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US-12624330-B2 - Cell culture method, product producing method, and cell culture device

US12624330B2US 12624330 B2US12624330 B2US 12624330B2US-12624330-B2

Abstract

Provide are a cell culture method which includes using a culture vessel which contains a cell suspension containing cells and discharging a gas including 30% by volume or more of oxygen into the cell suspension from a sparger disposed in the culture vessel to culture the cells, in which an average hole diameter in a gas discharge portion in the sparger is 1 μm or more and 300 μm or less, and a surface area A (m 2 ) in the gas discharge portion in the sparger, a volume X (m 3 ) of the cell suspension in the culture vessel, and a flow rate Q (m 3 /min) of the gas that is discharged from the sparger satisfy a specific relationship, a product producing method including the cell culture method, and a cell culture device that enables the cell culture method.

Inventors

  • Shinichi Nakai
  • Kosuke Taniguchi
  • Nobuyuki Haraguchi
  • Tsukasa Ishihara

Assignees

  • FUJIFILM CORPORATION

Dates

Publication Date
20260512
Application Date
20201125
Priority Date
20180627

Claims (17)

  1. 1 . A cell culture method comprising: using a culture vessel which contains a cell suspension; and discharging a gas including 30% by volume or more of oxygen into the cell suspension from a sparger disposed in the culture vessel to culture the cells, wherein a plurality of spargers are included as the sparger, an average hole diameter of a gas discharge portion in each of the spargers is 1 m or more and 300 m or less, and a surface area A (m 2 ) of the gas discharge portion in the sparger, a volume X (m 3 ) of the cell suspension in the culture vessel, and a flow rate Q (m 3 /min) of the gas that is discharged from the sparger satisfy Expression 1-1, Expression 1-2, and Expression 1-3, wherein 0.004≤A/X≤0.025, Expression 1-1 X≥0.5, and Expression 1-2 0.001≤Q/X≤0.05 Expression 1-3 wherein a cell concentration in the cell suspension is 4×10 7 cells/mL or more.
  2. 2 . The cell culture method according to claim 1 , wherein a bubble diameter distribution in a gas discharge outlet of the sparger is measured by replacing the cell suspension in the culture vessel with a measurement solution which is pure water containing 1 g/L of poloxamer 188, 7 g/L of sodium chloride, and sending 2 g/L of sodium hydrogen carbonate, an air gas having the same flow rate as the Q (m 3 /min) in the culture from the sparger to the measurement solution, where a volume-average diameter Dv (μm) of bubbles of the air gas satisfies Expression 2-1, wherein 50≤Dv≤800 Expression 2-1.
  3. 3 . The cell culture method according to claim 1 , wherein a bubble diameter distribution in a gas discharge outlet of the sparger is measured by replacing the cell suspension in the culture vessel with a measurement solution which is pure water containing 1 g/L of poloxamer 188, 7 g/L of sodium chloride, and 2 g/L of sodium hydrogen carbonate, and sending an air gas having the same flow rate as the Q (m 3 /min) in the culture from the sparger to the measurement solution, where a proportion of a cumulative volume of air bubbles having a bubble diameter of 20 m or more and 500 m or less is 30% by volume or more of a total volume of the bubbles in the bubble diameter distribution.
  4. 4 . The cell culture method according to claim 1 , wherein an oxygen transfer capacity coefficient kLa (hr −1 ) by the sparger in the culture is 22 or more.
  5. 5 . The cell culture method according to claim 1 , wherein a bubble diameter distribution in a gas discharge outlet of the sparger is measured by replacing the cell suspension in the culture vessel with a measurement solution which is pure water containing 1 g/L of poloxamer 188, 7 g/L of sodium chloride, and 2 g/L of sodium hydrogen carbonate, and sending an air gas having the same flow rate as the Q (m 3 /min) in the culture from the sparger to the measurement solution, where the Q (m 3 /min), the A (m 2 ), a density ρL (kg/m 3 ) of the measurement solution, a density ρg (kg/m 3 ) of the air gas, a viscosity μL (kg/m/s) of the measurement solution, and a gravitational acceleration g (m/s 2 ) satisfy Expression 3-1, wherein 0.1<(Q/A/60)/[{3×10 −8 ×(ρL−ρg)×g}/(18×μL)}]≤5 Expression 3-1.
  6. 6 . The cell culture method according to claim 1 , wherein in the culture, the Q (m 3 /min) and the A (m 2 ) satisfy Expression 4-1, wherein 0.1≤Q/A≤5 Expression 4-1.
  7. 7 . The cell culture method according to claim 1 , wherein a bubble diameter distribution in a gas discharge outlet of the sparger is measured by replacing the cell suspension in the culture vessel with a measurement solution which is pure water containing 1 g/L of poloxamer 188, 7 g/L of sodium chloride, and 2 g/L of sodium hydrogen carbonate, and sending an air gas having the same flow rate as the Q (m 3 /min) in the culture from the sparger to the measurement solution, where a liquid level ZL (m) from an inner bottom surface of the culture vessel to an upper surface of the cell suspension in the culture, a height Zs (m) from the inner bottom surface of the culture vessel to a sparger installation surface, a volume average bubble diameter Dv (μm) of bubbles in the bubble diameter distribution, a density ρL (kg/m 3 ) of the measurement solution, a density ρg (kg/m 3 ) of the air gas, a gravitational acceleration g (m/s 2 ), and a viscosity μL (kg/m/s) of the measurement solution satisfy Expression 5-1, wherein 2<(ZL−Zs)/{Dv 2 ×10 −12 ×(ρL−ρg)×g/(18×μL)}≤300 Expression 5-1.
  8. 8 . The cell culture method according to claim 1 , wherein the X (m 3 ), a liquid level ZL (m) from an inner bottom surface of the culture vessel to an upper surface of the cell suspension, and a height Zs (m) from the inner bottom surface of the culture vessel to a sparger installation surface satisfy Expression 6-1 and Expression 6-2, wherein Zs/ZL≤0.5, and Expression 6-1 0.5≤ZL/(4×X/ZL/3.14) 0.5 ≤4 Expression 6-2.
  9. 9 . The cell culture method according to claim 1 , wherein the cell culture is a perfusion culture.
  10. 10 . The cell culture method according to claim 1 , wherein the culture vessel is a single-use culture tank.
  11. 11 . The cell culture method according to claim 1 , wherein the sparger is a sintered metal sparger.
  12. 12 . The cell culture method according to claim 1 , wherein a shape of the gas discharge portion of the sparger is a circular plane shape, a polygonal plane shape, or a cylinder shape.
  13. 13 . The cell culture method according to claim 1 , wherein the sparger is configured to include a plurality of units each including one or more spargers, and the flow rate of the gas is adjusted for each of the units.
  14. 14 . The cell culture method according to claim 1 , wherein the volume X (m 3 ) of the cell suspension in the culture vessel satisfies Expression 1-2, wherein X≥1.1 Expression 1-2.
  15. 15 . The cell culture method according to claim 1 , wherein a total value of the surface area of the gas discharge portion in the sparger which satisfies the average hole diameter is 60% by area or more.
  16. 16 . A product producing method comprising: culturing cells with the cell culture method according to claim 1 , and obtaining a product produced by the cultured cells.
  17. 17 . The product producing method according to claim 16 , wherein the product is an antibody.

Description

CROSS-REFERENCE TO RELATED APPLICATION This application is a continuation application of International Application No. PCT/JP2019/020717, filed May 24, 2019, the disclosure of which is incorporated herein by reference in its entirety. Further, this application claims priority from Japanese Patent Application No. 2018-122322, filed Jun. 27, 2018, the disclosure of which is incorporated herein by reference in their entirety. BACKGROUND OF THE INVENTION 1. Field of the Invention The present disclosure relates to a cell culture method, a product producing method, and a cell culture device. 2. Description of the Related Art Cell culture is performed for the purpose of increasing the number of cells having useful properties, causing cells to produce a product or the like. As the conventional cell culture method or device used for cell culture, JP1993-503848A (JP-H05-503848A), WO2011/070791A, and JP2016-536122A are mentioned. JP1993-503848A (JP-H05-503848A) discloses a method for supplying gaseous nutrients on a bubble form to cells in a bioreactor at least partially filled with a medium, which is characterized by the point that the retention time of the bubble is equal to the dissolution time of the gaseous nutrients in the medium. WO2011/070791A discloses a culture method for culturing cells or microorganisms by dissolving oxygen or carbon dioxide in a culture solution containing nutrients, including a process of culturing cells or microorganisms by supplying a gas including oxygen or carbon dioxide to a porous body to generate bubbles having a 50% diameter of 200 μm or less in a culture solution, the 50% diameter being obtained in the volume-based size distribution and the culture solution containing at least one of a protein hydrolyzate or a cytoprotective agent for protecting cells, thereby dissolving oxygen or carbon dioxide in the culture solution containing nutrients. JP2016-536122A discloses an aeration device as an aeration device used at the time of cell culture, which includes a base member and a plurality of aeration elements that are installed attachably and detachably to the base member, each of the aeration elements including a gas permeable material and an inlet which is configured to connect to a gas source and being in fluid communication with the gas permeable material. SUMMARY OF THE INVENTION Cell culture may be performed while discharging a gas including oxygen into a cell suspension containing cells. It is speculated that the discharge causes oxygen to be supplied to the cell suspension. In a case of performing cell culture, there is a demand to increase the cell concentration in the cell suspension as high as possible. Particularly, in the production of biomedical drugs such as in a case where a product (for example, antibody) is produced by cell culture, it is important to increase the cell concentration in the cell suspension in order to improve the productivity of the product. For this reason, it is necessary to dissolve a large amount of oxygen in the cell suspension and supply a sufficient amount of oxygen to cells having a high cell concentration. As a method of discharging oxygen to a cell suspension, a method in which a sparger is installed in the culture vessel is known. In order to discharge a sufficient amount of oxygen to cells, a technique of dissolving a large amount of oxygen in a cell suspension was used as a general technique. In this method, an average hole diameter in a gas discharge portion in a sparger is minimized to improve the solubility of oxygen and the flow rate of the gas including oxygen is increased (refer to, for example, JGC Technical Journal, Vol. 01, No. 01 (2011), Application of microbubbles to culture). However, the inventors of the present invention noticed that in a case where the cell suspension volume is large (for example, the cell suspension volume is 0.5 m3 or more) as in the case such as a mass production scale, it is difficult to culture cells at a high cell concentration with the method such as the method described above, since a sufficient amount of oxygen for cell culture cannot be dissolved in the cell suspension and the damage to cells is large. This is presumably because oxygen is difficult to be dissolved in the cell suspension by the above method, the oxygen transfer capacity coefficient kLa (hereinafter, may be referred to as “kLa”) is not so high, the damage to cells due to the discharged bubbles is increased as the average hole diameter of the gas discharge portion in the sparger is decreased, and the damage to cells is increased due to the discharge of a gas including a large amount of oxygen. Specifically, it is speculated as follows; when a gas including oxygen is discharged from the sparger, cells are damaged by the energy transferred when bubbles collide with the cells or the bubbles break themselves, but the energy increases as the sparger hole diameter is decreased, that is, as the bubbles having small diameters are present, an