WO2021199529A1 - 細胞培養装置及び培養方法 - Google Patents

細胞培養装置及び培養方法 Download PDF

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Publication number
WO2021199529A1
WO2021199529A1 PCT/JP2020/047727 JP2020047727W WO2021199529A1 WO 2021199529 A1 WO2021199529 A1 WO 2021199529A1 JP 2020047727 W JP2020047727 W JP 2020047727W WO 2021199529 A1 WO2021199529 A1 WO 2021199529A1
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Prior art keywords
culture
culture tank
cell
cells
separation tube
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Ceased
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PCT/JP2020/047727
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English (en)
French (fr)
Japanese (ja)
Inventor
優史 丸山
優至 佐藤
靖彦 多田
悠午 鈴木
雅弘 岡野定
尚武 陳
亮介 高橋
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Resonac Corp
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Showa Denko Materials Co Ltd
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Priority to JP2022511531A priority Critical patent/JPWO2021199529A1/ja
Publication of WO2021199529A1 publication Critical patent/WO2021199529A1/ja
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M1/00—Apparatus for enzymology or microbiology
    • C12M1/34—Measuring or testing with condition measuring or sensing means, e.g. colony counters
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M3/00—Tissue, human, animal or plant cell, or virus culture apparatus
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/0006—Modification of the membrane of cells, e.g. cell decoration
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues

Definitions

  • the present disclosure relates to a cell culture device and a culture method.
  • Japanese Patent Application Laid-Open No. 6-209761 describes in a perfusion culture (perfusion culture) that a culture medium in which cell metabolites are accumulated is used in the culture tank while continuously supplying a fresh culture solution into the culture tank.
  • a culture device that discharges and collects the cells to the outside is disclosed.
  • this culture device rapidly attenuates the disturbance of the culture solution due to stirring in the sedimentation separation tube. It is equipped with a sedimentation separation tube that can form a clear settling zone.
  • the cell support to which the cells are attached may be drawn into the separation tube.
  • the cell support When the cell support is drawn into the separation tube, it easily invades the upper part of the separation tube as the culture solution is discharged. In order to recover the cells, it is sometimes necessary to stop the perfusion and collect the cell support that has entered the inside of the separation tube. It is conceivable to slow down the perfusion rate so that it is not drawn into the separation tube, but it takes time to replace the medium and is inefficient.
  • An object of the present disclosure is to provide a cell culture apparatus capable of efficiently culturing cells. Another object of the present disclosure is to provide a culturing method capable of culturing cells efficiently.
  • This disclosure relates to the following embodiments.
  • a culture tank having a capacity of 0.5 L to 50 L, a separation tube extending from the inside to the outside of the culture tank and having an inner diameter of 0.5 cm to 5 cm, and a separation tube connected to the separation tube.
  • a pump, an intake port and an exhaust port provided in the culture tank, a medium supply port provided in the culture tank, and at least a part of the separation tube are provided, and the separation tube is adjusted to 30 ° C. to 45 ° C.
  • a cell culture device comprising a adjusting tool and a device for culturing.
  • a method of culturing including doing.
  • (6) The culture method according to (5), wherein the capacity of the culture tank is 0.5 L to 50 L.
  • the present disclosure it is possible to provide a cell culture device capable of efficiently culturing cells. Further, according to the present disclosure, it is possible to provide a culture method for efficiently culturing cells.
  • FIG. 1 is a schematic view showing an example of a cell culture apparatus.
  • FIG. 2 is a schematic view showing another example of the cell culture apparatus.
  • FIG. 3 is a schematic view showing another example of the cell culture apparatus.
  • FIG. 1 is a schematic view showing an example of a cell culture apparatus according to the present embodiment.
  • the cell culture apparatus 20 according to the present embodiment includes a culture tank 1 for culturing target cells, a separation tube 3, a pump 4, and a regulator (temperature regulator) 8.
  • a regulator temperature regulator 8.
  • FIG. 1 the routes connecting the intake port 5, the exhaust port 6, the medium supply port 7, and the recovery tank 9, which will be described later, are shown by lines for simplification.
  • the cell culture device 20 is for culturing cells, and here the cells may be those attached to a cell support. Although the cells are shown in circles in FIG. 1 for convenience, there are no particular restrictions on the shape of the cells attached to the cell support. The cells and cell supports will be described later.
  • the culture tank 1 contains a liquid medium 2c in an amount corresponding to the size of the culture tank 1.
  • the medium will be described later.
  • the size of the culture tank 1 is not particularly limited, and can be, for example, 0.5 L or more, 1 L or more, 2 L or more, 5 L or more, 10 L or more, 20 L or more, or 50 L or more, and in other embodiments, 80 L or more. As described above, a culture tank having a larger size may be used.
  • the cell culture apparatus 20 may be provided with a heater for keeping the temperature of the medium 2c contained in the culture tank 1 at a constant temperature, for example, around 37 ° C.
  • a stirrer 10 for stirring the medium when the internal medium is contained is provided inside the culture tank 1, a stirrer 10 for stirring the medium when the internal medium is contained.
  • the stirring speed of the stirrer 10 can be appropriately selected depending on the shape of the stirrer, the size of the culture tank 1, and the like.
  • an intake port 5 for supplying a mixed gas containing oxygen, carbon dioxide, etc. into the culture tank 1 an exhaust port 6 for discharging the mixed gas from the culture tank 1, and a liquid medium 2c are cultured.
  • a medium supply port 7 for supplying into the tank 1 is provided.
  • the intake port 5 is provided with a removable filter 11.
  • the filter 11 may be provided in only one of the intake port 5 and the exhaust port 6, or may be provided in both the intake port 5 and the exhaust port 6. If there is another structure that does not allow foreign matter to enter the intake port 5 and / or the exhaust port 6, the intake port 5 and / or the exhaust port 6 may not be provided with the filter 11.
  • the shape of the pores of the filter 11 may be any shape as long as it can prevent foreign substances such as microorganisms and impurities from entering the culture tank 1, and examples thereof include a circular shape, an elliptical shape, a rectangular shape, and a polygonal shape.
  • the hole diameter of the filter 11 means the diameter when the shape of the hole of the filter 11 is circular, and when the shape of the hole of the filter is other than circular, it is arbitrary to pass through the center point of the hole in the cross section of the hole. Means the length of the longest straight line of.
  • the pore size of the filter 11 can be, for example, in the range of 0.01 ⁇ m to 30.0 ⁇ m, and can be 0.2 ⁇ m or less.
  • the filter 11 can be, for example, a sterile filter.
  • the separation tube 3 extends from the inside of the culture tank 1 to the outside.
  • the inner diameter of the separation tube 3 is not particularly limited as long as the temperature of the culture supernatant passing through the inside can be adjusted by the adjusting tool 8 described later, and can be appropriately selected depending on the material of the separation tube 3 and the like.
  • the separation tube 3 according to one embodiment has an inner diameter of 0.5 cm to 5 cm or 1 cm to 4 cm.
  • the material of the separation tube 3 is not particularly limited, and examples thereof include glass, SUS, and resin.
  • the culture supernatant refers to a medium obtained after culturing cells for a certain period of time and accumulating metabolites of the cells.
  • the opening surface of the separation tube 3 at the outer end of the culture tank 1 is connected to the pump 4 in order to discharge the culture supernatant to the outside of the culture tank 1.
  • the culture supernatant is stored in the recovery tank 9 by the pump 4.
  • the pump 4 is not particularly limited as long as the culture supernatant can be discharged to the recovery tank 9, and for example, a positive displacement pump such as a peristaltic pump can be used. Further, the culture supernatant contained in the recovery tank 9 may be used for analysis of the internal state of the culture tank 1, and the culture supernatant recovered in the recovery tank 9 may be used for other purposes.
  • the opening surface of the separation tube 3 at the inner side end of the culture tank 1 is located in the medium 2c.
  • the distance D between the opening surface at the inner side end of the culture tank 1 of the separation tube 3 and the inner bottom surface of the culture tank 1 can be a distance that allows the culture supernatant to be discharged and does not cause precipitation, clogging, or the like.
  • the size of the culture tank 1, the cell density, the discharge rate and the like can be selected.
  • the distance D can be, for example, 0.1 cm or more, 0.5 cm or more, or 1.0 cm or more.
  • the upper limit of the distance D may be a position where the inner side end of the culture tank 1 of the separation tube 3 does not rise above the liquid level of the medium 2c housed in the culture tank 1 by the drive of the pump 4. It can be appropriately selected depending on the amount of liquid contained therein.
  • the inner side end of the culture tank 1 of the separation tube 3 is 80%, 85%, 90%, or 95% of the distance from the inner bottom surface of the culture tank 1 to the liquid level of the liquid contained in the culture tank 1. Can be the position.
  • the separation tube 3 is provided with an adjusting tool 8 in a portion extending to the outside of the culture tank 1.
  • the adjusting tool 8 can adjust the separation tube 3 to a constant temperature, for example, 30 ° C. to 45 ° C., 33 ° C. to 42 ° C., or 35 ° C. to 40 ° C.
  • a liquid flow is generated in the downward direction (direction from the outside of the culture tank to the inside of the culture tank) near the inner wall of the separation tube 3, and an upward direction (in the vicinity of the center).
  • the speed of the liquid flow (in the direction from the inside of the culture tank to the outside of the culture tank) may increase, and heat convection may occur.
  • the separation tube 3 is provided with the adjuster 8 to adjust the temperature difference between the vicinity of the inner wall of the separation tube 3 and the vicinity of the center to prevent the occurrence of heat convection. can.
  • the temperature of the separation tube 3 adjusted by the adjusting tool 8 is not limited to the above-mentioned temperature range as long as the temperature difference between the vicinity of the inner wall of the separation tube 3 and the vicinity of the center of the liquid arranged inside can be reduced. It can be appropriately selected depending on the temperature of the liquid contained inside the culture tank 1, the temperature of the space inside the culture tank 1, the temperature outside the culture tank 1, the temperature of the place where the cell culture device 20 is installed, and the like.
  • the regulator 8 may be provided in at least a part of the separation tube 3.
  • the regulator 8 is one of the separation tubes 3 located outside the culture tank 1. It may be provided in the whole area (the entire area of the separation tube 3 between the culture tank 1 and the pump 4) unlike the drawing, and may not come into contact with the liquid in the culture tank 1.
  • the separation pipe 3 located in the region may be provided in the entire area or a part thereof, or may be provided in two or more regions as a combination of one or more of these regions.
  • One or more adjusters 8 may be provided in each region.
  • the adjusting tool 8 may be any as long as it keeps or heats the separation tube 3, and examples thereof include a heat insulating material and a heater.
  • the generation of heat convection can be suppressed in the separation tube 3. Therefore, even if cells, for example, cells attached to a cell support are cultured in the culture tank 1, the cells are not easily drawn into the separation tube 3, and the cells and the culture supernatant can be efficiently separated. Further, in the case of culturing cells by pulling out only the culture supernatant through the separation tube 3 while adding the medium, that is, in the case of perfusion culture, the speed is faster than that of the culturing device without the adjusting tool 8. The culture supernatant can be withdrawn at, and fresh medium can be added at a faster rate.
  • ⁇ Culture method> The culture method according to this embodiment will be described.
  • the culturing method will be described with reference to the cell culturing device 20 shown in FIG. 1, but the culturing method according to the present embodiment is not limited to the method using the cell culturing device 20 shown in FIG. No.
  • the cells adhering to the cell support are suspended and cultured in the culture tank 1 provided with the separation tube 3 extending from the inside to the outside, and the culture supernatant in the culture tank 1 is cultivated. It includes discharging the culture supernatant to the outside of the culture tank 1 through the separation tube 3 and adjusting the temperature of the culture supernatant to 30 to 45 ° C. when passing through the separation tube 3.
  • the cells that can be cultured using the cell culture apparatus according to the embodiment according to the present disclosure are not particularly limited as long as they are cells capable of suspension culture, and may be floating cells or adhesive cells. good.
  • the cells may be those in which a plurality of cells come into contact with each other to form an aggregate during suspension culture, or they may adhere to a culture carrier to form a complex of cells and a culture carrier, and the cells may be simply formed during culture. It may float in the suspension as a single cell.
  • the adhesive cell may be any adhesive cell that can adhere to the cell support, and examples thereof include somatic cells and stem cells.
  • somatic cells for example, endothelial cells, epidermal cells, epithelial cells, myocardial cells, myoblasts, nerve cells, bone cells, osteoblasts, fibroblasts, fat cells, hepatocytes, renal cells, pancreatic cells, adrenal cells , Dental cells, gingival cells, bone membrane cells, skin cells, dendritic cells, macrophages and the like.
  • Adhesive cells are preferably animal-derived cells, and more preferably mammalian-derived cells. Mammals include, for example, humans, monkeys, chimpanzees, cows, pigs, horses, sheep, goats, rabbits, rats, mice, guinea pigs, dogs, cats and the like. Adhesive cells may be, for example, cells derived from tissues such as skin, liver, kidney, muscle, bone, blood vessel, blood, nervous tissue and the like.
  • Adhesive cells can be used alone or in combination of two or more.
  • the adherent cell may be a stem cell.
  • stem cells include somatic stem cells such as mesenchymal stem cells, hematopoietic stem cells, neural stem cells, bone marrow stem cells, and germline stem cells, and can be mesenchymal stem cells or bone marrow mesenchymal stem cells. ..
  • Mesenchymal stem cells broadly mean somatic stem cells that are present in various tissues of an individual and can be differentiated into all or some of mesenchymal cells such as osteoblasts, chondrocytes and adipocytes. do.
  • the stem cells may further include induced pluripotent stem cells (iPS cells) and embryonic stem cells (ES cells).
  • iPS cells induced pluripotent stem cells
  • ES cells embryonic stem cells
  • the floating cell may be any cell. Examples thereof include, but are not limited to, Chinese hamster ovary cells (CHO cells), Vero cells, Jurkat, HL60, FM3A and the like.
  • the culture When cells are suspended and cultured, the culture is carried out as a cell suspension having a cell concentration of 1 ⁇ 10 3 to 1 ⁇ 10 8 cells / mL, although it depends on the type of cells, the presence or absence of a cell support, and the like. You can start.
  • the medium used for culturing cells preferably contains inorganic salts, amino acids, sugars, and water.
  • the medium may further contain any component such as serum, vitamins, hormones, antibiotics, growth factors, adhesion factors and the like.
  • a medium known as a basal medium for cell culture can be used as the medium.
  • any medium known to be used for culturing selected cells can be used without particular limitation.
  • DMEM Denbecco modified Eagle's medium
  • MEM Eagle's minimum essential medium
  • ⁇ MEM medium Eagle's minimum essential medium ⁇ modified type
  • the medium used for culturing can be free of heterologous components.
  • the medium containing no heterologous component include a serum-free medium, a limited culture medium (Chemically defined medium), and the like.
  • the medium containing no heterologous component is a substitute additive for serum (for example, Knockout Serum Replacement (KSR) (manufactured by Invitrogen), Chemically-defined Lipid concentrated (manufactured by Gibco), Glutamax (manufactured by Gibco), instead of the serum derived from animals. Company), etc.) can be included.
  • KSR Knockout Serum Replacement
  • the cell support that can be used for cell culture is also called a microcarrier, and takes the form of beads having a diameter of several hundred ⁇ m, for example.
  • the cells can be obtained in a high culture area and high density by adhering the cells on beads floating in the medium and proliferating the cells.
  • the material of the cell support may be an organic substance, an inorganic substance, or a composite material thereof, and may be either soluble or insoluble.
  • organic substances include polystyrene, polyester, polyurethane, polyethylene, polypropylene, polyvinyl alcohol, (meth) acrylic polymers, (meth) acrylamide polymers, silicone polymers, epoxy resins, synthetic polymers such as urethane resins, cellulose, and the like. Natural polymers such as dextran, collagen, polygalacturonic acid, polyargicic acid, and gelatin can be mentioned, but are not limited thereto.
  • the inorganic substance include, but are not limited to, glass, ceramics, metals, alloys, metal oxides and the like.
  • the carrier one containing at least polystyrene is used.
  • Cell detachment from the cell support can be performed by enzymatically degrading the bond between the carrier and the cell. If the cell support is, for example, alginic acid, the enzyme used for degradation is trypsin.
  • a cell adhesion peptide such as collagen, laminin, fibronectin, or vitronectin or a partial peptide thereof may be bound to the cell support in order to improve the adhesion between the cell and the carrier.
  • cells are cultured by accommodating a predetermined amount of medium, cell support, and cells in the culture tank 1.
  • the cells adhere to the cell support in the medium, and by driving the stirrer 10 in the culture tank 1, the cells grow and proliferate while floating in the medium together with the cell support.
  • the discharge rate of the culture supernatant by the pump 4 may be equal to or lower than the sedimentation rate of the cells 2b (and the cell support 2a) that enter when the culture supernatant is passed through the separation tube 3, for example.
  • the linear velocity is 2.5 cm / min or less.
  • the temperature is adjusted to 30 ° C. to 45 ° C. when the culture supernatant is passed through the separation tube 3 discharged to the outside of the culture tank 1.
  • the culture supernatant can be adjusted to the temperature range by using the adjusting tool 8 for at least a part of the separation tube 3 outside the culture tank 1.
  • the regulator 8 can reduce the temperature difference between the temperature of the culture supernatant near the inner wall of the separation tube 3 and the temperature of the culture supernatant near the center of the separation tube 3, thereby suppressing heat convection. Can be done. As a result, even if cells, for example, cells attached to a cell support are cultured in the culture tank 1, the cells are less likely to be drawn into the separation tube 3, and the cells and the culture supernatant can be efficiently separated. Further, when culturing cells by pulling out only the culture supernatant through the separation tube 3 while adding the medium, the culture supernatant is pulled out at a faster speed as compared with the culture apparatus not provided with the adjusting tool 8. It is possible to add fresh medium at a faster rate. In this way, by discharging the culture supernatant and supplying the fresh medium faster, the cell density in the medium can be increased, and the cell culture can be performed efficiently.
  • the cell culture device 20 shown in FIG. 1 has been used for explanation, but the generation of heat convection inside the separation tube 3 is suppressed to reduce the contribution of the downward liquid flow, and the cells adhere to the cell support.
  • the configuration of the cell culture device 20 is not limited as long as the drawing of the cells into the separation tube 3 can be suppressed.
  • an embodiment including a separation tube in which the diameter of the separation tube 3 is smaller than the position outside the culture tank 1, a separation tube in which the direction of the separation tube 3 is changed horizontally from the position outside the culture tank 1 An embodiment to be provided, or an embodiment in which these are combined may be used.
  • a culture tank having a capacity of 0.5 L to 50 L, a separation tube extending from the inside to the outside of the culture tank and having an inner diameter of 0.5 cm to 5 cm, and a separation tube.
  • a pump connected to the culture tank, an intake port and an exhaust port provided in the culture tank, a medium supply port provided in the culture tank, and at least a part of the separation tube, and the separation tube is set to 30 ° C. to 45 ° C.
  • the intake port may include a filter.
  • FIG. 2 shows a schematic view showing an example of the cell culture apparatus according to this embodiment. Note that, in FIG. 2, the same reference reference numerals may be given to the configuration requirements common to those in FIG. 1, and the description may be omitted.
  • the cell culture device 22 shown in FIG. 2 includes a culture tank 1, a separation tube 3, a pump 4, an intake port 5, an exhaust port 6, a medium supply port 7, a regulator 8, and a medium component concentration measuring device. A 16 and an observation device 18 are provided.
  • the culture tank 1 the separation tube 3, the pump 4, the intake port 5, the exhaust port 6, the medium supply port 7, and the adjusting tool 8, the ones described in FIG. 1 above may be used.
  • the concentration measuring device 16 measures the concentration of at least one or more medium components in the medium in the culture tank.
  • the concentration measuring device 16 is not particularly limited, and for example, a measuring device based on near-infrared spectroscopy (NIR), Raman spectroscopy, enzyme electrode method, or the like can be used. Thereby, the change in the concentration of the medium component can be monitored by measuring the medium component.
  • the concentration of the medium component may be quantified using a calibration model.
  • the calibration model means a model in which the medium component concentration is calculated based on the measured values acquired by the concentration measuring device such as the spectral intensity, the current value, and the voltage value.
  • the method described in Anal. Chem. 2020, 92, 2946-2952 can be used, but the method is not limited thereto.
  • the observation device 18 observes the state of cells in the culture tank.
  • the observing device 18 is not particularly limited as long as it is a device capable of observing the state of cells, and may be an imaging device such as a camera, or an electrical measuring device including an electrical measuring unit. It may be both, or it may be another device.
  • the electric measurement unit can be connected to, for example, two or more conductive parts provided in the culture tank, and a direct current or an alternating current can be applied between the two or more electrodes, and the electric capacity or conductivity can be set to voltage. By changing the current, frequency, etc., it is possible to observe the inside of the incubator.
  • the cell culture device 30 shown in FIG. 3 includes a culture tank 1, a medium supply path 32, a discharge flow path 34, a NIR measuring unit 36 which is a concentration measuring device, and a capacitance sensor probe 38 of an electric measuring device. ..
  • a culture tank 1 includes a culture tank 1, a medium supply path 32, a discharge flow path 34, a NIR measuring unit 36 which is a concentration measuring device, and a capacitance sensor probe 38 of an electric measuring device. ..
  • the members not particularly described those described in FIGS. 1 and 2 may be used, and the members described in FIGS. 1 and 2 may be applied to the cell culture apparatus 30 shown in FIG.
  • the medium supply path 32 conducts from the storage unit 32a containing the fresh medium to the culture tank 1, and the fresh medium is supplied from the storage unit 32a to the culture tank 1 by driving the pump 32b.
  • the opening of the medium supply path 32 on the culture tank 1 side functions as a medium supply port.
  • the discharge flow path 34 conducts from the culture tank 1 to the accommodating portion 34a accommodating the excretion medium, and discharges the medium discharged by the drive of the pump 34b from the culture tank 1 to the accommodating portion 34a.
  • the portion of the discharge channel 34 extending from the inside to the outside of the culture tank 1 also functions as a separation tube.
  • the culture medium 39c and the cells 39d adhered to the support are housed in the culture tank 1.
  • the culture tank 1 may further include a stirrer 40 such as a stirrer blade, whereby the medium can be agitated during cell culture.
  • FIG. 3 shows a cell culture device 30 capable of monitoring by NIR.
  • the cell culture apparatus 30 is provided with a medium supply path 32 for supplying a fresh medium to the culture tank 1 and a discharge flow path 34 for discharging the used medium (culture supernatant) from the culture tank 1.
  • the culture tank 1 side of the discharge flow path 34 also serves as a separation pipe 3 (see FIG. 1).
  • a measuring unit 36 by NIR is provided on the discharge flow path 34, and the concentration of the component in the medium discharged from the culture tank 1, for example, the concentration of glutamine in the medium component is measured.
  • the drive of the pump provided in the cell culture device 30 is controlled based on the measurement result.
  • the pump whose drive is controlled may be a pump 32b for the culture medium supply path 32, a pump 34b for the discharge flow path 34, or a combination thereof.
  • the amount of fresh medium supplied to the culture tank 1 is adjusted according to the monitored concentration change of the medium component, and the amount of the medium component in the culture tank 1 is optimized.
  • the concentration of the medium component to be measured can be adjusted within a predetermined range. This makes it possible to efficiently carry out suspension culture of cells while monitoring changes in the concentration of medium components.
  • the pump 32b of the culture medium supply path 32 can be controlled based on the information obtained by the measuring unit 36.
  • the cell culture device 30 is provided with a capacitance sensor probe 38 as an electrical measurement device, and the number of cells (cell concentration) in the culture tank 1 is electrically measured.
  • the driving of the stirrer 10 in the culture tank 1 is controlled based on the measurement result, and the number of cells in the culture tank 1 is optimized. As a result, suspension culture of cells can be efficiently performed while monitoring the number of cells in the culture tank 1.
  • the cell number may be monitored in real time by a monitor for the result of electrical measurement.
  • the measuring unit 36 concentrate measuring apparatus
  • the sensor probe 38 observing apparatus control the driving of the pump and the stirring blade, respectively, and adjust the concentration of the medium component in the culture tank 1 and adjust the concentration.
  • the cell state in the culture tank 1 is not limited to this as long as it can be controlled.
  • the cell culture device 30 includes both a concentration measuring device and an observing device, but either one may be used.
  • the measuring unit 36 may measure the concentration of the medium component in the medium supplied to the culture tank 1. As a result, it is possible to more accurately monitor the change in the concentration of the medium component in the culture tank 1 from the concentration of the medium component in the medium supplied to the culture tank 1 and the concentration of the medium component discharged from the culture tank 1. Therefore, the concentration of the medium component in the culture tank 1 can be adjusted more precisely.
  • the cell culture device 30 includes one concentration measuring device 16 and one cell observing device 18, but one or more of them may be independently provided. Further, although the cell culture device 30 includes both the concentration measuring device 16 and the cell observing device 18, only the concentration measuring device 16 or the cell observing device 18 may be used. Further, the cell culture device 30 may be provided with an adjusting tool for preventing the entrainment of cells at the time of discharging the medium, and may not be provided with the adjusting tool as long as it has another entrainment prevention device. .. Examples of other entrainment prevention devices include a filter provided near the opening of the discharge channel on the culture tank side, a baffle for controlling convection in the culture tank, and the like.
  • another embodiment of the present disclosure includes a culture tank having a capacity of 0.5 L to 50 L, a separation tube extending from the inside to the outside of the culture tank and having an inner diameter of 0.5 cm to 5 cm. It is provided with a pump connected to a separation tube, an intake port and an exhaust port provided in the culture tank, a medium supply port provided in the culture tank, and at least one of a medium component concentration measuring device and a cell observation device.
  • Cell culture apparatus can be provided.
  • Yet another embodiment of the present disclosure is to suspend and culture cells adhering to a cell support in a culture tank provided with a separation tube having an inner diameter of 0.5 cm to 5 cm extending from the inside to the outside. It is possible to provide a culture method including discharging the culture supernatant in the culture tank to the outside of the culture tank through a separation tube, and monitoring at least one of the cell number and the medium component.
  • the culture supernatant may be adjusted to a temperature of 30 ° C. to 45 ° C. as it passes through at least a part of the separation tube discharged to the outside of the culture tank.
  • the capacity of this culture tank may be 0.5 L to 50 L.
  • the culturing method according to this embodiment can be carried out using the cell culturing apparatus shown in FIGS. 2 and 3, and the detailed method can be carried out according to the above method, but the method is not limited thereto.
  • a method for monitoring at least one of the cell number and the medium component for example, the above-mentioned medium component concentration measuring device, cell observing device, or a combination thereof can be used.
  • Any of the above-mentioned cell culture devices can perform perfusion culture. Any of the above-mentioned culture methods can be applied to the method of performing perfusion culture.
  • the numerical range indicated by using "-" indicates a range including the numerical values before and after "-" as the minimum value and the maximum value, respectively.
  • the upper limit value or the lower limit value of the numerical range of one step can be arbitrarily combined with the upper limit value or the lower limit value of the numerical range of another step.
  • the materials exemplified in the present specification may be used alone or in combination of two or more.
  • the content of each component in the composition is the total amount of the plurality of substances present in the composition unless otherwise specified, when a plurality of substances corresponding to each component are present in the composition. Means.
  • the term "process” is included in this term not only in an independent process but also in the case where the desired action of the process is achieved even if it cannot be clearly distinguished from other processes.
  • Example 1 Cells were cultured using the cell culture device described above. Specifically, human mesenchymal stem cells were cultured in a culture tank having a capacity of 2 L. The medium was 1.0 L ⁇ MEM supplemented with 10% FBS. Soluble microcarriers (Corning), which is a cell support, were added to the medium together with the cells, and the temperature was maintained at 37 ° C. The stirring blade provided in the culture tank was rotated at 140 rpm.
  • the distance D between the opening surface at the inner end of the culture tank and the inner bottom surface of the culture tank is about 9 cm, and the separation tube outside the culture tank is provided with a heat insulating material as an adjusting tool.
  • the opening surface of the separation tube at the inner end of the culture tank was about 1 cm from the liquid level of the medium 2c.
  • Example 2 The cells were cultured in the same manner as in Example 1 except that the distance D was changed to about 5 cm.
  • the opening surface of the separation tube at the inner end of the culture tank was about 5 cm from the liquid level of the medium 2c.
  • Example 1 The cells were cultured in the same manner as in Example 1 except that the heat insulating material as an adjusting tool was not provided.
  • Example 1 The culture efficiencies in Example 1, Example 2 and Comparative Example 1 were evaluated based on the amount of cell supports drawn into the recovery tank by driving the pump. The cells were attached to the cell support recovered in the recovery tank. In the cell culture apparatus of Example 1, Example 2 and Comparative Example 1, the drain rate was changed as shown in Table 1, and the cell support reached the recovery tank when the culture supernatant was discharged at each drain rate. Concentration (g / L) was measured. The results are shown in Table 1. Note that "-" in Table 1 means that the measurement has not been performed.
  • Example 1 From Table 1, in Examples 1 and 2 in which the heat insulating material was provided in the separation tube, the cell supports collected in the recovery tank at the same drain rate as compared with Comparative Example 1 in which the heat insulating material was not provided in the separation tube. It can be seen that the amount of is low. Further, for example, as a result of comparison at a drain rate at which 0.253 g / L of cell support was recovered, Comparative Example 1 was 1.5 mL / min, but Example 1 was more than doubled to 3.7 mL / min. Therefore, it was found that in Example 2, the culture supernatant could be discharged while being well separated from the cell support even at a faster rate than in Example 1.
  • Example 1 and Example 2 were separated more efficiently in Example 1 and Example 2 than in Comparative Example 1. It was also found that the closer the opening of the separation tube is to the inner bottom surface of the culture tank, the more efficiently the cells and the culture supernatant can be separated. Therefore, cells can be efficiently cultured by using this cell culture device. Further, in the above-described embodiment, cell culture can also be performed even when a device for measuring the concentration of the medium by near-infrared spectroscopy is provided in the separation tube.
  • the code of the drawing is as follows. 1 culture tank; 2a cell support; 2b cells; 2c medium; 3 separation tube; 4 pump; 5 intake port; 6 exhaust port; 7 medium supply port; 8 regulator; 9 recovery tank; 10 stirrer (stirring blade); 11 Filter; 16 Concentration measuring device; 18 Observation device; 20 Cell culture device; 22 Cell culture device; 30 Cell culture device; 32 Medium supply path; 34 Discharge channel; 36 Measuring unit; 38 Sensor probe.

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