding new nutrients to the nutrient storage tank.

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

6. The culture medium regeneration system according to claim 5, wherein, The culture medium regeneration system also includes a discharge nozzle for discharging culture medium from the culture section toward the semipermeable membrane. The outlet nozzle is set at an angle relative to the vertical direction.

7. The culture medium regeneration system according to any one of claims 1 to 6, wherein, The regeneration culture medium contains an adsorbent that adsorbs the metabolites.

8. The culture medium regeneration system according to claim 7, wherein, The adsorbent targets at least one of the following as metabolic waste products: lactic acid, ammonia, glutamic acid, isovaleric acid, butyric acid, and citric acid.

9. A culture medium regeneration system, wherein, The culture medium regeneration system comprises: The culture medium circulation circuit is used to circulate the culture medium and metabolites contained in the culture section. Nutrient supply pathway, which circulates the culture medium containing nutrients for regeneration; A culture medium regeneration module is connected to the culture medium circulation path and the nutrient supply path, and contains a semi-permeable membrane that allows the nutrients and metabolites to pass through. as well as The control unit adjusts the circulation flow rate of the culture medium circulating in the culture medium circulation path and the circulation flow rate of the regeneration culture medium circulating in the nutrient supply path. The culture medium regeneration system changes the rate of change of at least one of the concentration of the metabolites and the concentration of the nutrients in the culture section by adjusting the circulation flow rate of the culture medium circulating in the culture medium circulation path and the circulation flow rate of the regeneration culture medium circulating in the nutrient supply path.

10. The culture medium regeneration system according to claim 9, wherein, The culture medium regeneration system increases at least one of the rate at which the concentration of the metabolites in the culture section decreases and the rate at which the concentration of the nutrients in the culture section increases by increasing the circulation flow rate of the regeneration culture medium flowing in the nutrient supply path and the circulation flow rate of the culture medium circulating in the culture medium circulation path.

11. A culture medium regeneration system, wherein, The culture medium regeneration system comprises: A culture medium circulation circuit that circulates the culture medium contained in the culture section along with the cell aggregates; and A discharge nozzle that discharges the culture medium from the culture section. The outlet nozzle is set at an angle relative to the vertical direction. The culture medium regeneration system, based on the tilt angle of the outlet nozzle, inhibits cell aggregates from being discharged from the culture section into the culture medium circulation path and increases the circulation flow rate.

12. A culture medium regeneration system, wherein, The culture medium regeneration system increases at least one of the rate at which the concentration of the metabolites in the culture section decreases and the rate at which the concentration of the nutrients in the culture section increases by increasing the circulation flow rate in the culture medium circulation path.

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