WO2019239780A1 - Cell culture method, cell culture device, and method for producing product - Google Patents

Cell culture method, cell culture device, and method for producing product Download PDF

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Publication number
WO2019239780A1
WO2019239780A1 PCT/JP2019/019361 JP2019019361W WO2019239780A1 WO 2019239780 A1 WO2019239780 A1 WO 2019239780A1 JP 2019019361 W JP2019019361 W JP 2019019361W WO 2019239780 A1 WO2019239780 A1 WO 2019239780A1
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cell
solution
cells
culture
concentration
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French (fr)
Japanese (ja)
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真一 中居
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富士フイルム株式会社
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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
    • C12M1/02—Apparatus for enzymology or microbiology with agitation means; with heat exchange means
    • 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
    • C12M1/00—Apparatus for enzymology or microbiology
    • C12M1/42—Apparatus for the treatment of microorganisms or enzymes with electrical or wave energy, e.g. magnetism, sonic waves
    • 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
    • C12M3/02—Tissue, human, animal or plant cell, or virus culture apparatus with means providing suspensions
    • 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
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor

Definitions

  • the present disclosure relates to a cell culture method, a cell culture apparatus, and a product manufacturing method.
  • the cell culture is performed for the purpose of increasing the number of cells having useful properties and causing the cells to produce a product.
  • Japanese Patent Application Laid-Open No. 2008-263847 discloses a container for storing a cell suspension containing a plurality of types of cells having different dielectrophoretic characteristics, an electrode disposed in the container, and a periphery of the electrode
  • a cell separation device that includes an electric field control unit that applies a voltage so as to generate a gradient of the electric field strength, and collects any cell on the electrode.
  • Japanese Patent Publication No. 2017-502666 discloses a bioreactor having an acoustic cell separation device and a filter.
  • a living cell When a living cell is cultured, a certain percentage of the living cells are generally killed, resulting in a mixture of living and dead cells.
  • the dead cells When dead cells coexist with living cells, the dead cells inhibit the growth of living cells, inhibit the metabolism of living cells, etc., resulting in a decrease in the growth of living cells and the production of products produced by living cells. Degradation of quality occurs.
  • live cells and dead cells are similar in characteristics such as size, and it is generally difficult to separate dead cells and live cells contained in a culture solution.
  • the present inventor has conceived of separating dead cells and living cells by using a dielectrophoresis technique as described in JP-A-2008-263847.
  • a living cell since the cell membrane separates the extracellular space and the intracellular space, the dielectric constant of the liquid in the living cell is different from the dielectric constant of the liquid outside the living cell.
  • the cell membrane of dead cells can only block or be insufficient between the outside and inside of the cell, and as a result, the liquid inside the dead cell can be a liquid similar to or similar to the liquid existing outside the dead cell. Since the liquid has a close composition, the dielectric constant of the liquid inside the dead cell is almost the same as the dielectric constant of the liquid existing outside the dead cell. For this reason, the present inventor expected that dead cells and living cells can be efficiently separated by using the dielectrophoresis technique.
  • Culturing at a high cell concentration is preferable because the amount of viable cells obtained is increased, and when the cells are cells that produce a substance, the amount of the product obtained is increased.
  • the cell concentration increases, there arises a problem that the separation efficiency of live cells and dead cells by dielectrophoresis decreases as described above.
  • the cell suspension is directly fed from the cell suspension container to a treatment tank for performing dielectrophoresis, and the separation efficiency of living cells and dead cells is increased when the cell concentration in the cell suspension container is increased. Will fall.
  • the present inventor does not directly subject the cell suspension in the culture vessel to the treatment by dielectrophoresis, but performs the treatment by dielectrophoresis after performing the treatment by reducing the cell concentration in the cell suspension. The technical idea that this problem can be solved was obtained.
  • the problems to be solved by the embodiments of the present invention include a cell culture method and a cell culture apparatus for removing dead cells in a culture vessel while culturing cells at a high concentration, and a culture vessel while culturing cells at a high concentration. It is to provide a method for producing a product using a cell culture method for removing dead cells.
  • Means for solving the above problems include the following aspects. ⁇ 1> Culturing cells in a cell suspension contained in a culture vessel; The cell suspension is extracted from the culture vessel, and the extracted cell suspension is divided into a first solution having a cell concentration higher than the cell concentration in the cell suspension, and the cell suspension. Performing a first cell separation treatment to separate into a second solution having a cell concentration lower than the cell concentration in the liquid; Returning the first solution to the culture vessel; The second solution is subjected to a second cell separation process for separating live cells and dead cells by dielectrophoresis, and the ratio of the number of live cells to the number of dead cells in the second solution is as follows.
  • a cell culture method comprising: ⁇ 2> ⁇ 1> The cell culturing method according to ⁇ 1>, wherein the first cell separation treatment is a cell separation treatment in which cells are captured in a standing wave belly or node using a standing wave sound wave.
  • ⁇ 3> The cell culture method according to ⁇ 1> or ⁇ 2>, wherein the second cell separation treatment includes using an electrode to migrate a living cell in a direction in which the cell is separated from the electrode.
  • the cell suspension extracted from the culture vessel is fed by a pump that floats in the cell suspension and feeds the cell suspension by rotating a rotating blade having a magnet by a magnetic force.
  • ⁇ 5> The second solution is fed by a pump that feeds the second solution by rotating a rotating blade that floats in the second solution and has a magnet by a magnetic force.
  • ⁇ 1> to ⁇ The cell culture method according to any one of 4>.
  • the total cell concentration of live cells and dead cells in the second solution is a concentration of 0.1% or more and 50% or less with respect to the total cell concentration of live cells and dead cells in the cell suspension in the culture container.
  • the cell culture method according to any one of ⁇ 1> to ⁇ 5>, wherein ⁇ 7> The cell culture method according to any one of ⁇ 1> to ⁇ 6>, wherein the viable cell concentration in the fourth solution is 50% or less of the viable cell concentration in the second solution.
  • the living cell concentration in the cell suspension in the culture container is 20 ⁇ 10 6 cells / ml or more and 150 ⁇ 10 6 cells / ml or less, according to any one of ⁇ 1> to ⁇ 7> Cell culture method.
  • the living cell concentration in the cell suspension in the culture vessel is 80% or more and 99.9% or less of the total cell concentration of the living cells and dead cells in the cell suspension in the culture vessel.
  • ⁇ 10> Any one of ⁇ 1> to ⁇ 9>, wherein the total cell concentration of the living cells and dead cells in the second solution is 0.5 ⁇ 10 6 cells / ml or more and 50 ⁇ 10 6 cells / ml or less.
  • ⁇ 11> The cell culture according to any one of ⁇ 1> to ⁇ 10>, wherein the dead cell concentration in the fourth solution is 0.01 ⁇ 10 6 cells / ml or more and 10 ⁇ 10 6 cells / ml or less.
  • Method. ⁇ 12> The living cell concentration in the fourth solution is 20% or less with respect to the total cell concentration of living cells and dead cells in the fourth solution, according to any one of ⁇ 1> to ⁇ 11> Cell culture method.
  • ⁇ 15> Culturing cells that produce the product using the cell culture method according to any one of ⁇ 1> to ⁇ 14>; Recovering the product from the fourth solution;
  • a method for producing a product comprising: ⁇ 16> The method for producing a product according to ⁇ 15>, wherein the product is an antibody.
  • a culture vessel containing the cell suspension; The cell suspension extracted from the culture vessel is divided into a first solution having a cell concentration higher than the cell concentration in the cell suspension, and cells lower than the cell concentration in the cell suspension.
  • the second solution is subjected to a second cell separation process for separating live cells and dead cells by dielectrophoresis, and the ratio of the number of live cells to the number of dead cells in the second solution is as follows.
  • the first cell separation processing unit is a cell separation processing unit that captures cells in a standing wave belly or node using a standing wave sound wave.
  • the second cell separation processing unit includes an electrode, and the second cell separation processing unit migrates live cells in a direction separating the electrode from the electrode.
  • the cell suspension is sent from the culture vessel by rotating magnetically rotating a rotating blade provided between the first cell separation processing unit and suspended in the cell suspension and having a magnet.
  • the cell culture device according to any one of ⁇ 17> to ⁇ 19>, further comprising a pump.
  • a rotating blade provided between the first cell separation processing unit and the second cell separation processing unit floating in the second solution and having a magnet by the magnetic force, the second The cell culture device according to any one of ⁇ 17> to ⁇ 20>, further comprising a pump for feeding the solution.
  • a cell culture method and a cell culture apparatus for removing dead cells in a culture vessel while culturing cells at a high concentration, and dead cells in a culture vessel while culturing cells at a high concentration
  • a method for producing a product using the cell culture method to be removed is provided.
  • a numerical range indicated by using “to” means a range including the numerical values described before and after “to” as the minimum value and the maximum value, respectively.
  • an upper limit value or a lower limit value described in a numerical range may be replaced with an upper limit value or a lower limit value in another numerical range.
  • the upper limit value or the lower limit value described in a certain numerical range may be replaced with the values shown in the examples.
  • a combination of two or more preferred embodiments is a more preferred embodiment.
  • the amount of each component means the total amount of a plurality of types of substances unless there is a specific case when there are a plurality of types of substances corresponding to each component.
  • the term “process” includes not only an independent process but also a process that cannot be clearly distinguished from other processes as long as the intended purpose of the process is achieved.
  • the cell culture method includes: Culturing cells in a cell suspension contained in a culture vessel; The cell suspension is extracted from the culture vessel, and the extracted cell suspension is divided into a first solution having a cell concentration higher than the cell concentration in the cell suspension, and the cell suspension. Performing a first cell separation treatment to separate into a second solution having a cell concentration lower than the cell concentration in the liquid; Returning the first solution to the culture vessel; The second solution is subjected to a second cell separation process for separating live cells and dead cells by dielectrophoresis, and the ratio of the number of live cells to the number of dead cells in the second solution is as follows.
  • a cell culture method comprising:
  • the cell suspension extracted from the culture vessel is divided into a first solution having a cell concentration higher than the cell concentration in the cell suspension, and the cell suspension.
  • a first cell separation process is performed for separation into a second solution having a cell concentration lower than the cell concentration.
  • the second solution is subjected to a second cell separation process by dielectrophoresis.
  • the cell concentration in the second solution subjected to dielectrophoresis is lower than the cell concentration in the cell suspension accommodated in the culture vessel, and the separation of live cells and dead cells by dielectrophoresis is performed. Efficiency is improved.
  • the first solution, the second solution, the third solution, and the first solution are distinguished from the cell suspension in the culture vessel and the cell suspension extracted from the culture vessel. Although the term “four solutions” is used, these solutions are actually cell suspensions.
  • the cells cultured in the cell culture method according to the present disclosure are not particularly limited, and examples thereof include eukaryotic cells such as animal cells, plant cells, and yeast, and prokaryotic cells such as Bacillus subtilis and Escherichia coli.
  • the cells may be ES cells, IPS cells, various stem cells, and the like.
  • the cell cultured in the cell culture method according to the present disclosure may be a cell that produces a product. When cells that produce a product are cultured, the product is produced by the cell, and if this is recovered, substance production using the cell can be performed.
  • the cells used as the cells for producing the product are not particularly limited, and may be any of eukaryotic cells such as animal cells, plant cells, and yeast, and prokaryotic cells such as Bacillus subtilis and Escherichia coli.
  • Animal cells such as CHO cells, BHK-21 cells, C127 cells, hybridoma cells, NS0 cells, and SP2 / 0-Ag14 cells are preferred, and CHO cells are known because many analyzes have been performed and genetic engineering techniques have been established. Is more preferable. Even if the cells do not originally produce the desired product or the production volume is small, for example, by introducing an expression vector such as a plasmid encoding a protein necessary for producing the product into the cell, the desired product can be obtained. Can be produced efficiently.
  • the product produced by the cells in the present disclosure is not particularly limited as long as it is a substance produced by the cells in the culture solution, and examples thereof include alcohols, enzymes, antibiotics, nucleic acids, recombinant proteins, and antibodies. Substances. Among them, the product is preferably a recombinant protein or an antibody, more preferably an antibody.
  • the type of antibody is not particularly limited.
  • Examples of antibodies to be produced include not only monoclonal antibodies derived from animals such as humans, mice, rats, hamsters, rabbits, monkeys, but also artificially modified antibodies such as chimeric antibodies, humanized antibodies, and bispecific antibodies. included.
  • concentration of the cell in the cell suspension accommodated in the culture container is not specifically limited. However, considering that the higher the cell concentration in the solution subjected to dielectrophoresis, the lower the efficiency of separating live and dead cells by dielectrophoresis, the cell suspension contained in the culture vessel The higher the cell concentration is, the more prominent the effect of the cell culture method according to the present disclosure is. In addition, the higher the cell concentration in the cell suspension contained in the culture vessel, the larger the cell volume, and the greater the production volume of the product when the cells produce the product. However, it is preferable that the cell concentration is high.
  • the concentration of the cells in the cell suspension accommodated in the culture vessel is preferably 20 ⁇ 10 6 cells / ml or more and 150 ⁇ 10 6 cells / ml or less.
  • the concentration of the cells in the cell suspension accommodated in the culture vessel is 20 ⁇ 10 6 cells / ml or more, the amount of cells can be increased, and for example, the cells produce the product. In some cases, the output of the product can be increased.
  • the concentration of the cells in the cell suspension accommodated in the culture vessel is 150 ⁇ 10 6 cells / ml or less
  • the concentration of cells in the cell suspension accommodated in the culture vessel is more preferably 30 ⁇ 10 6 cells / ml or more and 120 ⁇ 10 6 cells / ml or less, and 40 ⁇ 10 6 cells / ml or more and 100 ⁇ 10 6 cells.
  • / Ml or less is more preferable, and 50 ⁇ 10 6 cells / ml or more and 100 ⁇ 10 6 cells / ml or less is more preferable.
  • a liquid medium usually used for cell culture can be used.
  • OptiCHO Lifetechnologies, 12681011
  • medium Dulbecco's modified Eagle medium (DMEM), Eagle minimum essential medium (MEM), RPMI-1640 medium, RPMI-1641 medium, F-12K medium, Ham F12 medium, Iscob modified method Dulbecco's medium (IMDM), McCoy's 5A medium, Leibovitz L-15 medium, and EX-CELL TM 300 series (JRH Biosciences)
  • CHO-S-SFMII Invitrogen
  • CHO-SF Sigma-Aldrich
  • CD-CHO Invitrogen
  • IS CHO-V Irvine Scientific
  • PF-ACF-CHO Sigma-Aldrich
  • Serum such as fetal calf serum (FCS) may be added to the medium.
  • the medium may be a serum-free medium, such as a fully synthetic medium.
  • the medium may be supplemented with additional components such as amino acids, salts, sugars, vitamins, hormones, growth factors, buffers, antibiotics, lipids, trace elements, plant protein hydrolysates and the like.
  • the pH of the medium varies depending on the cells to be cultured, but is generally pH 6.0 to 8.0, preferably pH 6.8 to 7.6, and more preferably pH 7.0 to 7.4.
  • the culture temperature is generally 30 ° C. to 40 ° C., preferably 32 ° C. to 37 ° C., more preferably 36 ° C. to 37 ° C., and the culture temperature may be changed during the culture.
  • the culture may be performed in an atmosphere having a CO 2 concentration of 0 to 40%, and is preferably performed in an atmosphere having a CO 2 concentration of 2 to 10%.
  • medium exchange, aeration, and stirring can be added as necessary.
  • the cells can be cultured using a general culture apparatus (also referred to as a bioreactor) or other suitable container as a culture container.
  • a general culture apparatus also referred to as a bioreactor
  • the culture apparatus fermenter tank culture apparatus, air lift culture apparatus, culture flask culture apparatus, spinner flask culture apparatus, microcarrier culture apparatus, fluidized bed culture apparatus, holofiber culture apparatus, roller bottle
  • a mold culture apparatus, a filling tank type culture apparatus, or the like can be used.
  • the culture scale may be, for example, from 0.1 L to 5000 L, preferably from 0.5 L to 3000 L, more preferably from 1 L to 2500 L, and most preferably from 1500 L to 2200 L.
  • the larger the culture scale the more cultures can be performed at one time.
  • the amount of the culture solution is 5000 L or less, the size of the first cell separation processing unit and the size of the second cell separation processing unit necessary for efficient removal of dead cells can be suppressed. Setting the amount of the culture solution to 5000 L or less is preferable from the viewpoint of suppressing the installation area of the first cell separation processing unit and the second cell separation processing unit and the apparatus cost.
  • the cell culture method according to the present disclosure may include stirring the cell suspension in the culture vessel with a stirring blade, or may include gas aeration with a sparger.
  • the cell culture is preferably perfusion culture.
  • Perfusion culture is a culture method in which a fresh medium is added and at the same time the spent medium is removed. With perfusion culture, it is also possible to achieve high cell concentrations exceeding 1 ⁇ 10 8 cells / mL.
  • a typical perfusion culture begins with a batch culture start-up that lasts for one or two days, after which fresh feed medium is added to the culture continuously, stepwise and / or intermittently and spent medium is removed simultaneously. To do.
  • the spent medium refers to a medium containing waste products released from cells whose nutrients are reduced or depleted as a result of being used for cell culture.
  • spent media can be removed while maintaining cell concentration using methods such as sedimentation, centrifugation or filtration.
  • Perfusion may be any form of continuous, gradual, intermittent or a combination thereof.
  • Extraction of the cell suspension from the culture vessel can usually be performed using a pump, but other available liquid feeding means may be used.
  • the cell suspension extracted from the culture vessel is subjected to a first cell separation process. Extraction of the cell suspension from the culture vessel may be performed intermittently or continuously, but it is preferably performed continuously from the viewpoint of keeping the system state stable.
  • the culture container and the first cell separation processing unit may be directly connected, and in that case, a pump may be installed in the flow path for returning the first solution to the culture container.
  • the liquid feeding means for extracting the cell suspension from the culture container and supplying it to the first cell separation processing unit may be in the form of a direct connection between the culture container and the first cell separation processing unit. And a flow path connecting the first cell separation processing unit.
  • the installation position of liquid feeding means such as a pump for generating a flow of the cell suspension from the culture container to the first cell separation processing unit is not particularly limited as long as the flow can be formed.
  • the extraction position of the cell suspension in the culture vessel is not particularly limited as long as it is below the liquid surface of the cell suspension, and can be, for example, near the bottom of the culture vessel.
  • the extracted cell suspension is divided into a first solution having a cell concentration higher than the cell concentration in the cell suspension and lower than the cell concentration in the cell suspension.
  • the treatment is not particularly limited as long as the treatment is separated into a second solution having a cell concentration, and examples thereof include centrifugation treatment, standing wave sound waves, cell separation treatment using a filter, and the like.
  • the first cell separation process is preferably a cell separation process using standing wave sound waves.
  • the cells can be separated into a supernatant liquid having a cell concentration decreased from that before the treatment and a solution having a cell concentration increased from that before the treatment by sedimenting the cells by centrifugal force.
  • a filter with a pore size close to the cell diameter is selected and filtered to obtain a filtrate with a cell concentration reduced from that before the treatment, and a concentrate with a cell concentration increased from that before the treatment. And can be separated. Filtration by a filter can be performed by, for example, a cross flow method.
  • the cells are trapped in standing wave nodes or abdomen using standing wave sound waves, and the aggregated cells are allowed to settle down in the direction of gravity, so that the cell concentration is higher than before treatment. It can be separated into a reduced solution and a solution with an increased cell concentration than before the treatment. Whether an object is trapped by a standing wave node or a belly depends on factors such as the compressibility and density of the object as compared to the surrounding liquid.
  • the supply rate of the cell suspension to the first cell separation process may be set according to the processing capacity of the first cell separation process, and is not particularly limited.
  • the treatment conditions for the first cell separation treatment are such that the total cell concentration of the live and dead cells in the second solution produced by the first cell separation treatment is such that the live and dead cells in the cell suspension in the culture vessel It is preferable to set the concentration to be 0.1% or more and 50% or less with respect to the total cell concentration. It is suppressed that the number of cells subjected to the second cell separation treatment becomes smaller than the processing capacity of the second cell separation treatment by being 0.1% or more, and dead cells are obtained by the second cell separation treatment. Is more efficiently removed.
  • the total cell concentration of live cells and dead cells in the second solution is more preferably 1% or more and 30% or less with respect to the total cell concentration of live cells and dead cells in the cell suspension in the culture container, More preferably, it is 3% or more and 20% or less.
  • the cell separation process using standing wave sound waves will be described more specifically.
  • the treatment with sound waves of standing waves captures the cells in standing wave nodes or abdomen using standing wave sound waves and settles the aggregated cells downward in the direction of gravity, so that the cell concentration and the cell concentration are increased.
  • a process for producing a reduced solution In cell separation processing using standing-wave sound waves, a cell suspension is generally supplied from below in the vertical direction to a standing-wave field of sound waves formed in a liquid.
  • the cells are trapped in the standing wave nodes or abdomen while passing through and the solution with reduced cell concentration is discharged vertically above the standing wave field, while the trapped cells aggregate together. When it reaches a certain size, it settles down and is collected in a collection portion provided vertically below and discharged as a liquid with an increased cell concentration.
  • the vertical direction here may not be strictly the vertical direction, and may be deviated from the vertical direction within a range in which the first liquid and the second liquid having different cell concentrations can be generated.
  • the deviation angle may be, for example, within 30 °, preferably within 20 °, and more preferably within 10 °.
  • cell separation treatment apparatuses using standing wave sound waves that can be used for the first cell separation treatment are also commercially available.
  • products of the Biosep series manufactured by Applikon Biotechnology such as Biosep 1L / Day, Biosep 10L / day, Biosep 50L / day, Biosep 200L / day, Biosep 1000L / day, and the like can be used.
  • An example of the configuration of the cell separation processing unit using standing-wave sound waves is shown in FIG.
  • a cell separation processing unit 300 (hereinafter referred to as “sonic standing wave type cell separation processing unit 300”) using standing-wave sound waves shown in FIG. 2 converts the cell suspension to be processed into a sound wave standing wave.
  • a port 340 for discharging, a port 330 for discharging the first solution having an increased cell concentration, a transducer T for generating a sound wave as a standing wave, and a sound wave reflecting plate M are provided.
  • the cell suspension supplied from the introduction unit 320 to the chamber 350 is exposed to the standing wave S formed by the transducer T and the sound wave reflector M, and the living cells and dead cells in the cell suspension are fixed. It is captured by the node of standing wave S.
  • a cell suspension having a reduced cell concentration corresponding to the trapped cells is discharged from the port 340.
  • the cell suspension settled from the chamber 350 and the cell concentration increased from the port 330 is indicated by the arrow A8. To be discharged.
  • the configuration of the cell separation processing unit using standing-wave sound waves that can be used in the present disclosure is not limited to the configuration illustrated in FIG. 2, but is more than the cell concentration in the cell suspension extracted from the culture vessel. If it is a configuration that can be separated into a first solution having a high cell concentration and a second solution having a cell concentration lower than the cell concentration in the cell suspension extracted from the culture vessel Any configuration can be used. Further, two or more first cell separation processing units may be provided as necessary.
  • the first solution having a cell concentration higher than the cell concentration in the cell suspension resulting from the first cell separation treatment is returned to the culture vessel through the first circulation channel. For this reason, the living cells contained in the first solution are efficiently collected in the culture container.
  • the movement of the first solution toward the culture vessel usually occurs according to the flow formed by the liquid feeding means for extracting the cell suspension from the culture vessel. If necessary, the first solution is cultured.
  • a separate liquid feeding means for example, a pump) for returning the container to the container may be provided.
  • the second solution having a cell concentration lower than the cell concentration in the cell suspension resulting from the first cell separation treatment is a second cell that separates live and dead cells by dielectrophoresis. It is subjected to separation processing. By the second cell separation process, the second solution is transformed into a third solution having a higher ratio of live cells / dead cells than in the second solution, and live in the second solution. And a fourth solution having a live / dead cell ratio lower than the cell / dead cell ratio.
  • the ratio of living cells / dead cells is the ratio of the number of living cells to the number of dead cells, and can be obtained by dividing the number of living cells by the number of dead cells. It can also be determined by dividing the cell concentration by the dead cell concentration.
  • the second cell separation process is a process of separating live cells and dead cells using dielectrophoresis.
  • Dielectrophoresis is a phenomenon in which, for example, an object placed in an alternating electric field moves in a direction in which the electric field strength increases or in the opposite direction according to its dielectric characteristics.
  • the migration characteristics of the object depend on the frequency of the applied AC electric field. Since dead cells and live cells have different dielectric properties as described above, the electrophoretic characteristics of dead cells and live cells can be made different by appropriately setting the voltage application conditions. It becomes possible to separate live cells.
  • the efficiency of separating live and dead cells tends to decrease as the cell concentration of the supplied cell suspension increases.
  • the voltage is increased, damage to living cells increases.
  • the cell concentration in the second solution is reduced from the cell concentration in the cell suspension in the culture container by the first cell separation treatment, Separation efficiency with dead cells can be improved. For this reason, it is not necessary to raise the voltage excessively.
  • the second cell separation process will be described by way of an example in which an alternating voltage is applied between adjacent rod-shaped electrodes using electrodes in which rod-shaped electrodes are arranged in parallel as shown in FIGS.
  • a live cell has a migration characteristic in which the electric field strength is weakened, and a dead cell has no mobility or a mobility that has weak mobility. Electrophoresis is performed in a direction away from the electrode without approaching the periphery of the electrode.
  • the rod-shaped electrode group acts like a kind of shielding plate for living cells.
  • dead cells only move along the flow of the liquid and are not affected or hardly affected by the AC voltage applied to the rod-shaped electrode group.
  • the rod-shaped electrode group does not function as a shielding plate for dead cells.
  • the cell product also exhibits electrophoretic properties with no or low mobility.
  • FIG. 3 shows an example of electrode arrangement for the second cell separation processing unit.
  • An AC voltage controlled by the voltage controller 430 is applied from the AC power source 420 between the rod-shaped electrode 410a and the rod-shaped electrode 410b.
  • a plurality of rod-shaped electrodes 410a and rod-shaped electrodes 410b are formed, and are combined in a comb shape at the longitudinal ends.
  • an electric field is formed around the rod-like electrode 410a and the rod-like electrode 410b.
  • FIG. 4 schematically shows the operation of the second cell separation processing unit when such an electrode group is arranged in the second cell separation processing unit 40 in FIG.
  • FIG. 4 is a schematic view showing a cross section orthogonal to the longitudinal direction of the rod-shaped electrode.
  • a strong electric field region E is formed around each of the rod-like electrodes 410a and 410b.
  • the live cells 440 contained in the second solution supplied from the flow path 53 in the direction indicated by the arrow A13 cannot approach the strong electric field region E due to the dielectrophoretic force, as indicated by the arrow A10.
  • the strong electric field region E cannot move to the collection channel 55 side.
  • the dead cell 450 and the product (for example, antibody) 460 are not affected by the dielectrophoretic force or the degree thereof is small. It moves to the direction shown by arrow A11 and arrow A12, is discharged
  • the living cells 440 that do not easily pass through the strong electric field region E are discharged to the circulation channel 54 along the flow in the direction indicated by the arrow A9 of the fluid, and move in the direction indicated by the arrow A14. As a result, a fourth solution in which the living cells 440 are completely removed or reduced is obtained. Since the second cell treatment unit shown in FIG. 4 is separated by the flow of the solution and the electrophoretic force, it is not limited to the arrangement in which the downward direction in FIG. 4 is downward in the vertical direction, and is arranged in an arbitrary direction. be able to.
  • the second solution comprises a third solution having a live cell / dead cell ratio that is higher than a live cell / dead cell ratio in the second solution, and a live cell / dead cell ratio in the second solution.
  • a fourth solution having a lower live / dead cell ratio.
  • each rod-shaped electrode may be, for example, 50 ⁇ m to 1 mm, and the distance between the rod-shaped electrodes (center distance) may be, for example, 100 ⁇ m to 2 mm.
  • the rod-shaped electrode may be supported by disposing a support material such as plastic in a direction crossing (for example, orthogonal to) each rod-shaped electrode.
  • the configuration shown in FIG. 3 and FIG. 1 is an example, and the arrangement, shape, and the like of the electrode are the third having a living cell / dead cell ratio higher than the living cell / dead cell ratio in the second solution.
  • separation into a fourth solution having a ratio of living cells / dead cells lower than the ratio of living cells / dead cells in the second solution is possible.
  • What is necessary is just to design suitably according to a composition, the shape of the 2nd cell separation process part etc. which perform a 2nd cell separation process.
  • a pair of rod-shaped electrodes that are arranged in parallel may be arranged so as to intersect (for example, orthogonally) to form mesh electrodes.
  • each electrode it is preferable to set the shape and arrangement of each electrode so that the electric field formed by the electrode group forms a strong electric field region on the assumed shielding surface for separating live cells and dead cells.
  • the second cell separation processing unit two or more electrode rows may be arranged, and two or more cell separation processing units may be provided as necessary.
  • the living cells can exhibit such migration characteristics.
  • the stronger the applied voltage the stronger the electrophoretic characteristics tend to be, and the higher the separation efficiency between live and dead cells.
  • the applied voltage is excessively strong, the damage to living cells tends to increase.
  • the frequency and voltage of the applied AC voltage are preferably set so as to satisfy the following. That is, the viable cell concentration in the fourth solution is preferably 50% or less of the viable cell concentration in the second solution, more preferably 20% or less, and a concentration of 10% or less. More preferably, the concentration is 5% or less.
  • the lower limit value of the ratio of the viable cell concentration in the fourth solution to the viable cell concentration in the second solution can be 0%. That is, the living cells may be completely removed in the fourth solution. Alternatively, even if a very small amount of living cells are lost, it can be sufficiently compensated by the growth of living cells in the cell suspension in the culture vessel, so that the lower limit value can be set to 0.01% or more.
  • the ratio of the viable cell concentration in the fourth solution to the viable cell concentration in the second solution can be set to 50% or less, the removal amount of viable cells can be further reduced, and the cell suspension in the culture vessel can be reduced. There is a tendency that a decrease in the cell concentration of the suspension and a decrease in the productivity of the product can be avoided more effectively.
  • the viable cell concentration in the fourth solution is preferably 20% or less, more preferably 10% or less, more preferably 5%, relative to the total cell concentration of the live cells and dead cells in the fourth solution. More preferably, it is more preferably 2% or less.
  • the ratio of the concentration of living cells in the solution to the total cell concentration of living cells and dead cells represents the ratio of the number of living cells in all cells (live cells and dead cells) in the solution. It can also be called rate.
  • the lower limit value of the ratio of the viable cell concentration in the fourth solution to the total cell concentration of the live cells and dead cells in the fourth solution can be 0%. That is, the living cells may be completely removed in the fourth solution.
  • the lower limit value can be set to 0.01% or more.
  • the ratio of the living cell concentration in the fourth solution to the total cell concentration of the living cells and dead cells in the fourth solution is 20% or less, the removal amount of living cells can be further reduced, There is a tendency that a decrease in product productivity in the culture vessel can be avoided more effectively.
  • the second cell separation processing unit that performs the second cell separation processing may be directly connected to the first cell separation processing unit, and a flow path exists between the second cell separation processing unit and the first cell separation processing unit. Also good.
  • liquid feeding means such as a pump is provided in the flow path to separate the second solution into the second cell separation. The liquid can be sent to the processing section.
  • a liquid feeding means is provided for the third solution, and according to the liquid feeding of the third solution, the first The second solution can be supplied to the second cell separation processing unit.
  • the total cell concentration of live cells and dead cells in the second solution is preferably 0.5 ⁇ 10 6 cells / ml or more and 50 ⁇ 10 6 cells / ml or less, and 2.0 ⁇ 10 6 cells / ml or more. It is more preferably 30 ⁇ 10 6 cells / ml or less, and further preferably 5.0 ⁇ 10 6 cells / ml or more and 20 ⁇ 10 6 cells / ml or less.
  • the total cell concentration of the live cells and dead cells in the second solution is 0.5 ⁇ 10 6 cells / ml or more, the number of cells subjected to the second cell separation treatment is It is suppressed that the processing capacity becomes smaller, and dead cells are more efficiently removed by the second cell separation process.
  • the efficiency of selective separation of dead cells in the second cell separation treatment is more effectively reduced.
  • the decrease in product productivity in the culture vessel due to the removal of living cells together with dead cells tends to be more effectively avoided.
  • the third solution obtained by the second cell separation treatment and having a living cell / dead cell ratio higher than the living cell / dead cell ratio in the second solution is cultured through the second circulation channel. Returned to container. For this reason, the living cells contained in the second solution are not wasted.
  • the movement of the third solution toward the culture vessel usually occurs according to the flow formed by the liquid feeding means for sending the second solution to the second cell separation processing unit.
  • Separate liquid feeding means for example, a pump
  • the increase in the ratio of live cells / dead cells in the third solution is the number of live cells / dead cells in the fourth solution.
  • the disclosure also encompasses embodiments where the increase in the live / dead cell ratio in the third solution is negligible, although it can be subtle compared to the decrease in the ratio.
  • a fourth solution obtained by the second cell separation treatment and having a living cell / dead cell ratio lower than the living cell / dead cell ratio in the second solution is a culture vessel, the first cell separation treatment and It is removed from the circulatory system including the second cell separation process.
  • dead cells can be removed from the circulatory system, and inhibition of the growth of living cells in the culture vessel, reduction in quality of products produced by living cells, and the like can be reduced.
  • the concentration of dead cells in the fourth solution is preferably 0.01 ⁇ 10 6 cells / ml or more and 10 ⁇ 10 6 cells / ml or less, and 0.05 ⁇ 10 6 cells / ml or more and 5 ⁇ 10 6 cells / ml. More preferably, it is not more than ml, and more preferably not less than 0.1 ⁇ 10 6 cells / ml and not more than 2 ⁇ 10 6 cells / ml.
  • concentration of dead cells in the fourth solution is 0.01 ⁇ 10 6 cells / ml or more, dead cells originally contained in the cell suspension accommodated in the culture container are more efficiently removed. be able to.
  • the dead cell concentration in the fourth solution By setting the dead cell concentration in the fourth solution to 10 ⁇ 10 6 cells / ml or less, the number of living cells removed together with dead cells can be further reduced, and the product productivity in the culture vessel can be reduced. Reduction can be reduced more effectively.
  • the removal of the fourth solution can be performed by, for example, a liquid feeding means such as a pump provided in the flow path for removing the fourth solution.
  • the fourth solution removes dead cells from the fourth solution without removing all of them from the culture vessel, the circulation system including the first cell separation treatment and the second cell separation treatment. If desired, the product may be collected and the remaining liquid returned to the culture vessel.
  • the amount of liquid in the culture vessel decreases.
  • a fresh medium may be added to the circulation system (for example, added to the culture vessel). Such addition can be performed by monitoring the amount of liquid in the culture vessel.
  • the pump can be selected from generally used pumps without particular limitation, such as an ATF (Alternating Tangential Flow Filtration) diaphragm type reciprocating pump, floating in a cell suspension
  • a pump hereinafter also simply referred to as a magnetic levitation pump
  • a peristaltic pump and the like that send a cell suspension by rotating a rotating blade having a magnet by a magnetic force.
  • a magnetic levitation type pump for the circulation system including at least the culture vessel, the first cell separation processing unit, and the second cell separation processing unit.
  • the magnetic levitation pump can reduce damage to living cells by appropriately setting the shear stress.
  • the magnetic levitation type pump can send liquid at a constant flow rate, when used in the cell culture method according to the present disclosure including the first cell separation process and the second cell separation process, the culture stability is improved. To improve the stability of the quality, quantity, etc. of the collected product even when used in the production method of the product according to the present disclosure described later. Can do.
  • the cell suspension extracted from the culture vessel may be fed by a magnetic levitation pump.
  • the second solution may be fed by a magnetic levitation pump.
  • FIG. 5 is a schematic sectional view showing an example of the configuration of the magnetic levitation pump.
  • the magnetic levitation pump 200 includes a case 210 having a suction port 211 for sucking a cell suspension and a discharge port 212 for discharging the cell suspension, a rotating blade 220 accommodated in the case 210, and a rotating blade 220.
  • a rotor magnet 221 provided at the bottom, a stator 230 that rotates the rotor magnet 221, and a coil 240 wound around the stator 230 are configured. By passing a current through the coil 240, a magnetic force is generated in the stator 230.
  • the rotating blades 220 suspended in the cell suspension in the case 210 are rotated by the magnetic force generated by the stator 230.
  • D represents the diameter of the rotary blade 220
  • L represents the distance between the tip of the rotary blade 220 and the inner wall of the case 210 surrounding the rotary blade 220.
  • the magnetic levitation pump can be used, for example, to send a cell suspension extracted from a culture vessel, and can also be used to send a second solution.
  • the flow rate of the cell suspension extracted from the culture vessel and the flow rate of the first to fourth solutions are appropriately determined according to the amount of the cell suspension stored in the culture vessel, the desired dead cell removal rate, etc. Can be set. For example, let X be the amount of cell suspension in the culture vessel expressed in liters, Y be the flow rate expressed in liters / minute of the second solution subjected to the second cell separation process, It is preferable that both the following inequality A and inequality B are satisfied, where Z is the flow rate of the fourth solution generated by the cell separation treatment expressed in liters / minute.
  • Z and X preferably satisfy the following inequality A2, more preferably satisfy the following inequality A3, and more preferably satisfy the following inequality A4.
  • Inequality A2 0.6 ⁇ 1440 ⁇ Z / X ⁇ 4.0
  • Inequality A3 0.8 ⁇ 1440 ⁇ Z / X ⁇ 3.0
  • Inequality A4 1.0 ⁇ 1440 ⁇ Z / X ⁇ 2.5
  • Y and Z preferably satisfy the following inequality B2, more preferably satisfy the following inequality B3, and more preferably satisfy the following inequality B4.
  • the viable cell concentration in the cell suspension in the culture container is preferably 80% or more and 99.9% or less with respect to the total cell concentration of the living cells and dead cells in the cell suspension in the culture container. .
  • the viable cell concentration in the cell suspension in the culture vessel is 80% or more with respect to the total cell concentration of the living cells and dead cells in the cell suspension in the culture vessel. Growth inhibition and production production inhibition can be more effectively suppressed. 99.9% is the upper limit that can be reached in the culture technique.
  • the living cell concentration in the cell suspension in the culture vessel is more preferably 85% or more and 99% or less, with respect to the total cell concentration of the living cells and dead cells in the cell suspension in the culture vessel, 90% It is more preferably 99% or less and more preferably 92% or more and 99% or less.
  • the concentration of living cells and the concentration of dead cells in each part described in the above description, and the total cell concentration of living cells and dead cells are measured using a cell suspension or solution as a sample in each part to be measured. It is obtained by collecting and measuring using BECKMAN COULTER Vi-CELL XR. Since Vi-CELL XR uses trypan blue staining, it is possible to count live and dead cells. According to Vi-CELL XR, not only the concentration of live cells and dead cells, and the total cell concentration of live cells and dead cells, but also the number of live cells, dead cells, and live and dead cells in the sample. The total number can also be determined.
  • a method for producing a product according to the present disclosure is as follows: Culturing cells producing the product using the cell culture method according to the present disclosure; Recovering the product from the fourth solution; Is a method for producing a product.
  • Culturing cells producing the product using the cell culture method according to the present disclosure since culturing is performed using the cell culture method according to the present disclosure, dead cells are efficiently removed from the cell suspension in the culture container, and living cells Therefore, the productivity of the product can be increased, and the quality of the product can be improved.
  • the cells that produce the product exemplified above can be used as the cells that produce the product, and the products exemplified above can be used as the product.
  • the product is preferably an antibody.
  • the product Since the product is generally much smaller in size than the cell, the product exhibits the following behavior in the cell culture method according to the present disclosure. That is, when the cell suspension is extracted from the culture vessel, the product contained in the cell suspension is also extracted. In the first cell separation treatment, since the product has a smaller size than the cells, it is directly contained in the second solution without a significant decrease in concentration. The product contained in the second liquid is not easily affected by the strong electric field in the second cell separation process, and part of the product is transferred to the fourth solution without being greatly affected by the shielding by the electric field. For this reason, the product can be recovered from the fourth solution.
  • the product may be recovered simply by recovering the fourth solution, for example, recovering the fourth solution in the tank.
  • the recovered fourth solution is preferably subjected to a treatment for separating the product from dead cells.
  • dead cells can be removed from the solution by a filter or centrifugation.
  • the solution from which dead cells have been removed can be subjected to further processing.
  • the product contained in the fourth solution can be purified by a purification treatment.
  • the resulting product can be purified to high purity.
  • the separation and purification of the product may be performed using the separation and purification methods used in ordinary polypeptides. For example, if a chromatography column such as affinity chromatography, filter, ultrafiltration, salting out, dialysis, SDS polyacrylamide gel electrophoresis, isoelectric focusing etc. is appropriately selected and combined, the polypeptide is separated and purified.
  • the methods that can be used for separation and production are not limited to these.
  • the concentration of the obtained polypeptide can be measured by measuring absorbance, enzyme-linked immunosorbent assay (ELISA), or the like. *
  • the cell culture device is: A culture vessel containing the cell suspension; The cell suspension extracted from the culture vessel is divided into a first solution having a cell concentration higher than the cell concentration in the cell suspension, and cells lower than the cell concentration in the cell suspension. A second solution having a concentration, a first cell separation treatment unit for performing a first cell separation treatment to be separated, A first circulation channel for returning the first solution to the culture vessel; The second solution is subjected to a second cell separation process for separating live cells and dead cells by dielectrophoresis, and the ratio of the number of live cells to the number of dead cells in the second solution is as follows.
  • the first cell separation processing unit performs the first cell separation processing.
  • the first cell separation process includes a centrifugal separation process, a cell separation process using standing wave sound waves, and the like, and correspondingly, the first cell separation processing unit includes a centrifuge, A cell separation processing unit using standing wave sound waves can be used.
  • the first cell separation processing unit is preferably a cell separation processing unit using standing wave sound waves.
  • the culture vessel and the first cell separation processing unit may be directly connected or may be communicated with each other through a flow path.
  • a liquid feeding device can be provided in the flow channel between the culture vessel and the first cell separation processing unit, and it is particularly preferable to provide a magnetic levitation pump.
  • the first cell separation processing unit and the second cell separation processing unit may be directly connected or may be communicated with each other through a flow path.
  • a liquid feeding device can be provided in the flow path between the first cell separation processing section and the second cell separation processing section, and in particular, a magnetic levitation pump is provided. It is preferable.
  • the second cell separation processing unit performs a second cell separation process by dielectrophoresis on the second solution.
  • the second cell separation processing unit can be configured to include an electrode electrically connected to an AC power source. It is preferable that the second cell separation processing unit migrates the living cells in a direction to separate them from the electrodes.
  • FIG. 1 is a diagram illustrating an example of a configuration of a cell culture device 100 that can be applied to the production method for a product according to the present disclosure.
  • the cell culture device 100 is accommodated in the culture vessel 10 that accommodates and cultures cells that produce antibodies as an example of the product together with the culture medium, the flow channel 56 that supplies the fresh culture medium into the culture vessel 10, and the culture vessel 10.
  • the first cell separation process is performed on the cell suspension extracted from the culture vessel 10 and the flow path 51 that is extracted from the cell suspension and sent to the sonic standing wave cell separation processing unit 30. Return the sonic standing wave cell separation processing unit 30 as the first cell separation processing unit and the first solution discharged from the sonic standing wave cell separation processing unit 30 to the culture vessel 10.
  • a circulation channel 52 a channel 53 for sending the second solution with reduced cell concentration discharged from the sonic standing wave cell separation processing unit 30 to the second cell separation processing unit 40, and a second solution The second cell separation process by dielectrophoresis Cell separation processing unit 40, a circulation channel 54 for returning the third solution with an increased ratio of living cells / dead cells discharged from the second cell separation processing unit 40 to the culture vessel, and a second cell separation process And a recovery channel 55 for sending the fourth solution having a reduced ratio of living cells / dead cells discharged from the section 40 for recovery.
  • Arrow A1 indicates the direction of the fresh medium flow in the flow path 56
  • arrow A2 indicates the direction of the cell suspension flow in the flow path 51
  • arrow A3 indicates the direction of the first solution flow in the circulation flow path 52
  • the arrow A4 indicates the direction of the second solution flow in the flow path 53
  • the arrow A5 indicates the direction of the third solution flow in the circulation flow path 54
  • the arrow A6 indicates the fourth direction in the recovery flow path 55. Shows the direction of the solution flow.
  • a stirring device having a stirring blade 11 is provided inside the culture vessel 10. By rotating the stirring blade 11, the medium contained in the culture vessel 10 is stirred, and the uniformity of the medium is maintained.
  • a pump P1 is provided, and a cell suspension is extracted from the culture vessel and fed to the sonic standing wave cell separation processing unit 30.
  • the cell suspension introduced into the sonic standing wave type cell separation processing unit 30 is exposed to the standing wave S of the sonic wave formed by the transducer T and the sonic wave reflector M, and the node or belly of the standing wave S is exposed.
  • the cells are trapped.
  • a second solution having a cell concentration reduced from that of the cell suspension extracted from the culture container is obtained, and the solution is fed to the second cell separation processing unit 40 by the pump P2 provided in the flow path 53. Is done.
  • the cells trapped in the nodes or the abdomen of the standing wave S aggregate and settle, and enter the circulation channel 52 as a first solution having a higher cell concentration than the cell suspension extracted from the culture vessel. Is discharged and returned to the culture vessel 10.
  • the electrode 41 exerts a dielectrophoretic force
  • a dead cell and a part of the antibody as a product are generated by a voltage applied to the electrode 41. It passes through the formed strong electric field region, and is discharged to the recovery channel 55 by the pump P3 as a fourth solution having a living cell / dead cell ratio lower than the living cell / dead cell ratio in the second solution. .
  • Dead cells and the remainder of the antibody as product and living cells are passed through the circulation channel 54 as a third solution having a higher ratio of living cells / dead cells than the ratio of living cells / dead cells in the second solution. It returns to the culture vessel 10 through. As a result, since the amount of the cell suspension in the culture vessel 10 is reduced by the amount of the fourth solution discharged to the recovery channel 55, the pump P4 is driven to pass through the channel 56. The culture medium 10 is replenished with fresh medium.
  • the fourth solution discharged to the recovery channel 55 passes through a filter 57 having a pore size that does not allow dead cells to pass but allows the antibody as a product to pass through. Dead cells are collected in the filter 57.
  • the solution containing the antibody as a product that has passed through the filter 57 is supplied to a further purification processing section (not shown).
  • the culture vessel 10, the channel 51, the sonic standing wave cell separation processing unit 30, the circulation channel 52, the channel 53, the second cell separation processing unit 40, and the circulation channel 54 form a circulation system. Yes. For this reason, live cells repeatedly pass through these portions. Therefore, it is preferable to use pumps P1 and P2 that are less damaging to living cells, and it is particularly preferable to use a magnetic levitation pump.
  • the cell culture method and the cell culture apparatus for removing dead cells in the culture container while culturing the cells at a high concentration, and the cells in the culture container while culturing the cells at a high concentration A method for producing a product using a cell culture method for removing dead cells can be provided.
  • the produced product can be used, for example, in biopharmaceuticals and regenerative medicine.

Abstract

Provided are a cell culture method, a method for producing products using the cell culture method, and a cell culture device for conducting the cell culture method. This cell culture method involves: culturing cells in a cell suspension stored in a culture vessel; performing first cell separation processing in which the cell suspension is extracted from the culture vessel, and is separated into a first solution having a high cell concentration and a second solution having a low cell concentration; returning the first solution into the culture vessel; performing a second cell separation processing on the second solution, in which dielectrophoresis is conducted to separate live cells from dead cells, and the second solution is separated into a third solution having a high live cell-to-dead cell ratio and a forth solution having a low live cell-to-dead cell ratio; returning the third solution into the culture vessel; and recovering the fourth solution.

Description

細胞培養方法、細胞培養装置、及び生産物の製造方法CELL CULTURE METHOD, CELL CULTURE DEVICE, AND PRODUCT PRODUCTION METHOD
　本開示は、細胞培養方法、細胞培養装置、及び生産物の製造方法に関する。 The present disclosure relates to a cell culture method, a cell culture apparatus, and a product manufacturing method.
　細胞の培養は、有用な性質を有する細胞を増加させるため、細胞に生産物を生産させるため等の目的で行われている。 The cell culture is performed for the purpose of increasing the number of cells having useful properties and causing the cells to produce a product.
　細胞の分離に関係する技術として、特開２００８－２６３８４７号公報は、誘電泳動特性の異なる複数種の細胞を含む細胞浮遊液を貯留する容器と、容器内に配置される電極と、電極の周囲に電界強度の勾配を生じさせるように電圧を加える電界制御部とを備え、電極に、いずれかの細胞を捕集させる細胞分離装置を開示している。 As a technique related to cell separation, Japanese Patent Application Laid-Open No. 2008-263847 discloses a container for storing a cell suspension containing a plurality of types of cells having different dielectrophoretic characteristics, an electrode disposed in the container, and a periphery of the electrode A cell separation device that includes an electric field control unit that applies a voltage so as to generate a gradient of the electric field strength, and collects any cell on the electrode.
　また、複雑な遠心操作無しに生体分子を高効率で回収することを可能とする技術として、特表２０１７－５０２６６６号公報には、音響細胞分離装置及びフィルタを有するバイオリアクターが開示されている。 Also, as a technology that enables highly efficient recovery of biomolecules without complicated centrifugation, Japanese Patent Publication No. 2017-502666 discloses a bioreactor having an acoustic cell separation device and a filter.
　生細胞を培養すると、一般的にはある程度の割合の生細胞は死滅し、生細胞と死細胞が混在した状態となる。死細胞が生細胞と共存していると、死細胞による生細胞の成長の阻害、生細胞の代謝の阻害などが起こり、その結果、生細胞の成長の低下、生細胞が生産する生産物の品質の低下などが生じる。しかし、生細胞と死細胞はサイズ等の特性が類似しており、培養液中に含まれる死細胞と生細胞とを分離することは一般には困難である。 When a living cell is cultured, a certain percentage of the living cells are generally killed, resulting in a mixture of living and dead cells. When dead cells coexist with living cells, the dead cells inhibit the growth of living cells, inhibit the metabolism of living cells, etc., resulting in a decrease in the growth of living cells and the production of products produced by living cells. Degradation of quality occurs. However, live cells and dead cells are similar in characteristics such as size, and it is generally difficult to separate dead cells and live cells contained in a culture solution.
　本発明者は、特開２００８－２６３８４７号公報に記載されているような誘電泳動技術を用いることで、死細胞と生細胞を分離することを着想した。生細胞においては、細胞膜が細胞外空間と細胞内空間とを隔てているため、生細胞内の液体の誘電率は生細胞外の液体の誘電率と異なる値となっている。一方、死細胞の細胞膜は細胞外と細胞内を遮断することができないか又は不十分にしかできず、その結果、死細胞内の液体は死細胞外に存在する液体と同様の液体又はこれに近い組成の液体となるため、死細胞内の液体の誘電率は死細胞外に存在する液体の誘電率と概ね同一となる。このため、誘電泳動技術を用いれば死細胞と生細胞とを効率よく分離できると本発明者は予想した。 The present inventor has conceived of separating dead cells and living cells by using a dielectrophoresis technique as described in JP-A-2008-263847. In a living cell, since the cell membrane separates the extracellular space and the intracellular space, the dielectric constant of the liquid in the living cell is different from the dielectric constant of the liquid outside the living cell. On the other hand, the cell membrane of dead cells can only block or be insufficient between the outside and inside of the cell, and as a result, the liquid inside the dead cell can be a liquid similar to or similar to the liquid existing outside the dead cell. Since the liquid has a close composition, the dielectric constant of the liquid inside the dead cell is almost the same as the dielectric constant of the liquid existing outside the dead cell. For this reason, the present inventor expected that dead cells and living cells can be efficiently separated by using the dielectrophoresis technique.
　しかし実際には、細胞懸濁液に対して誘電泳動を行っても、生細胞と死細胞の分離の効率が低い場合があることが判明した。本発明者は、研究の結果、誘電泳動に供する細胞懸濁液中の細胞濃度と生細胞と死細胞の分離の効率とが相関しており、誘電泳動に供する細胞懸濁液中の細胞濃度が低い方が、誘電泳動による生細胞と死細胞の分離の効率が向上する傾向があるとの技術思想を得た。 However, in practice, it has been found that even when dielectrophoresis is performed on a cell suspension, the efficiency of separating live and dead cells may be low. As a result of research, the present inventor has found that the cell concentration in the cell suspension subjected to dielectrophoresis correlates with the efficiency of separation of live and dead cells, and the cell concentration in the cell suspension subjected to dielectrophoresis The technical idea was that the lower the tendency, the higher the efficiency of separating live and dead cells by dielectrophoresis.
　高い細胞濃度で培養を行うことは、得られる生細胞量が増加し、また、細胞が物質を生産する細胞である場合には、得られる生産物の量が増大することから好ましい。一方で、細胞濃度が高くなると、上記のとおり、誘電泳動による生細胞と死細胞の分離効率が低下するという問題が生じる。特開２００８－２６３８４７号公報においては、細胞浮遊液が細胞浮遊容器から誘電泳動を行う処理槽に直接送液されており、細胞浮遊容器内の細胞濃度を高くすると生細胞と死細胞の分離効率が低下してしまう。本発明者は、培養容器内の細胞懸濁液をそのまま誘電泳動による処理に供するのではなく、細胞懸濁液中の細胞濃度を減少させる処理に供してから誘電泳動による処理を行うことで上記の問題が解決できるという技術思想を得た。 Culturing at a high cell concentration is preferable because the amount of viable cells obtained is increased, and when the cells are cells that produce a substance, the amount of the product obtained is increased. On the other hand, when the cell concentration increases, there arises a problem that the separation efficiency of live cells and dead cells by dielectrophoresis decreases as described above. In Japanese Patent Application Laid-Open No. 2008-263847, the cell suspension is directly fed from the cell suspension container to a treatment tank for performing dielectrophoresis, and the separation efficiency of living cells and dead cells is increased when the cell concentration in the cell suspension container is increased. Will fall. The present inventor does not directly subject the cell suspension in the culture vessel to the treatment by dielectrophoresis, but performs the treatment by dielectrophoresis after performing the treatment by reducing the cell concentration in the cell suspension. The technical idea that this problem can be solved was obtained.
　本発明の実施形態が解決しようとする課題は、高濃度で細胞を培養しながら培養容器内の死細胞を除去する細胞培養方法及び細胞培養装置、並びに高濃度で細胞を培養しながら培養容器内の死細胞を除去する細胞培養方法を用いた生産物の製造方法を提供することである。 The problems to be solved by the embodiments of the present invention include a cell culture method and a cell culture apparatus for removing dead cells in a culture vessel while culturing cells at a high concentration, and a culture vessel while culturing cells at a high concentration. It is to provide a method for producing a product using a cell culture method for removing dead cells.
　上記課題を解決するための手段には、以下の態様が含まれる。
＜１＞
　培養容器内に収容された細胞懸濁液中で細胞を培養することと、
　上記培養容器から上記細胞懸濁液を抜き出して、抜き出された上記細胞懸濁液を、上記細胞懸濁液中における細胞濃度よりも高い細胞濃度を有する第一の溶液と、上記細胞懸濁液中における細胞濃度よりも低い細胞濃度を有する第二の溶液と、に分離する第一の細胞分離処理を行うことと、
　上記第一の溶液を上記培養容器に戻すことと、
　上記第二の溶液に対して、誘電泳動により生細胞と死細胞とを分離する第二の細胞分離処理を施して、上記第二の溶液中における生細胞の個数の死細胞の個数に対する比である生細胞／死細胞比よりも高い生細胞／死細胞比を有する第三の溶液と、上記第二の溶液中における生細胞／死細胞比よりも低い生細胞／死細胞比を有する第四の溶液と、に上記第二の溶液を分離することと、
　上記第三の溶液を上記培養容器に戻すことと、
　上記第四の溶液を回収することと、
　を含む細胞培養方法。
＜２＞
　上記第一の細胞分離処理が、定在波の音波を用いて定在波の腹又は節に細胞を捕捉する細胞分離処理である、＜１＞に記載の細胞培養方法。
＜３＞
　上記第二の細胞分離処理が、電極を用いて、生細胞を上記電極から引き離す方向に泳動させることを含む、＜１＞又は＜２＞に記載の細胞培養方法。
＜４＞
　上記培養容器から抜き出された上記細胞懸濁液が、上記細胞懸濁液中に浮遊し且つ磁石を有する回転羽根を磁力により回転させることで上記細胞懸濁液を送液するポンプにより送液される、＜１＞～＜３＞のうちいずれか１つに記載の細胞培養方法。
＜５＞
　上記第二の溶液が、上記第二の溶液中に浮遊し且つ磁石を有する回転羽根を磁力により回転させることで上記第二の溶液を送液するポンプにより送液される、＜１＞～＜４＞のうちいずれか１つに記載の細胞培養方法。
＜６＞
　上記第二の溶液における生細胞及び死細胞の合計細胞濃度が、上記培養容器内の上記細胞懸濁液における生細胞及び死細胞の合計細胞濃度に対し、０．１％以上５０％以下の濃度である、＜１＞～＜５＞のうちいずれか１つに記載の細胞培養方法。
＜７＞
　上記第四の溶液における生細胞濃度が、上記第二の溶液における生細胞濃度の５０％以下の濃度である、＜１＞～＜６＞のうちいずれか１つに記載の細胞培養方法。
＜８＞
　上記培養容器内の上記細胞懸濁液における生細胞濃度が２０×１０６ｃｅｌｌｓ／ｍｌ以上１５０×１０６ｃｅｌｌｓ／ｍｌ以下である、＜１＞～＜７＞のうちいずれか１つに記載の細胞培養方法。
＜９＞
　上記培養容器内の上記細胞懸濁液における生細胞濃度が、上記培養容器内の上記細胞懸濁液における生細胞及び死細胞の合計細胞濃度に対し、８０％以上９９．９％以下の濃度である、＜１＞～＜８＞のうちいずれか１つに記載の細胞培養方法。
＜１０＞
　上記第二の溶液における生細胞及び死細胞の合計細胞濃度が、０．５×１０６ｃｅｌｌｓ／ｍｌ以上５０×１０６ｃｅｌｌｓ／ｍｌ以下である、＜１＞～＜９＞のうちいずれか１つに記載の細胞培養方法。
＜１１＞
　上記第四の溶液における死細胞濃度が、０．０１×１０６ｃｅｌｌｓ／ｍｌ以上１０×１０６ｃｅｌｌｓ／ｍｌ以下である、＜１＞～＜１０＞のうちいずれか１つに記載の細胞培養方法。
＜１２＞
　上記第四の溶液における生細胞濃度が、上記第四の溶液における生細胞及び死細胞の合計細胞濃度に対して２０％以下である、＜１＞～＜１１＞のうちいずれか１つに記載の細胞培養方法。
＜１３＞
　上記培養容器内の上記細胞懸濁液のリットルで表した液量をＸとし、上記第二の細胞分離処理に供される上記第二の溶液のリットル／分で表した流量をＹとし、上記第二の細胞分離処理により生じる上記第四の溶液のリットル／分で表した流量をＺとしたときに、以下の不等式Ａ及び不等式Ｂの両方が満たされる、＜１＞～＜１２＞のうちいずれか１つに記載の細胞培養方法。
　不等式Ａ　　０．３≦１４４０×Ｚ／Ｘ≦５．０
　不等式Ｂ　　２≦Ｙ／Ｚ≦１００
＜１４＞
　上記細胞懸濁液に含まれる細胞がＣＨＯ細胞である、＜１＞～＜１３＞のうちいずれか１つに記載の細胞培養方法。
＜１５＞
　生産物を生産する細胞を＜１＞～＜１４＞のうちいずれか１つに記載の細胞培養方法を用いて培養することと、
　上記第四の溶液から生産物を回収することと、
　を含む、生産物の製造方法。
＜１６＞
　上記生産物が抗体である、＜１５＞に記載の生産物の製造方法。
＜１７＞
　細胞懸濁液を収容する培養容器と、
　上記培養容器から抜き出された上記細胞懸濁液を、上記細胞懸濁液中における細胞濃度よりも高い細胞濃度を有する第一の溶液と、上記細胞懸濁液中における細胞濃度よりも低い細胞濃度を有する第二の溶液と、に分離する第一の細胞分離処理を行う第一の細胞分離処理部と、
　上記第一の溶液を上記培養容器に戻す第一の循環流路と、
　上記第二の溶液に対して、誘電泳動により生細胞と死細胞とを分離する第二の細胞分離処理を施して、上記第二の溶液中における生細胞の個数の死細胞の個数に対する比である生細胞／死細胞比よりも高い生細胞／死細胞比を有する第三の溶液と、上記第二の溶液中における生細胞／死細胞比よりも低い生細胞／死細胞比を有する第四の溶液と、に上記第二の溶液を分離する第二の細胞分離処理部と、
　上記第三の溶液を上記培養容器に戻す第二の循環流路と、
　上記第四の溶液を回収するための、回収流路と、
　を含む、細胞培養装置。
＜１８＞
　上記第一の細胞分離処理部が、定在波の音波を用いて定在波の腹又は節に細胞を捕捉する細胞分離処理部である、＜１７＞に記載の細胞培養装置。
＜１９＞
　上記第二の細胞分離処理部が電極を含み、上記第二の細胞分離処理部は、生細胞を上記電極から引き離す方向に泳動させる、＜１７＞又は＜１８＞に記載の細胞培養装置。
＜２０＞
　上記培養容器から上記第一の細胞分離処理部の間に設けられた、上記細胞懸濁液中に浮遊し且つ磁石を有する回転羽根を磁力により回転させることで上記細胞懸濁液を送液するポンプをさらに含む、＜１７＞～＜１９＞のうちいずれか１つに記載の細胞培養装置。
＜２１＞
　上記第一の細胞分離処理部と上記第二の細胞分離処理部との間に設けられた、上記第二の溶液中に浮遊し且つ磁石を有する回転羽根を磁力により回転させることで上記第二の溶液を送液するポンプをさらに含む、＜１７＞～＜２０＞のうちいずれか１つに記載の細胞培養装置。 Means for solving the above problems include the following aspects.
<1>
Culturing cells in a cell suspension contained in a culture vessel;
The cell suspension is extracted from the culture vessel, and the extracted cell suspension is divided into a first solution having a cell concentration higher than the cell concentration in the cell suspension, and the cell suspension. Performing a first cell separation treatment to separate into a second solution having a cell concentration lower than the cell concentration in the liquid;
Returning the first solution to the culture vessel;
The second solution is subjected to a second cell separation process for separating live cells and dead cells by dielectrophoresis, and the ratio of the number of live cells to the number of dead cells in the second solution is as follows. A third solution having a higher ratio of live cells / dead cells than a ratio of live cells / dead cells, and a fourth solution having a ratio of live cells / dead cells lower than the ratio of live cells / dead cells in the second solution. Separating the second solution into a solution of
Returning the third solution to the culture vessel;
Recovering the fourth solution;
A cell culture method comprising:
<2>
<1> The cell culturing method according to <1>, wherein the first cell separation treatment is a cell separation treatment in which cells are captured in a standing wave belly or node using a standing wave sound wave.
<3>
The cell culture method according to <1> or <2>, wherein the second cell separation treatment includes using an electrode to migrate a living cell in a direction in which the cell is separated from the electrode.
<4>
The cell suspension extracted from the culture vessel is fed by a pump that floats in the cell suspension and feeds the cell suspension by rotating a rotating blade having a magnet by a magnetic force. The cell culture method according to any one of <1> to <3>.
<5>
The second solution is fed by a pump that feeds the second solution by rotating a rotating blade that floats in the second solution and has a magnet by a magnetic force. <1> to < The cell culture method according to any one of 4>.
<6>
The total cell concentration of live cells and dead cells in the second solution is a concentration of 0.1% or more and 50% or less with respect to the total cell concentration of live cells and dead cells in the cell suspension in the culture container. The cell culture method according to any one of <1> to <5>, wherein
<7>
The cell culture method according to any one of <1> to <6>, wherein the viable cell concentration in the fourth solution is 50% or less of the viable cell concentration in the second solution.
<8>
The living cell concentration in the cell suspension in the culture container is 20 × 10 6 cells / ml or more and 150 × 10 6 cells / ml or less, according to any one of <1> to <7> Cell culture method.
<9>
The living cell concentration in the cell suspension in the culture vessel is 80% or more and 99.9% or less of the total cell concentration of the living cells and dead cells in the cell suspension in the culture vessel. The cell culture method according to any one of <1> to <8>.
<10>
Any one of <1> to <9>, wherein the total cell concentration of the living cells and dead cells in the second solution is 0.5 × 10 6 cells / ml or more and 50 × 10 6 cells / ml or less. The cell culture method described in 1.
<11>
The cell culture according to any one of <1> to <10>, wherein the dead cell concentration in the fourth solution is 0.01 × 10 6 cells / ml or more and 10 × 10 6 cells / ml or less. Method.
<12>
The living cell concentration in the fourth solution is 20% or less with respect to the total cell concentration of living cells and dead cells in the fourth solution, according to any one of <1> to <11> Cell culture method.
<13>
The amount of liquid expressed in liters of the cell suspension in the culture vessel is X, the flow rate expressed in liters / minute of the second solution used for the second cell separation treatment is Y, and When the flow rate expressed in liters / minute of the fourth solution generated by the second cell separation treatment is Z, both the following inequality A and inequality B are satisfied, among <1> to <12> The cell culture method according to any one of the above.
Inequality A 0.3 ≦ 1440 × Z / X ≦ 5.0
Inequality B 2 ≦ Y / Z ≦ 100
<14>
The cell culture method according to any one of <1> to <13>, wherein the cells contained in the cell suspension are CHO cells.
<15>
Culturing cells that produce the product using the cell culture method according to any one of <1> to <14>;
Recovering the product from the fourth solution;
A method for producing a product, comprising:
<16>
The method for producing a product according to <15>, wherein the product is an antibody.
<17>
A culture vessel containing the cell suspension;
The cell suspension extracted from the culture vessel is divided into a first solution having a cell concentration higher than the cell concentration in the cell suspension, and cells lower than the cell concentration in the cell suspension. A second solution having a concentration, a first cell separation treatment unit for performing a first cell separation treatment to be separated,
A first circulation channel for returning the first solution to the culture vessel;
The second solution is subjected to a second cell separation process for separating live cells and dead cells by dielectrophoresis, and the ratio of the number of live cells to the number of dead cells in the second solution is as follows. A third solution having a higher ratio of live cells / dead cells than a ratio of live cells / dead cells, and a fourth solution having a ratio of live cells / dead cells lower than the ratio of live cells / dead cells in the second solution. A second cell separation processing unit for separating the second solution into
A second circulation channel for returning the third solution to the culture vessel;
A recovery channel for recovering the fourth solution;
A cell culture device.
<18>
The cell culture device according to <17>, wherein the first cell separation processing unit is a cell separation processing unit that captures cells in a standing wave belly or node using a standing wave sound wave.
<19>
The cell culture device according to <17> or <18>, wherein the second cell separation processing unit includes an electrode, and the second cell separation processing unit migrates live cells in a direction separating the electrode from the electrode.
<20>
The cell suspension is sent from the culture vessel by rotating magnetically rotating a rotating blade provided between the first cell separation processing unit and suspended in the cell suspension and having a magnet. The cell culture device according to any one of <17> to <19>, further comprising a pump.
<21>
By rotating a rotating blade provided between the first cell separation processing unit and the second cell separation processing unit floating in the second solution and having a magnet by the magnetic force, the second The cell culture device according to any one of <17> to <20>, further comprising a pump for feeding the solution.
　本発明の実施形態によれば、高濃度で細胞を培養しながら培養容器内の死細胞を除去する細胞培養方法及び細胞培養装置、並びに高濃度で細胞を培養しながら培養容器内の死細胞を除去する細胞培養方法を用いた生産物の製造方法が提供される。 According to the embodiments of the present invention, a cell culture method and a cell culture apparatus for removing dead cells in a culture vessel while culturing cells at a high concentration, and dead cells in a culture vessel while culturing cells at a high concentration A method for producing a product using the cell culture method to be removed is provided.
本開示に係る細胞培養方法及び生産物の製造方法の実施に適用可能な細胞培養装置の構成の一例を示す図である。It is a figure which shows an example of a structure of the cell culture apparatus applicable to implementation of the cell culture method which concerns on this indication, and the manufacturing method of a product. 定在波の音波を用いた第一の細胞分離処理部の構成の一例を示す模式図である。It is a schematic diagram which shows an example of a structure of the 1st cell separation process part using the acoustic wave of standing wave. 誘電泳動のための電極配置の一例を示す模式図である。It is a schematic diagram which shows an example of the electrode arrangement | positioning for dielectrophoresis. 第二の細胞分離処理部における誘電泳動の作用を説明するための概念図である。It is a conceptual diagram for demonstrating the effect | action of the dielectrophoresis in a 2nd cell separation process part. 磁気浮上型ポンプの構成の一例を示す図である。It is a figure which shows an example of a structure of a magnetic levitation type pump.
　以下、本開示に係る細胞培養方法、細胞培養装置、及び生産物の製造方法について説明する。但し、本開示に係る実施形態は以下の実施形態に何ら限定されるものではなく、適宜、変更を加えて実施することができる。 Hereinafter, a cell culture method, a cell culture apparatus, and a product manufacturing method according to the present disclosure will be described. However, the embodiments according to the present disclosure are not limited to the following embodiments, and can be implemented with appropriate modifications.
　本開示において「～」を用いて示された数値範囲は、「～」の前後に記載される数値をそれぞれ最小値及び最大値として含む範囲を意味する。
　本開示に段階的に記載されている数値範囲において、ある数値範囲で記載された上限値又は下限値は、他の段階的な記載の数値範囲の上限値又は下限値に置き換えてもよい。また、本開示に記載されている数値範囲において、ある数値範囲で記載された上限値又は下限値は、実施例に示されている値に置き換えてもよい。
　本開示において、２以上の好ましい態様の組み合わせは、より好ましい態様である。
　本開示において、各成分の量は、各成分に該当する物質が複数種存在する場合には、特に断らない限り、複数種の物質の合計量を意味する。
　本開示において、「工程」との語は、独立した工程だけではなく、工程の所期の目的が達成される限りは、他の工程と明確に区別できない工程をも含む。 In the present disclosure, a numerical range indicated by using “to” means a range including the numerical values described before and after “to” as the minimum value and the maximum value, respectively.
In a numerical range described in stages in the present disclosure, an upper limit value or a lower limit value described in a numerical range may be replaced with an upper limit value or a lower limit value in another numerical range. Further, in the numerical ranges described in the present disclosure, the upper limit value or the lower limit value described in a certain numerical range may be replaced with the values shown in the examples.
In the present disclosure, a combination of two or more preferred embodiments is a more preferred embodiment.
In the present disclosure, the amount of each component means the total amount of a plurality of types of substances unless there is a specific case when there are a plurality of types of substances corresponding to each component.
In the present disclosure, the term “process” includes not only an independent process but also a process that cannot be clearly distinguished from other processes as long as the intended purpose of the process is achieved.
　本開示に係る細胞培養方法は、
　培養容器内に収容された細胞懸濁液中で細胞を培養することと、
　上記培養容器から上記細胞懸濁液を抜き出して、抜き出された上記細胞懸濁液を、上記細胞懸濁液中における細胞濃度よりも高い細胞濃度を有する第一の溶液と、上記細胞懸濁液中における細胞濃度よりも低い細胞濃度を有する第二の溶液と、に分離する第一の細胞分離処理を行うことと、
　上記第一の溶液を上記培養容器に戻すことと、
　上記第二の溶液に対して、誘電泳動により生細胞と死細胞とを分離する第二の細胞分離処理を施して、上記第二の溶液中における生細胞の個数の死細胞の個数に対する比である生細胞／死細胞比よりも高い生細胞／死細胞比を有する第三の溶液と、上記第二の溶液中における生細胞／死細胞比よりも低い生細胞／死細胞比を有する第四の溶液と、に上記第二の溶液を分離することと、
　上記第三の溶液を上記培養容器に戻すことと、
　上記第四の溶液を回収することと、
　を含む細胞培養方法である。 The cell culture method according to the present disclosure includes:
Culturing cells in a cell suspension contained in a culture vessel;
The cell suspension is extracted from the culture vessel, and the extracted cell suspension is divided into a first solution having a cell concentration higher than the cell concentration in the cell suspension, and the cell suspension. Performing a first cell separation treatment to separate into a second solution having a cell concentration lower than the cell concentration in the liquid;
Returning the first solution to the culture vessel;
The second solution is subjected to a second cell separation process for separating live cells and dead cells by dielectrophoresis, and the ratio of the number of live cells to the number of dead cells in the second solution is as follows. A third solution having a higher ratio of live cells / dead cells than a ratio of live cells / dead cells, and a fourth solution having a ratio of live cells / dead cells lower than the ratio of live cells / dead cells in the second solution. Separating the second solution into a solution of
Returning the third solution to the culture vessel;
Recovering the fourth solution;
A cell culture method comprising:
　本開示に係る細胞培養方法においては、培養容器から抜き出された細胞懸濁液を、細胞懸濁液中における細胞濃度よりも高い細胞濃度を有する第一の溶液と、細胞懸濁液中における細胞濃度よりも低い細胞濃度を有する第二の溶液と、に分離する第一の細胞分離処理を行う。そして、第二の溶液に対して誘電泳動による第二の細胞分離処理を施す。このため、誘電泳動が施される第二の溶液における細胞濃度は、培養容器内に収容された細胞懸濁液中における細胞濃度よりも低くなり、誘電泳動による生細胞と死細胞等の分離の効率が向上する。また、細胞懸濁液中における細胞濃度よりも高い細胞濃度を有する第一の溶液、及び第二の溶液中における生細胞／死細胞比よりも高い生細胞／死細胞比を有する第三の溶液は、いずれも培養容器に戻されるため、培養容器から抜き出された細胞懸濁液中に含まれていた生細胞を培養容器内に効率的に戻すことができる。例えば、第二の溶液の流量が第一の溶液の流量と比べて少ない場合などには、第一の溶液中における細胞濃度の増加は第二の溶液中における細胞濃度の減少に比べてわずかなものとなりうるが、本開示は第一の溶液中における細胞濃度の増加がわずかである実施形態をも包含する。
　なお、本開示中において、単に「細胞」と記載した場合には、生細胞と死細胞の両方を指し、単に「細胞濃度」と記載した場合には、生細胞と死細胞の合計濃度を指す。また、細胞について「濃度」とは、個数密度を指す。また、本開示においては、培養容器内の細胞懸濁液及び培養容器から抜き出された細胞懸濁液との区別のために第一の溶液、第二の溶液、第三の溶液、及び第四の溶液の語を用いるが、これらの溶液もその実態は細胞懸濁液である。 In the cell culture method according to the present disclosure, the cell suspension extracted from the culture vessel is divided into a first solution having a cell concentration higher than the cell concentration in the cell suspension, and the cell suspension. A first cell separation process is performed for separation into a second solution having a cell concentration lower than the cell concentration. Then, the second solution is subjected to a second cell separation process by dielectrophoresis. For this reason, the cell concentration in the second solution subjected to dielectrophoresis is lower than the cell concentration in the cell suspension accommodated in the culture vessel, and the separation of live cells and dead cells by dielectrophoresis is performed. Efficiency is improved. A first solution having a cell concentration higher than the cell concentration in the cell suspension; and a third solution having a living cell / dead cell ratio higher than the living cell / dead cell ratio in the second solution. Since all are returned to the culture container, the living cells contained in the cell suspension extracted from the culture container can be efficiently returned to the culture container. For example, when the flow rate of the second solution is small compared to the flow rate of the first solution, the increase in cell concentration in the first solution is slight compared to the decrease in cell concentration in the second solution. While this may be the case, the present disclosure also encompasses embodiments in which there is a slight increase in cell concentration in the first solution.
In the present disclosure, when simply described as “cell”, it refers to both live and dead cells, and when simply described as “cell concentration”, it refers to the total concentration of live and dead cells. . Further, “concentration” for cells refers to the number density. Further, in the present disclosure, the first solution, the second solution, the third solution, and the first solution are distinguished from the cell suspension in the culture vessel and the cell suspension extracted from the culture vessel. Although the term “four solutions” is used, these solutions are actually cell suspensions.
　本開示に係る細胞培養方法において培養する細胞は、特に限定されず、例えば、動物細胞、植物細胞、酵母などの真核細胞、及び枯草菌、大腸菌などの原核細胞が挙げられる。細胞は、ＥＳ細胞、ＩＰＳ細胞、各種幹細胞などであってもよい。
　本開示に係る細胞培養方法において培養する細胞は、生産物を生産する細胞であってもよい。生産物を生産する細胞を培養すると、細胞により生産物が生産され、これを回収すれば細胞を用いた物質生産を行うことができる。生産物を生産する細胞として用いられる細胞は、特に限定されず、動物細胞、植物細胞、酵母などの真核細胞、及び枯草菌、大腸菌などの原核細胞のいずれであってもよい。ＣＨＯ細胞、ＢＨＫ－２１細胞、Ｃ１２７細胞、ハイブリドーマ細胞、ＮＳ０細胞及びＳＰ２／０－Ａｇ１４細胞などの動物細胞が好ましく、解析が多数行われ、遺伝子工学的な手法が確立している点でＣＨＯ細胞がより好ましい。所望の生産物を細胞が元々生産しない又は生産量が少ない場合であっても、例えば、生産物を生産するのに必要な蛋白質をコードするプラスミドなどの発現ベクターを細胞に導入することで、所望の生産物を効率よく生産させることができる。本開示における細胞によって生産される生産物は、上記の細胞が培養液中に生産する物質であれば、特に限定されず、例えば、アルコール、酵素、抗生物質、核酸、組換えタンパク質、抗体などの物質が挙げられる。中でも生産物として、好ましくは、組み換えタンパク質又は抗体であり、より好ましくは抗体である。 The cells cultured in the cell culture method according to the present disclosure are not particularly limited, and examples thereof include eukaryotic cells such as animal cells, plant cells, and yeast, and prokaryotic cells such as Bacillus subtilis and Escherichia coli. The cells may be ES cells, IPS cells, various stem cells, and the like.
The cell cultured in the cell culture method according to the present disclosure may be a cell that produces a product. When cells that produce a product are cultured, the product is produced by the cell, and if this is recovered, substance production using the cell can be performed. The cells used as the cells for producing the product are not particularly limited, and may be any of eukaryotic cells such as animal cells, plant cells, and yeast, and prokaryotic cells such as Bacillus subtilis and Escherichia coli. Animal cells such as CHO cells, BHK-21 cells, C127 cells, hybridoma cells, NS0 cells, and SP2 / 0-Ag14 cells are preferred, and CHO cells are known because many analyzes have been performed and genetic engineering techniques have been established. Is more preferable. Even if the cells do not originally produce the desired product or the production volume is small, for example, by introducing an expression vector such as a plasmid encoding a protein necessary for producing the product into the cell, the desired product can be obtained. Can be produced efficiently. The product produced by the cells in the present disclosure is not particularly limited as long as it is a substance produced by the cells in the culture solution, and examples thereof include alcohols, enzymes, antibiotics, nucleic acids, recombinant proteins, and antibodies. Substances. Among them, the product is preferably a recombinant protein or an antibody, more preferably an antibody.
　動物細胞に抗体を生産させる場合、抗体の種類は特に限定されず、例えば、抗ＩＬ－６レセプター抗体、抗ＩＬ－６抗体、抗グリピカン－３抗体、抗ＣＤ３抗体、抗ＣＤ２０抗体、抗ＧＰＩＩｂ／ＩＩＩａ抗体、抗ＴＮＦ抗体、抗ＣＤ２５抗体、抗ＥＧＦＲ抗体、抗Ｈｅｒ２／ｎｅｕ抗体、抗ＲＳＶ抗体、抗ＣＤ３３抗体、抗ＣＤ５２抗体、抗ＩｇＥ抗体、抗ＣＤ１１ａ抗体、抗ＶＥＧＦ抗体、抗ＶＬＡ４抗体などが挙げられる。生産させる抗体の例には、ヒト、マウス、ラット、ハムスター、ウサギ、サル等の動物由来のモノクローナル抗体だけでなく、キメラ抗体、ヒト化抗体、二重特異性抗体など人為的に改変した抗体も含まれる。 When producing an antibody in animal cells, the type of antibody is not particularly limited. For example, anti-IL-6 receptor antibody, anti-IL-6 antibody, anti-glypican-3 antibody, anti-CD3 antibody, anti-CD20 antibody, anti-GPIIb / IIIa antibody, anti-TNF antibody, anti-CD25 antibody, anti-EGFR antibody, anti-Her2 / neu antibody, anti-RSV antibody, anti-CD33 antibody, anti-CD52 antibody, anti-IgE antibody, anti-CD11a antibody, anti-VEGF antibody, anti-VLA4 antibody, etc. Can be mentioned. Examples of antibodies to be produced include not only monoclonal antibodies derived from animals such as humans, mice, rats, hamsters, rabbits, monkeys, but also artificially modified antibodies such as chimeric antibodies, humanized antibodies, and bispecific antibodies. included.
　培養容器に収容された細胞懸濁液中における細胞の濃度は特に限定されない。ただし、誘電泳動に供される溶液中の細胞濃度が高い方が、誘電泳動による生細胞と死細胞の分離の効率が低下しやすいことを考慮すると、培養容器に収容された細胞懸濁液中における細胞の濃度が高い方が本開示に係る細胞培養方法の効果がより顕著に発揮される傾向となる。また、培養容器に収容された細胞懸濁液中における細胞の濃度が高い方が、細胞量が大きくなること、及び細胞が生産物を生産する場合には生産物の生産量が大きくなることからも、細胞の濃度が高いことは好ましい。ただし、細胞濃度が過剰に高くなると第一の細胞分離処理により生じる第一の溶液と第二の溶液との間での細胞濃度の差が減少し、また第二の細胞分離処理における生細胞と死細胞の分離の効率が低下する傾向がある。これらのことから、培養容器に収容された細胞懸濁液中における細胞の濃度は、２０×１０６ｃｅｌｌｓ／ｍｌ以上１５０×１０６ｃｅｌｌｓ／ｍｌ以下が好ましい。培養容器に収容された細胞懸濁液中における細胞の濃度が２０×１０６ｃｅｌｌｓ／ｍｌ以上であることで、細胞量を大きくすることができ、また、例えば細胞が生産物を生産する細胞である場合に生産物の生産量を大きくすることができる。培養容器に収容された細胞懸濁液中における細胞の濃度が１５０×１０６ｃｅｌｌｓ／ｍｌ以下であることで、第一の細胞分離処理及び第二の細胞分離処理をより効果的に行うことができる。培養容器に収容された細胞懸濁液中における細胞の濃度は、３０×１０６ｃｅｌｌｓ／ｍｌ以上１２０×１０６ｃｅｌｌｓ／ｍｌ以下がより好ましく、４０×１０６ｃｅｌｌｓ／ｍｌ以上１００×１０６ｃｅｌｌｓ／ｍｌ以下がさらに好ましく、５０×１０６ｃｅｌｌｓ／ｍｌ以上１００×１０６ｃｅｌｌｓ／ｍｌ以下が一層好ましい。 The density | concentration of the cell in the cell suspension accommodated in the culture container is not specifically limited. However, considering that the higher the cell concentration in the solution subjected to dielectrophoresis, the lower the efficiency of separating live and dead cells by dielectrophoresis, the cell suspension contained in the culture vessel The higher the cell concentration is, the more prominent the effect of the cell culture method according to the present disclosure is. In addition, the higher the cell concentration in the cell suspension contained in the culture vessel, the larger the cell volume, and the greater the production volume of the product when the cells produce the product. However, it is preferable that the cell concentration is high. However, if the cell concentration becomes excessively high, the difference in cell concentration between the first solution and the second solution generated by the first cell separation treatment is reduced, and the cell concentration in the second cell separation treatment is reduced. There is a tendency for the efficiency of separation of dead cells to decrease. For these reasons, the concentration of the cells in the cell suspension accommodated in the culture vessel is preferably 20 × 10 6 cells / ml or more and 150 × 10 6 cells / ml or less. When the concentration of the cells in the cell suspension accommodated in the culture vessel is 20 × 10 6 cells / ml or more, the amount of cells can be increased, and for example, the cells produce the product. In some cases, the output of the product can be increased. When the concentration of the cells in the cell suspension accommodated in the culture vessel is 150 × 10 6 cells / ml or less, the first cell separation treatment and the second cell separation treatment can be more effectively performed. it can. The concentration of cells in the cell suspension accommodated in the culture vessel is more preferably 30 × 10 6 cells / ml or more and 120 × 10 6 cells / ml or less, and 40 × 10 6 cells / ml or more and 100 × 10 6 cells. / Ml or less is more preferable, and 50 × 10 6 cells / ml or more and 100 × 10 6 cells / ml or less is more preferable.
　細胞の培養に用いる培地としては、細胞の培養に通常使用されている液体培地を用いることができる。例えば、ＯｐｔｉＣＨＯ（Ｌｉｆｅｔｅｃｈｎｏｌｏｇｉｅｓ社、１２６８１０１１）培地、ダルベッコ変法イーグル培地（ＤＭＥＭ）、イーグル最小必須培地（ＭＥＭ）、ＲＰＭＩ－１６４０培地、ＲＰＭＩ－１６４１培地、Ｆ－１２Ｋ培地、ハムＦ１２培地、イスコブ変法ダルベッコ培地（ＩＭＤＭ）、マッコイ５Ａ培地、ライボビッツＬ－１５培地、およびＥＸ－ＣＥＬＬ（商標）３００シリーズ（ＪＲＨ Ｂｉｏｓｃｉｅｎｃｅｓ社）、ＣＨＯ－Ｓ－ＳＦＭＩＩ（Ｉｎｖｉｔｒｏｇｅｎ社）、ＣＨＯ－ＳＦ（Ｓｉｇｍａ－Ａｌｄｒｉｃｈ社）、ＣＤ－ＣＨＯ（Ｉｎｖｉｔｒｏｇｅｎ社）、　ＩＳ　ＣＨＯ－Ｖ（Ｉｒｖｉｎｅ　Ｓｃｉｅｎｔｉｆｉｃ社）、ＰＦ－ＡＣＦ－ＣＨＯ　（Ｓｉｇｍａ－Ａｌｄｒｉｃｈ社）などを使用することができる。 As a medium used for cell culture, a liquid medium usually used for cell culture can be used. For example, OptiCHO (Lifetechnologies, 12681011) medium, Dulbecco's modified Eagle medium (DMEM), Eagle minimum essential medium (MEM), RPMI-1640 medium, RPMI-1641 medium, F-12K medium, Ham F12 medium, Iscob modified method Dulbecco's medium (IMDM), McCoy's 5A medium, Leibovitz L-15 medium, and EX-CELL ™ 300 series (JRH Biosciences), CHO-S-SFMII (Invitrogen), CHO-SF (Sigma-Aldrich) , CD-CHO (Invitrogen), IS CHO-V (Irvine Scientific), PF-ACF-CHO (Sigma-Aldrich), etc. Can.
　培地には牛胎児血清（ＦＣＳ）等の血清を添加してもよい。あるいは、培地は無血清培地、例えば完全合成培地であってもよい。
　培地には、アミノ酸、塩、糖類、ビタミン、ホルモン、増殖因子、緩衝液、抗生物質、脂質、微量元素、植物タンパク質の加水分解物などの追加成分を補充してもよい。 Serum such as fetal calf serum (FCS) may be added to the medium. Alternatively, the medium may be a serum-free medium, such as a fully synthetic medium.
The medium may be supplemented with additional components such as amino acids, salts, sugars, vitamins, hormones, growth factors, buffers, antibiotics, lipids, trace elements, plant protein hydrolysates and the like.
　培地のｐＨは培養する細胞により異なるが、一般的にはｐＨ６．０～８．０であり、好ましくはｐＨ６．８～７．６であり、より好ましくはｐＨ７．０～７．４である。
　培養温度は、一般的には３０℃～４０℃であり、好ましくは３２℃～３７℃であり、より好ましくは３６℃～３７℃であり、培養中に培養温度を変更してもよい。 The pH of the medium varies depending on the cells to be cultured, but is generally pH 6.0 to 8.0, preferably pH 6.8 to 7.6, and more preferably pH 7.0 to 7.4.
The culture temperature is generally 30 ° C. to 40 ° C., preferably 32 ° C. to 37 ° C., more preferably 36 ° C. to 37 ° C., and the culture temperature may be changed during the culture.
　培養は、ＣＯ２濃度が０～４０％の雰囲気下で行ってもよく、ＣＯ２濃度が２～１０％の雰囲気下で行うことが好ましい。
　培養においては、必要に応じて培地の交換、通気、攪拌を加えることができる。 The culture may be performed in an atmosphere having a CO 2 concentration of 0 to 40%, and is preferably performed in an atmosphere having a CO 2 concentration of 2 to 10%.
In culture, medium exchange, aeration, and stirring can be added as necessary.
　細胞の培養は、一般的な培養装置（バイオリアクターとも言う）、またはそれ以外の好適な容器を培養容器として用いて行うことができる。培養装置としては、発酵槽型タンク培養装置、エアーリフト型培養装置、カルチャーフラスコ型培養装置、スピナーフラスコ型培養装置、マイクロキャリアー型培養装置、流動層型培養装置、ホロファイバー型培養装置、ローラーボトル型培養装置、充填槽型培養装置等を用いることができる。 The cells can be cultured using a general culture apparatus (also referred to as a bioreactor) or other suitable container as a culture container. As the culture apparatus, fermenter tank culture apparatus, air lift culture apparatus, culture flask culture apparatus, spinner flask culture apparatus, microcarrier culture apparatus, fluidized bed culture apparatus, holofiber culture apparatus, roller bottle A mold culture apparatus, a filling tank type culture apparatus, or the like can be used.
　培養スケールは、例えば、０．１Ｌ以上５０００Ｌ以下としてもよく、０．５Ｌ以上３０００Ｌ以下が好ましく、１Ｌ以上２５００Ｌ以下がより好ましく、１５００Ｌ以上２２００Ｌ以下が最も好ましい。培養スケールは大きいほど、一度に多くの培養を行うことが可能となる。一方、培養液量を５０００Ｌ以下とした場合には、死細胞の効率的な除去に必要な第一の細胞分離処理部のサイズ及び第二の細胞分離処理部のサイズを抑えることができるため、培養液量を５０００Ｌ以下とすることは第一の細胞分離処理部及び第二の細胞分離処理部の設置面積並びに装置コストを抑える観点から好ましい。本開示に係る細胞培養方法は、培養容器内の細胞懸濁液を撹拌翼により撹拌することを含んでいてもよく、細胞懸濁液にスパージャーによりガス通気することを含んでいてもよい。 The culture scale may be, for example, from 0.1 L to 5000 L, preferably from 0.5 L to 3000 L, more preferably from 1 L to 2500 L, and most preferably from 1500 L to 2200 L. The larger the culture scale, the more cultures can be performed at one time. On the other hand, when the amount of the culture solution is 5000 L or less, the size of the first cell separation processing unit and the size of the second cell separation processing unit necessary for efficient removal of dead cells can be suppressed. Setting the amount of the culture solution to 5000 L or less is preferable from the viewpoint of suppressing the installation area of the first cell separation processing unit and the second cell separation processing unit and the apparatus cost. The cell culture method according to the present disclosure may include stirring the cell suspension in the culture vessel with a stirring blade, or may include gas aeration with a sparger.
　細胞の培養は灌流培養であることが好ましい。灌流培養は、新鮮な培地を添加し、同時に使用済み培地を除去する培養法である。灌流培養によれば、１×１０８細胞／ｍＬを超える高い細胞濃度を達成することも可能である。典型的な灌流培養は、１日間または２日間続くバッチ培養スタートアップで始まり、その後、培養物に新鮮な供給培地を連続的、段階的、および／または断続的に添加し、使用済み培地を同時に除去する。ここで、使用済み培地とは、細胞の培養に使用された結果、栄養分が減少又は枯渇し、細胞から放出された老廃物を含む培地のことを指す。灌流培養においては、沈降、遠心分離または濾過などの方法を用いて、細胞濃度を維持しながら使用済み培地を除去することができる。灌流は、連続的、段階的、断続的またはこれらの組み合わせの何れの形態でもよい。 The cell culture is preferably perfusion culture. Perfusion culture is a culture method in which a fresh medium is added and at the same time the spent medium is removed. With perfusion culture, it is also possible to achieve high cell concentrations exceeding 1 × 10 8 cells / mL. A typical perfusion culture begins with a batch culture start-up that lasts for one or two days, after which fresh feed medium is added to the culture continuously, stepwise and / or intermittently and spent medium is removed simultaneously. To do. Here, the spent medium refers to a medium containing waste products released from cells whose nutrients are reduced or depleted as a result of being used for cell culture. In perfusion culture, spent media can be removed while maintaining cell concentration using methods such as sedimentation, centrifugation or filtration. Perfusion may be any form of continuous, gradual, intermittent or a combination thereof.
　培養容器からの細胞懸濁液の抜き出しは、通常、ポンプを用いて行うことができるが、利用可能な他の送液手段を用いてもよい。培養容器から抜き出された細胞懸濁液は、第一の細胞分離処理に供される。培養容器からの細胞懸濁液の抜き出しは、間欠的に行っても連続的に行ってもよいが、系の状態を安定に保つ観点からは連続的に行うことが好ましい。
　培養容器と第一の細胞分離処理を行う第一の細胞分離処理部との間には流路が存在してもよく、その場合には培養容器と第一の細胞分離処理部との間の流路、及び第一の溶液を培養容器に戻すための流路（循環流路）のうち少なくとも一方にポンプを設けてもよい。あるいは、培養容器と第一の細胞分離処理部は直結していてもよく、その場合には第一の溶液を培養容器に戻すための流路にポンプを設置してもよい。言い換えれば、培養容器から細胞懸濁液を抜き出し第一の細胞分離処理部に供給する送液手段は、培養容器と第一の細胞分離処理部との直結という形態であってもよく、培養容器と第一の細胞分離処理部とを連結する流路という形態であってもよい。培養容器から第一の細胞分離処理部への細胞懸濁液の流れを生成するためのポンプ等の送液手段の設置位置は、流れを形成することができる限りにおいて特に限定されない。 Extraction of the cell suspension from the culture vessel can usually be performed using a pump, but other available liquid feeding means may be used. The cell suspension extracted from the culture vessel is subjected to a first cell separation process. Extraction of the cell suspension from the culture vessel may be performed intermittently or continuously, but it is preferably performed continuously from the viewpoint of keeping the system state stable.
There may be a flow path between the culture vessel and the first cell separation processing section for performing the first cell separation processing, in which case the flow path between the culture container and the first cell separation processing section is present. You may provide a pump in at least one among a flow path and the flow path (circulation flow path) for returning a 1st solution to a culture container. Alternatively, the culture container and the first cell separation processing unit may be directly connected, and in that case, a pump may be installed in the flow path for returning the first solution to the culture container. In other words, the liquid feeding means for extracting the cell suspension from the culture container and supplying it to the first cell separation processing unit may be in the form of a direct connection between the culture container and the first cell separation processing unit. And a flow path connecting the first cell separation processing unit. The installation position of liquid feeding means such as a pump for generating a flow of the cell suspension from the culture container to the first cell separation processing unit is not particularly limited as long as the flow can be formed.
　培養容器における細胞懸濁液の抜き出し位置は、細胞懸濁液の液面よりも下の位置であれば特に限定されず、例えば培養容器の底部近辺とすることができる。 The extraction position of the cell suspension in the culture vessel is not particularly limited as long as it is below the liquid surface of the cell suspension, and can be, for example, near the bottom of the culture vessel.
　第一の細胞分離処理は、抜き出された細胞懸濁液を、細胞懸濁液中における細胞濃度よりも高い細胞濃度を有する第一の溶液と、細胞懸濁液中における細胞濃度よりも低い細胞濃度を有する第二の溶液と、に分離する処理であれば特に限定されず、例えば遠心分離処理、定在波の音波、フィルタによる細胞分離処理などが挙げられる。連続運転適性、細胞へのダメージの少なさといった観点からは、第一の細胞分離処理は、定在波の音波による細胞分離処理であることが好ましい。
　遠心分離処理の場合は、遠心力によって細胞を沈降させることにより、処理前よりも細胞濃度が減少した上澄み液と、処理前よりも細胞濃度が増加した溶液と、に分離することができる。フィルタによる細胞分離処理の場合は、細胞の直径に近い孔径のフィルタを選定し、濾過することにより、処理前よりも細胞濃度が減少した濾過液と、処理前よりも細胞濃度が増加した濃縮液と、に分離することができる。フィルタによる濾過は例えばクロスフロー方式で行うことができる。定在波の音波による処理の場合は、定在波の音波により細胞を定在波の節又は腹に捕捉し、凝集した細胞を重力方向下方に沈降させることにより、処理前よりも細胞濃度が減少した溶液と処理前よりも細胞濃度が増加した溶液とに分離することができる。物体が定在波の節に捕捉されるか腹に捕捉されるかは、周囲の液体と比較した場合の物体の圧縮性、密度などの因子により決まる。 In the first cell separation treatment, the extracted cell suspension is divided into a first solution having a cell concentration higher than the cell concentration in the cell suspension and lower than the cell concentration in the cell suspension. The treatment is not particularly limited as long as the treatment is separated into a second solution having a cell concentration, and examples thereof include centrifugation treatment, standing wave sound waves, cell separation treatment using a filter, and the like. From the viewpoints of suitability for continuous operation and low damage to cells, the first cell separation process is preferably a cell separation process using standing wave sound waves.
In the case of the centrifugal separation treatment, the cells can be separated into a supernatant liquid having a cell concentration decreased from that before the treatment and a solution having a cell concentration increased from that before the treatment by sedimenting the cells by centrifugal force. In the case of cell separation treatment using a filter, a filter with a pore size close to the cell diameter is selected and filtered to obtain a filtrate with a cell concentration reduced from that before the treatment, and a concentrate with a cell concentration increased from that before the treatment. And can be separated. Filtration by a filter can be performed by, for example, a cross flow method. In the case of treatment with standing wave sound waves, the cells are trapped in standing wave nodes or abdomen using standing wave sound waves, and the aggregated cells are allowed to settle down in the direction of gravity, so that the cell concentration is higher than before treatment. It can be separated into a reduced solution and a solution with an increased cell concentration than before the treatment. Whether an object is trapped by a standing wave node or a belly depends on factors such as the compressibility and density of the object as compared to the surrounding liquid.
　第一の細胞分離処理への細胞懸濁液の供給速度は、第一の細胞分離処理の処理能力に応じて設定すればよく、特に限定されない。第一の細胞分離処理の処理条件は、第一の細胞分離処理により生じる第二の溶液における生細胞及び死細胞の合計細胞濃度が、培養容器内の細胞懸濁液における生細胞及び死細胞の合計細胞濃度に対し、０．１％以上５０％以下の濃度となるように設定することが好ましい。０．１％以上であることで第二の細胞分離処理に供される細胞数が第二の細胞分離処理の処理能力に対して小さくなることが抑制され、第二の細胞分離処理により死細胞がより効率的に除去される。５０％以下であることで、第二の細胞分離処理における死細胞の選択的な分離の効率の低下をより効果的に避けることができる傾向にある。第二の溶液における生細胞及び死細胞の合計細胞濃度は、培養容器内の細胞懸濁液における生細胞及び死細胞の合計細胞濃度に対し、１％以上３０％以下であることがより好ましく、３％以上２０％以下であることがさらに好ましい。 The supply rate of the cell suspension to the first cell separation process may be set according to the processing capacity of the first cell separation process, and is not particularly limited. The treatment conditions for the first cell separation treatment are such that the total cell concentration of the live and dead cells in the second solution produced by the first cell separation treatment is such that the live and dead cells in the cell suspension in the culture vessel It is preferable to set the concentration to be 0.1% or more and 50% or less with respect to the total cell concentration. It is suppressed that the number of cells subjected to the second cell separation treatment becomes smaller than the processing capacity of the second cell separation treatment by being 0.1% or more, and dead cells are obtained by the second cell separation treatment. Is more efficiently removed. By being 50% or less, there is a tendency that a decrease in the efficiency of selective separation of dead cells in the second cell separation treatment can be more effectively avoided. The total cell concentration of live cells and dead cells in the second solution is more preferably 1% or more and 30% or less with respect to the total cell concentration of live cells and dead cells in the cell suspension in the culture container, More preferably, it is 3% or more and 20% or less.
　定在波の音波による細胞分離処理についてより具体的に説明する。定在波の音波による処理は、定在波の音波により細胞を定在波の節又は腹に捕捉し、凝集した細胞を重力方向下方に沈降させることで細胞濃度が増加した溶液と細胞濃度が減少した溶液とを生成する処理である。定在波の音波による細胞分離処理では、一般に、液体中に形成された音波の定在波の場に、鉛直方向下方から細胞懸濁液を供給し、細胞懸濁液が定在波の場を通過する間に定在波の節又は腹に細胞を捕捉し、細胞濃度が減少した溶液を定在波の場の鉛直方向上方へと排出し、一方、捕捉された細胞は互いに凝集することである程度の大きさになると沈降して鉛直方向下方に設けられた回収部分に回収され細胞濃度が増加した液体として排出される。もちろん、ここでいう鉛直方向とは厳密に鉛直方向でなくてもよく、細胞濃度が異なる第一の液体と第二の液体を生成できる範囲で鉛直方向からずれていてもよい。ずれ角度は例えば３０°以内、好ましくは２０°以内、より好ましくは１０°以内としてもよい。 The cell separation process using standing wave sound waves will be described more specifically. The treatment with sound waves of standing waves captures the cells in standing wave nodes or abdomen using standing wave sound waves and settles the aggregated cells downward in the direction of gravity, so that the cell concentration and the cell concentration are increased. A process for producing a reduced solution. In cell separation processing using standing-wave sound waves, a cell suspension is generally supplied from below in the vertical direction to a standing-wave field of sound waves formed in a liquid. The cells are trapped in the standing wave nodes or abdomen while passing through and the solution with reduced cell concentration is discharged vertically above the standing wave field, while the trapped cells aggregate together. When it reaches a certain size, it settles down and is collected in a collection portion provided vertically below and discharged as a liquid with an increased cell concentration. Of course, the vertical direction here may not be strictly the vertical direction, and may be deviated from the vertical direction within a range in which the first liquid and the second liquid having different cell concentrations can be generated. The deviation angle may be, for example, within 30 °, preferably within 20 °, and more preferably within 10 °.
　本開示において第一の細胞分離処理のために使用可能な定在波の音波を用いた細胞分離処理装置は市販もされており、例えば、Ａｐｐｌｉｋｏｎ　Ｂｉｏｔｅｃｈｎｏｌｏｇｙ社製のＢｉｏｓｅｐシリーズの製品、例えばＢｉｏｓｅｐ　１Ｌ／ｄａｙ、Ｂｉｏｓｅｐ　１０Ｌ／ｄａｙ、Ｂｉｏｓｅｐ　５０Ｌ／ｄａｙ、Ｂｉｏｓｅｐ　２００Ｌ／ｄａｙ、Ｂｉｏｓｅｐ　１０００Ｌ／ｄａｙなどを用いることができる。
　定在波の音波を用いた細胞分離処理部の構成の一例を図２に示す。図２に示す定在波の音波を用いた細胞分離処理部３００（以下、「音波定在波型細胞分離処理部３００」と称する）は、処理対象となる細胞懸濁液を音波定在波型細胞分離処理部３００に導入するためのポート３１０、導入された細胞懸濁液をチャンバー３５０にチャンバー３５０の外周部下方から導入するための導入部３２０、細胞濃度が減少した第二の溶液を排出するためのポート３４０、細胞濃度が増加した第一の溶液を排出するためのポート３３０、定在波となる音波を発生するトランスデューサーＴ、及び音波反射板Ｍを備えている。導入部３２０からチャンバー３５０に供給された細胞懸濁液は、トランスデューサーＴと音波反射板Ｍとにより形成された定在波Ｓに曝され、細胞懸濁液中の生細胞及び死細胞は定在波Ｓの節に捕捉される。チャンバー３５０内には図２における矢印Ａ７で示される上向きへの流れが形成されており、捕捉された細胞の分、細胞濃度が減少した細胞懸濁液が、ポート３４０から排出される。また、節に捕捉された生細胞及び死細胞は互いに軽度に凝集し、サイズが増加するため、チャンバー３５０から沈降してポート３３０から細胞濃度が増加した細胞懸濁液として矢印Ａ８で示される方向に排出される。 In the present disclosure, cell separation treatment apparatuses using standing wave sound waves that can be used for the first cell separation treatment are also commercially available. For example, products of the Biosep series manufactured by Applikon Biotechnology, such as Biosep 1L / Day, Biosep 10L / day, Biosep 50L / day, Biosep 200L / day, Biosep 1000L / day, and the like can be used.
An example of the configuration of the cell separation processing unit using standing-wave sound waves is shown in FIG. A cell separation processing unit 300 (hereinafter referred to as “sonic standing wave type cell separation processing unit 300”) using standing-wave sound waves shown in FIG. 2 converts the cell suspension to be processed into a sound wave standing wave. Port 310 for introducing the cell separation processing unit 300, the introduction unit 320 for introducing the introduced cell suspension into the chamber 350 from below the outer periphery of the chamber 350, and the second solution having a reduced cell concentration A port 340 for discharging, a port 330 for discharging the first solution having an increased cell concentration, a transducer T for generating a sound wave as a standing wave, and a sound wave reflecting plate M are provided. The cell suspension supplied from the introduction unit 320 to the chamber 350 is exposed to the standing wave S formed by the transducer T and the sound wave reflector M, and the living cells and dead cells in the cell suspension are fixed. It is captured by the node of standing wave S. An upward flow indicated by an arrow A <b> 7 in FIG. 2 is formed in the chamber 350, and a cell suspension having a reduced cell concentration corresponding to the trapped cells is discharged from the port 340. In addition, since the living cells and dead cells trapped in the nodes are slightly aggregated and increase in size, the cell suspension settled from the chamber 350 and the cell concentration increased from the port 330 is indicated by the arrow A8. To be discharged.
　本開示において使用可能な定在波の音波を用いた細胞分離処理部の構成は、図２に示した構成に限定されず、培養容器から抜き出された細胞懸濁液中における細胞濃度よりも高い細胞濃度を有する第一の溶液と、培養容器から抜き出された細胞懸濁液中における細胞濃度よりも低い細胞濃度を有する第二の溶液と、に分離することが可能な構成であれば、任意の構成を用いることができる。また、第一の細胞分離処理部は、必要に応じて２つ以上設けてもかまわない。 The configuration of the cell separation processing unit using standing-wave sound waves that can be used in the present disclosure is not limited to the configuration illustrated in FIG. 2, but is more than the cell concentration in the cell suspension extracted from the culture vessel. If it is a configuration that can be separated into a first solution having a high cell concentration and a second solution having a cell concentration lower than the cell concentration in the cell suspension extracted from the culture vessel Any configuration can be used. Further, two or more first cell separation processing units may be provided as necessary.
　第一の細胞分離処理の結果生じた、細胞懸濁液中における細胞濃度よりも高い細胞濃度を有する第一の溶液は、第一の循環流路を通って培養容器に戻される。このため、第一の溶液に含まれる生細胞が培養容器内に効率的に回収される。第一の溶液の培養容器に向かっての移動は、通常は、培養容器から細胞懸濁液を抜き出すための送液手段によって形成された流れに従って生じるが、必要であれば第一の溶液を培養容器に戻すための別個の送液手段（例えばポンプ）を設けてもかまわない。 The first solution having a cell concentration higher than the cell concentration in the cell suspension resulting from the first cell separation treatment is returned to the culture vessel through the first circulation channel. For this reason, the living cells contained in the first solution are efficiently collected in the culture container. The movement of the first solution toward the culture vessel usually occurs according to the flow formed by the liquid feeding means for extracting the cell suspension from the culture vessel. If necessary, the first solution is cultured. A separate liquid feeding means (for example, a pump) for returning the container to the container may be provided.
　第一の細胞分離処理の結果生じた、細胞懸濁液中における細胞濃度よりも低い細胞濃度を有する第二の溶液は、さらに、誘電泳動により生細胞と死細胞とを分離する第二の細胞分離処理に供される。第二の細胞分離処理により、第二の溶液は、第二の溶液中における生細胞／死細胞比よりも高い生細胞／死細胞比を有する第三の溶液と、第二の溶液中における生細胞／死細胞比よりも低い生細胞／死細胞比を有する第四の溶液と、に分離される。なお、本開示において、生細胞／死細胞比とは、生細胞の個数の死細胞の個数に対する比であり、生細胞の個数を死細胞の個数で除すことにより求めることができ、例えば生細胞濃度を死細胞濃度で除すことでも求めることができる。 The second solution having a cell concentration lower than the cell concentration in the cell suspension resulting from the first cell separation treatment is a second cell that separates live and dead cells by dielectrophoresis. It is subjected to separation processing. By the second cell separation process, the second solution is transformed into a third solution having a higher ratio of live cells / dead cells than in the second solution, and live in the second solution. And a fourth solution having a live / dead cell ratio lower than the cell / dead cell ratio. In the present disclosure, the ratio of living cells / dead cells is the ratio of the number of living cells to the number of dead cells, and can be obtained by dividing the number of living cells by the number of dead cells. It can also be determined by dividing the cell concentration by the dead cell concentration.
　第二の細胞分離処理は誘電泳動を用いて生細胞と死細胞とを分離する処理である。誘電泳動とは、例えば交流電界の中に置かれた物体が、その誘電特性に応じて電界強度が強くなる方向又はその逆方向へと移動する現象である。物体の泳動特性（電界強度が強くなる方向に移動するか、電界強度が弱くなる方向に移動するか、あるいは特に移動性を示さないか）は、印加される交流電界の周波数等に依存する。死細胞と生細胞では上記のとおり誘電特性が異なるため、電圧の印加条件を適切に設定することで、死細胞と生細胞との泳動特性を異なるものとすることができ、これにより死細胞と生細胞とを分離することが可能になる。 The second cell separation process is a process of separating live cells and dead cells using dielectrophoresis. Dielectrophoresis is a phenomenon in which, for example, an object placed in an alternating electric field moves in a direction in which the electric field strength increases or in the opposite direction according to its dielectric characteristics. The migration characteristics of the object (whether it moves in the direction in which the electric field strength increases, moves in the direction in which the electric field strength decreases, or does not exhibit mobility) depend on the frequency of the applied AC electric field. Since dead cells and live cells have different dielectric properties as described above, the electrophoretic characteristics of dead cells and live cells can be made different by appropriately setting the voltage application conditions. It becomes possible to separate live cells.
　誘電泳動を用いた第二の細胞分離処理において、生細胞と死細胞とを分離する効率は、供給される細胞懸濁液の細胞濃度が高くなるについて低下する傾向がある。分離効率を高くするためには、印加する電圧を上昇させることが考えられるが、電圧を上昇させると生細胞に対するダメージが増加してしまう。しかし、本開示に係る細胞培養方法においては、第一の細胞分離処理により第二の溶液中の細胞濃度が培養容器内の細胞懸濁液中の細胞濃度よりも低減されており、生細胞と死細胞との分離効率を良好なものとすることができる。このため、電圧を過度に上昇させる必要が無い。 In the second cell separation process using dielectrophoresis, the efficiency of separating live and dead cells tends to decrease as the cell concentration of the supplied cell suspension increases. In order to increase the separation efficiency, it is conceivable to increase the voltage to be applied. However, if the voltage is increased, damage to living cells increases. However, in the cell culture method according to the present disclosure, the cell concentration in the second solution is reduced from the cell concentration in the cell suspension in the culture container by the first cell separation treatment, Separation efficiency with dead cells can be improved. For this reason, it is not necessary to raise the voltage excessively.
　第二の細胞分離処理について、図３及び図４に示すような、棒状電極が平行して配置された電極を用い、隣接する棒状電極間に交流電圧が印加される場合を一例として説明する。生細胞が電界強度が弱くなる方向に移動する泳動特性を示し、死細胞は移動性が無い又は移動性が弱い泳動特性を示すように交流電圧を印加すると、生細胞は電界強度が強くなる棒状電極周辺には近づけず、電極から引き離される方向に泳動される。言い換えれば、棒状電極群は生細胞に対しては一種の遮蔽板のような働きをする。一方、死細胞は液体の流れに沿って移動するだけであり、棒状電極群に印加される交流電圧による影響を受けない又は受けにくい。言い換えれば、棒状電極群は死細胞に対しては遮蔽板としては機能しない。この場合、細胞の生産物も移動性が無い又は移動性が弱い泳動特性を示す。 The second cell separation process will be described by way of an example in which an alternating voltage is applied between adjacent rod-shaped electrodes using electrodes in which rod-shaped electrodes are arranged in parallel as shown in FIGS. A live cell has a migration characteristic in which the electric field strength is weakened, and a dead cell has no mobility or a mobility that has weak mobility. Electrophoresis is performed in a direction away from the electrode without approaching the periphery of the electrode. In other words, the rod-shaped electrode group acts like a kind of shielding plate for living cells. On the other hand, dead cells only move along the flow of the liquid and are not affected or hardly affected by the AC voltage applied to the rod-shaped electrode group. In other words, the rod-shaped electrode group does not function as a shielding plate for dead cells. In this case, the cell product also exhibits electrophoretic properties with no or low mobility.
　具体的には、図３に第二の細胞分離処理部のための電極配置の一例を示す。棒状電極４１０ａと棒状電極４１０ｂとの間には、電圧制御部４３０によって制御された交流電圧が交流電源４２０から印加される。棒状電極４１０ａ及び棒状電極４１０ｂは、それぞれ複数形成され、その長手方向末端において櫛状の形状にまとめられている。交流電源４２０から棒状電極４１０ａ及び棒状電極４１０ｂに電圧が印加されることで、棒状電極４１０ａ及び棒状電極４１０ｂの周囲には電界が形成される。 Specifically, FIG. 3 shows an example of electrode arrangement for the second cell separation processing unit. An AC voltage controlled by the voltage controller 430 is applied from the AC power source 420 between the rod-shaped electrode 410a and the rod-shaped electrode 410b. A plurality of rod-shaped electrodes 410a and rod-shaped electrodes 410b are formed, and are combined in a comb shape at the longitudinal ends. By applying a voltage from the AC power source 420 to the rod-like electrode 410a and the rod-like electrode 410b, an electric field is formed around the rod-like electrode 410a and the rod-like electrode 410b.
　このような電極群を図１における第二の細胞分離処理部４０内に配置した場合の、第二の細胞分離処理部の作用を模式的に図４に示す。図４は、棒状電極の長手方向に直交する断面を表した模式図である。図４においては、各棒状電極４１０ａ及び４１０ｂの周囲には、強電界領域Ｅが形成されている。流路５３から矢印Ａ１３で示される方向に供給された第二の溶液中に含まれる生細胞４４０は、矢印Ａ１０で示すように、誘電泳動力により強電界領域Ｅには近づくことができず、強電界領域Ｅの回収流路５５側には移動することができない。一方、死細胞４５０及び生産物（例えば抗体）４６０は誘電泳動力の影響を受けない又はその程度が小さいため、流体の流れにしたがってその一部が強電界領域Ｅの回収流路５５側、つまり矢印Ａ１１及び矢印Ａ１２で示される方向、へと移動し、回収流路５５へと排出され、矢印Ａ１５で示される方向に移動する。一方、強電界領域Ｅを通過しにくい生細胞４４０は流体の矢印Ａ９で示される方向の流れに沿って循環流路５４へと排出され、矢印Ａ１４で示される方向に移動する。この結果、生細胞４４０が完全に除去又は低減された第四の溶液が得られる。図４に示す第二の細胞処理部については、溶液の流れと制電泳動力により分離がなされるため、図４における下方向が鉛直方向下向きである配置に限定されず、任意の向きに配置することができる。 FIG. 4 schematically shows the operation of the second cell separation processing unit when such an electrode group is arranged in the second cell separation processing unit 40 in FIG. FIG. 4 is a schematic view showing a cross section orthogonal to the longitudinal direction of the rod-shaped electrode. In FIG. 4, a strong electric field region E is formed around each of the rod- like electrodes 410a and 410b. The live cells 440 contained in the second solution supplied from the flow path 53 in the direction indicated by the arrow A13 cannot approach the strong electric field region E due to the dielectrophoretic force, as indicated by the arrow A10. The strong electric field region E cannot move to the collection channel 55 side. On the other hand, the dead cell 450 and the product (for example, antibody) 460 are not affected by the dielectrophoretic force or the degree thereof is small. It moves to the direction shown by arrow A11 and arrow A12, is discharged | emitted to the collection | recovery flow path 55, and moves to the direction shown by arrow A15. On the other hand, the living cells 440 that do not easily pass through the strong electric field region E are discharged to the circulation channel 54 along the flow in the direction indicated by the arrow A9 of the fluid, and move in the direction indicated by the arrow A14. As a result, a fourth solution in which the living cells 440 are completely removed or reduced is obtained. Since the second cell treatment unit shown in FIG. 4 is separated by the flow of the solution and the electrophoretic force, it is not limited to the arrangement in which the downward direction in FIG. 4 is downward in the vertical direction, and is arranged in an arbitrary direction. be able to.
　したがって図４に示すように、棒状電極群が、棒状電極群を含む平面の一方の面に第二の溶液が接するように、第二の溶液の流れに沿って方向に配置された場合、生細胞は棒状電極群を含む平面の他方の側には移動することができないか、あるいは移動が大きく制限される。これに対して死細胞、及び存在する場合は細胞による生産物は、第二の溶液の流れに沿って移動しつつ、その量の一部が棒状電極群を含む平面を通過して他方側、つまり矢印Ａ１１及び矢印Ａ１２で示される方向に、移動する。この結果、第二の溶液は、第二の溶液中における生細胞／死細胞比よりも高い生細胞／死細胞比を有する第三の溶液と、第二の溶液中における生細胞／死細胞比よりも低い生細胞／死細胞比を有する第四の溶液と、に分離される。 Therefore, as shown in FIG. 4, when the rod-shaped electrode group is arranged in the direction along the flow of the second solution so that the second solution is in contact with one surface of the plane including the rod-shaped electrode group, The cell cannot move to the other side of the plane including the rod-shaped electrode group, or the movement is greatly limited. On the other hand, dead cells, and if present, the product of the cells move along the flow of the second solution while a part of the amount passes through the plane containing the rod-shaped electrode group on the other side, That is, it moves in the directions indicated by the arrows A11 and A12. As a result, the second solution comprises a third solution having a live cell / dead cell ratio that is higher than a live cell / dead cell ratio in the second solution, and a live cell / dead cell ratio in the second solution. And a fourth solution having a lower live / dead cell ratio.
　各棒状電極の直径は、例えば５０μｍ～１ｍｍとしてもよく、棒状電極間の距離（中心間距離）は、例えば、１００μｍ～２ｍｍとしてもよい。棒状電極の径が小さくなると自立させることが難しくなる傾向になるため、各棒状電極と交差（例えば直交）する向きにプラスチック等の支持材を配置することで棒状電極を支持してもよい。 The diameter of each rod-shaped electrode may be, for example, 50 μm to 1 mm, and the distance between the rod-shaped electrodes (center distance) may be, for example, 100 μm to 2 mm. As the diameter of the rod-shaped electrode becomes small, it tends to be difficult to be self-supporting. Therefore, the rod-shaped electrode may be supported by disposing a support material such as plastic in a direction crossing (for example, orthogonal to) each rod-shaped electrode.
　もちろん、図３及び図１に示す構成は一例であって、電極の配置、形状等については、第二の溶液中における生細胞／死細胞比よりも高い生細胞／死細胞比を有する第三の溶液と、第二の溶液中における生細胞／死細胞比よりも低い生細胞／死細胞比を有する第四の溶液への分離が可能である限りにおいて特に限定されず、細胞種、培地の組成、第二の細胞分離処理を行う第二の細胞分離処理部の形状等に応じて適宜設計すればよい。
　例えば、それぞれ平行に配置された棒状電極２組を交差（例えば直交）させるように配置してメッシュ状の電極としたものを用いてもよい。電極群によって形成される電界が、生細胞と死細胞とを分離するための想定する遮蔽面において、強電界領域を形成するように各電極の形状及び配置を設定することが好ましい。第二の細胞分離処理部においては電極列は二列以上配置してもよく、また、必要に応じて２つ以上の細胞分離処理部を設けてもかまわない。 Of course, the configuration shown in FIG. 3 and FIG. 1 is an example, and the arrangement, shape, and the like of the electrode are the third having a living cell / dead cell ratio higher than the living cell / dead cell ratio in the second solution. As long as separation into a fourth solution having a ratio of living cells / dead cells lower than the ratio of living cells / dead cells in the second solution is possible. What is necessary is just to design suitably according to a composition, the shape of the 2nd cell separation process part etc. which perform a 2nd cell separation process.
For example, a pair of rod-shaped electrodes that are arranged in parallel may be arranged so as to intersect (for example, orthogonally) to form mesh electrodes. It is preferable to set the shape and arrangement of each electrode so that the electric field formed by the electrode group forms a strong electric field region on the assumed shielding surface for separating live cells and dead cells. In the second cell separation processing unit, two or more electrode rows may be arranged, and two or more cell separation processing units may be provided as necessary.
　上述のとおり、生細胞を電極から引き離す方向に泳動させることが好ましい。例えば、電極に印加される交流電圧の周波数を適切に設定することで、生細胞がこのような泳動特性を発揮するようにすることができる。また、印加される電圧の強さが強いほど泳動特性は強く発揮される傾向にあり、生細胞と死細胞との分離効率が向上する傾向にある。ただし、印加される電圧の強さが過剰に強い場合には、生細胞へのダメージが増大する傾向がある。印加される交流電圧の周波数及び電圧は、以下を満たすように設定することが好ましい。
　すなわち、第四の溶液における生細胞濃度は、第二の溶液における生細胞濃度の５０％以下の濃度とすることが好ましく、２０％以下の濃度とすることがより好ましく、１０％以下の濃度とすることがさらに好ましく、５％以下の濃度とすることが一層好ましい。第四の溶液における生細胞濃度の、第二の溶液における生細胞濃度に対する比の下限値は０％とすることができる。つまり、第四の溶液において生細胞は完全に除去されていてもよい。あるいは、ごく少量の生細胞が失われても培養容器内の細胞懸濁液中の生細胞の増殖により十分に補うことができるため、下限値を０．０１％以上に設定することもできる。
　なお、第四の溶液における生細胞濃度の、第二の溶液における生細胞濃度に対する比を５０％以下とすることで、生細胞の除去量をより低減することができ、培養容器内の細胞懸濁液の細胞濃度の減少及び生産物の生産性の低下をより効果的に避けることができる傾向にある。 As described above, it is preferable to migrate the living cells in a direction away from the electrodes. For example, by appropriately setting the frequency of the alternating voltage applied to the electrode, the living cells can exhibit such migration characteristics. In addition, the stronger the applied voltage, the stronger the electrophoretic characteristics tend to be, and the higher the separation efficiency between live and dead cells. However, when the applied voltage is excessively strong, the damage to living cells tends to increase. The frequency and voltage of the applied AC voltage are preferably set so as to satisfy the following.
That is, the viable cell concentration in the fourth solution is preferably 50% or less of the viable cell concentration in the second solution, more preferably 20% or less, and a concentration of 10% or less. More preferably, the concentration is 5% or less. The lower limit value of the ratio of the viable cell concentration in the fourth solution to the viable cell concentration in the second solution can be 0%. That is, the living cells may be completely removed in the fourth solution. Alternatively, even if a very small amount of living cells are lost, it can be sufficiently compensated by the growth of living cells in the cell suspension in the culture vessel, so that the lower limit value can be set to 0.01% or more.
By setting the ratio of the viable cell concentration in the fourth solution to the viable cell concentration in the second solution to 50% or less, the removal amount of viable cells can be further reduced, and the cell suspension in the culture vessel can be reduced. There is a tendency that a decrease in the cell concentration of the suspension and a decrease in the productivity of the product can be avoided more effectively.
　また、第四の溶液における生細胞濃度は、第四の溶液における生細胞及び死細胞の合計細胞濃度に対して２０％以下とすることが好ましく、１０％以下とすることがより好ましく、５％以下とすることがさらに好ましく、２％以下とすることが一層好ましい。溶液中における、生細胞濃度の、生細胞及び死細胞の合計細胞濃度に対する比は、溶液中における全細胞（生細胞及び死細胞）中における生細胞の個数割合を表しているといえ、生細胞率とも呼ぶことができる。第四の溶液における生細胞濃度の、第四の溶液における生細胞及び死細胞の合計細胞濃度に対する比の下限値は０％とすることができる。つまり、第四の溶液において生細胞は完全に除去されていてもよい。あるいは、ごく少量の生細胞が失われても培養容器内の細胞懸濁液中の生細胞の増殖により十分に補うことができるため、下限値を０．０１％以上に設定することもできる。
　なお、第四の溶液における生細胞濃度の、第四の溶液における生細胞及び死細胞の合計細胞濃度に対する比が２０％以下であることで、生細胞の除去量をより低減することができ、培養容器内での生産物生産性の低下をより効果的に避けることができる傾向にある。 The viable cell concentration in the fourth solution is preferably 20% or less, more preferably 10% or less, more preferably 5%, relative to the total cell concentration of the live cells and dead cells in the fourth solution. More preferably, it is more preferably 2% or less. The ratio of the concentration of living cells in the solution to the total cell concentration of living cells and dead cells represents the ratio of the number of living cells in all cells (live cells and dead cells) in the solution. It can also be called rate. The lower limit value of the ratio of the viable cell concentration in the fourth solution to the total cell concentration of the live cells and dead cells in the fourth solution can be 0%. That is, the living cells may be completely removed in the fourth solution. Alternatively, even if a very small amount of living cells are lost, it can be sufficiently compensated by the growth of living cells in the cell suspension in the culture vessel, so that the lower limit value can be set to 0.01% or more.
In addition, since the ratio of the living cell concentration in the fourth solution to the total cell concentration of the living cells and dead cells in the fourth solution is 20% or less, the removal amount of living cells can be further reduced, There is a tendency that a decrease in product productivity in the culture vessel can be avoided more effectively.
　第二の細胞分離処理を行う第二の細胞分離処理部は、第一の細胞分離処理部に直結していてもよく、第一の細胞分離処理部との間に流路が存在していてもよい。第一の細胞分離処理部と第二の細胞分離処理部との間に流路が存在する場合には、流路にポンプ等の送液手段を設けて第二の溶液を第二の細胞分離処理部に送液することができる。第一の細胞分離処理部と第二の細胞分離処理部とが直結している場合には、代わりに例えば第三の溶液について送液手段を設けて、第三の溶液の送液に従って、第二の溶液を第二の細胞分離処理部に供給することができる。 The second cell separation processing unit that performs the second cell separation processing may be directly connected to the first cell separation processing unit, and a flow path exists between the second cell separation processing unit and the first cell separation processing unit. Also good. When a flow path exists between the first cell separation processing section and the second cell separation processing section, liquid feeding means such as a pump is provided in the flow path to separate the second solution into the second cell separation. The liquid can be sent to the processing section. In the case where the first cell separation processing unit and the second cell separation processing unit are directly connected, instead, for example, a liquid feeding means is provided for the third solution, and according to the liquid feeding of the third solution, the first The second solution can be supplied to the second cell separation processing unit.
　第二の溶液における生細胞及び死細胞の合計細胞濃度は、０．５×１０６ｃｅｌｌｓ／ｍｌ以上５０×１０６ｃｅｌｌｓ／ｍｌ以下であることが好ましく、２．０×１０６ｃｅｌｌｓ／ｍｌ以上３０×１０６ｃｅｌｌｓ／ｍｌ以下であることがより好ましく、５．０×１０６ｃｅｌｌｓ／ｍｌ以上２０×１０６ｃｅｌｌｓ／ｍｌ以下であることがさら好ましい。
　第二の溶液における生細胞及び死細胞の合計細胞濃度が０．５×１０６ｃｅｌｌｓ／ｍｌ以上であることで、第二の細胞分離処理に供される細胞数が第二の細胞分離処理の処理能力に対して小さくなることが抑制され、第二の細胞分離処理により死細胞がより効率的に除去される。第二の溶液における生細胞及び死細胞の合計細胞濃度が５０×１０６ｃｅｌｌｓ／ｍｌ以下であることで、第二の細胞分離処理における死細胞の選択的な分離の効率の低下をより効果的に避けることができ、生細胞が死細胞と共に除去されることによる培養容器内での生産物生産性の低下をより効果的に避けることができる傾向にある。 The total cell concentration of live cells and dead cells in the second solution is preferably 0.5 × 10 6 cells / ml or more and 50 × 10 6 cells / ml or less, and 2.0 × 10 6 cells / ml or more. It is more preferably 30 × 10 6 cells / ml or less, and further preferably 5.0 × 10 6 cells / ml or more and 20 × 10 6 cells / ml or less.
When the total cell concentration of the live cells and dead cells in the second solution is 0.5 × 10 6 cells / ml or more, the number of cells subjected to the second cell separation treatment is It is suppressed that the processing capacity becomes smaller, and dead cells are more efficiently removed by the second cell separation process. When the total cell concentration of live cells and dead cells in the second solution is 50 × 10 6 cells / ml or less, the efficiency of selective separation of dead cells in the second cell separation treatment is more effectively reduced. The decrease in product productivity in the culture vessel due to the removal of living cells together with dead cells tends to be more effectively avoided.
　第二の細胞分離処理により得られた、第二の溶液中における生細胞／死細胞比よりも高い生細胞／死細胞比を有する第三の溶液は、第二の循環流路を通って培養容器に戻される。このため、第二の溶液に含まれる生細胞が無駄に捨てられることがない。第三の溶液の培養容器に向かっての移動は、通常は、第二の溶液を第二の細胞分離処理部に送るための送液手段によって形成された流れに従って生じるが、必要であれば第三の溶液を培養容器に戻すための別個の送液手段（例えばポンプ）を設けてもかまわない。例えば、第四の溶液の流量が第三の溶液の流量と比べて少ない場合などには、第三の溶液中における生細胞／死細胞比の増加は第四の溶液中における生細胞／死細胞比の減少に比べてわずかなものとなりうるが、本開示は第三の溶液中における生細胞／死細胞比の増加がわずかである実施形態をも包含する。 The third solution obtained by the second cell separation treatment and having a living cell / dead cell ratio higher than the living cell / dead cell ratio in the second solution is cultured through the second circulation channel. Returned to container. For this reason, the living cells contained in the second solution are not wasted. The movement of the third solution toward the culture vessel usually occurs according to the flow formed by the liquid feeding means for sending the second solution to the second cell separation processing unit. Separate liquid feeding means (for example, a pump) for returning the three solutions to the culture vessel may be provided. For example, when the flow rate of the fourth solution is small compared to the flow rate of the third solution, the increase in the ratio of live cells / dead cells in the third solution is the number of live cells / dead cells in the fourth solution. The disclosure also encompasses embodiments where the increase in the live / dead cell ratio in the third solution is negligible, although it can be subtle compared to the decrease in the ratio.
　第二の細胞分離処理により得られた、第二の溶液中における生細胞／死細胞比よりも低い生細胞／死細胞比を有する第四の溶液は、培養容器、第一の細胞分離処理及び第二の細胞分離処理を含む循環系からは除去される。これにより、死細胞を循環系から除去することができ、培養容器内における生細胞の成長への阻害、生細胞が生産する生産物の品質の低下等を低減することができる。 A fourth solution obtained by the second cell separation treatment and having a living cell / dead cell ratio lower than the living cell / dead cell ratio in the second solution is a culture vessel, the first cell separation treatment and It is removed from the circulatory system including the second cell separation process. Thereby, dead cells can be removed from the circulatory system, and inhibition of the growth of living cells in the culture vessel, reduction in quality of products produced by living cells, and the like can be reduced.
　第四の溶液における死細胞濃度は、０．０１×１０６ｃｅｌｌｓ／ｍｌ以上１０×１０６ｃｅｌｌｓ／ｍｌ以下であることが好ましく、０．０５×１０６ｃｅｌｌｓ／ｍｌ以上５×１０６ｃｅｌｌｓ／ｍｌ以下であることがより好ましく、０．１×１０６ｃｅｌｌｓ／ｍｌ以上２×１０６ｃｅｌｌｓ／ｍｌ以下であることがさらに好ましい。
　第四の溶液における死細胞濃度が０．０１×１０６ｃｅｌｌｓ／ｍｌ以上であることで、培養容器内に収容された細胞懸濁液に元々含まれていた死細胞をより効率的に除去することができる。第四の溶液における死細胞濃度を１０×１０６ｃｅｌｌｓ／ｍｌ以下とすることで、死細胞と共に除去される生細胞の数をより低減することができ、培養容器内での生産物生産性の低下をより効果的に低減できる。 The concentration of dead cells in the fourth solution is preferably 0.01 × 10 6 cells / ml or more and 10 × 10 6 cells / ml or less, and 0.05 × 10 6 cells / ml or more and 5 × 10 6 cells / ml. More preferably, it is not more than ml, and more preferably not less than 0.1 × 10 6 cells / ml and not more than 2 × 10 6 cells / ml.
When the concentration of dead cells in the fourth solution is 0.01 × 10 6 cells / ml or more, dead cells originally contained in the cell suspension accommodated in the culture container are more efficiently removed. be able to. By setting the dead cell concentration in the fourth solution to 10 × 10 6 cells / ml or less, the number of living cells removed together with dead cells can be further reduced, and the product productivity in the culture vessel can be reduced. Reduction can be reduced more effectively.
　第四の溶液の除去は、例えば、第四の溶液の除去のための流路に設けられたポンプ等の送液手段によって行うことができる。なお、第四の溶液は、その全てを培養容器、第一の細胞分離処理及び第二の細胞分離処理を含む循環系から除去しなくても、第四の溶液の中から死細胞を除去し、所望により生産物を回収して、残りの液体を培養容器に戻してもよい。 The removal of the fourth solution can be performed by, for example, a liquid feeding means such as a pump provided in the flow path for removing the fourth solution. The fourth solution removes dead cells from the fourth solution without removing all of them from the culture vessel, the circulation system including the first cell separation treatment and the second cell separation treatment. If desired, the product may be collected and the remaining liquid returned to the culture vessel.
　第四の溶液を除去することにより、培養容器内の液量が減少する。この減少分を補うために、新鮮培地を上記循環系に添加（例えば培養容器に添加）してもよい。このような添加は、培養容器内の液量をモニターして行うことができる。 除去 By removing the fourth solution, the amount of liquid in the culture vessel decreases. In order to compensate for this decrease, a fresh medium may be added to the circulation system (for example, added to the culture vessel). Such addition can be performed by monitoring the amount of liquid in the culture vessel.
　上記の各送液手段についてポンプを用いる場合、ポンプは一般的に用いられるポンプから特に制限無く選択することができ、例えばＡＴＦ（Alternating Tangential Flow Filtration）ダイアフラム式往復ポンプ、細胞懸濁液中に浮遊し且つ磁石を有する回転羽根を磁力により回転させることで細胞懸濁液を送液するポンプ（以下単に磁気浮上型ポンプとも称する）、ペリスタポンプなどが挙げられる。本開示に係る細胞培養方法においては、少なくとも培養容器、第一の細胞分離処理部及び第二の細胞分離処理部を含む循環系に用いるポンプについては、磁気浮上型ポンプを用いることが好ましい。磁気浮上型ポンプは、せん断応力を適切に設定することにより、生細胞に与えるダメージを小さくできるからである。また磁気浮上型ポンプは一定流速での送液が可能であるため、第一の細胞分離処理及び第二の細胞分離処理を含む本開示に係る細胞培養方法において用いた場合には、培養の安定性及び持続性をさらに向上することができ、また、後述する本開示に係る生産物の製造方法において用いた場合にも、回収された生産物の品質、量等の安定性をさらに向上することができる。例えば、培養容器から抜き出された細胞懸濁液を、磁気浮上型ポンプにより送液してもよい。また、第二の溶液を磁気浮上型ポンプにより送液してもよい。 When a pump is used for each of the above-described liquid feeding means, the pump can be selected from generally used pumps without particular limitation, such as an ATF (Alternating Tangential Flow Filtration) diaphragm type reciprocating pump, floating in a cell suspension In addition, a pump (hereinafter also simply referred to as a magnetic levitation pump), a peristaltic pump, and the like that send a cell suspension by rotating a rotating blade having a magnet by a magnetic force. In the cell culture method according to the present disclosure, it is preferable to use a magnetic levitation type pump for the circulation system including at least the culture vessel, the first cell separation processing unit, and the second cell separation processing unit. This is because the magnetic levitation pump can reduce damage to living cells by appropriately setting the shear stress. In addition, since the magnetic levitation type pump can send liquid at a constant flow rate, when used in the cell culture method according to the present disclosure including the first cell separation process and the second cell separation process, the culture stability is improved. To improve the stability of the quality, quantity, etc. of the collected product even when used in the production method of the product according to the present disclosure described later. Can do. For example, the cell suspension extracted from the culture vessel may be fed by a magnetic levitation pump. Further, the second solution may be fed by a magnetic levitation pump.
　図５は、磁気浮上型ポンプの構成の一例を示す概略断面図である。磁気浮上型ポンプ２００は、細胞懸濁液を吸入する吸入口２１１及び細胞懸濁液を排出する排出口２１２を有するケース２１０と、ケース２１０内に収容された回転羽根２２０と、回転羽根２２０の底部に設けられたロータ磁石２２１と、ロータ磁石２２１を回転させるステータ２３０と、ステータ２３０に巻回されたコイル２４０と、を含んで構成されている。コイル２４０に電流を流すことで、ステータ２３０に磁力が発生する。ケース２１０内において細胞懸濁液中に浮遊した回転羽根２２０は、ステータ２３０が発生させる磁力によって回転する。ステータ２３０が回転することで、細胞懸濁液は吸入口２１１から吸入され、排出口２１２から排出される。磁気浮上型ポンプ２００は、回転羽根２２０を回転駆動させるための回転軸を有していないため、洗浄処理及び滅菌処理を行う際の作業負担を軽減できる。なお、図１においてＤは回転羽根２２０の径を、Ｌは回転羽根２２０の先端と回転羽根２２０の周囲を囲うケース２１０の内壁との距離を表す。 FIG. 5 is a schematic sectional view showing an example of the configuration of the magnetic levitation pump. The magnetic levitation pump 200 includes a case 210 having a suction port 211 for sucking a cell suspension and a discharge port 212 for discharging the cell suspension, a rotating blade 220 accommodated in the case 210, and a rotating blade 220. A rotor magnet 221 provided at the bottom, a stator 230 that rotates the rotor magnet 221, and a coil 240 wound around the stator 230 are configured. By passing a current through the coil 240, a magnetic force is generated in the stator 230. The rotating blades 220 suspended in the cell suspension in the case 210 are rotated by the magnetic force generated by the stator 230. As the stator 230 rotates, the cell suspension is sucked from the suction port 211 and discharged from the discharge port 212. Since the magnetic levitation pump 200 does not have a rotating shaft for driving the rotary blade 220 to rotate, the work load when performing the cleaning process and the sterilization process can be reduced. In FIG. 1, D represents the diameter of the rotary blade 220, and L represents the distance between the tip of the rotary blade 220 and the inner wall of the case 210 surrounding the rotary blade 220.
　磁気浮上型ポンプは、例えば、培養容器から抜き出された細胞懸濁液を送液するために用いることができ、また、第二の溶液を送液するためにも用いることができる。 The magnetic levitation pump can be used, for example, to send a cell suspension extracted from a culture vessel, and can also be used to send a second solution.
　培養容器から抜き出される細胞懸濁液の流量並びに第一から第四の溶液の流量は、培養容器内に収納された細胞懸濁液の液量、所望の死細胞除去速度などに応じて適宜設定することができる。
　例えば、培養容器内の細胞懸濁液のリットルで表した液量をＸとし、第二の細胞分離処理に供される第二の溶液のリットル／分で表した流量をＹとし、第二の細胞分離処理により生じる第四の溶液のリットル／分で表した流量をＺとしたときに、以下の不等式Ａ及び不等式Ｂの両方が満たされるようにすることが好ましい。
　不等式Ａ　　０．３≦１４４０×Ｚ／Ｘ≦５．０
　不等式Ｂ　　２≦Ｙ／Ｚ≦１００
　０．３≦１４４０×Ｚ／Ｘの関係を満たすことで、死細胞の除去効率をさらに向上することができる。１４４０×Ｚ／Ｘ≦５．０を満たすことで、除去される培地の量をより少なくすることができ、コストをより下げることができる。２≦Ｙ／Ｚの関係を満たすことで、死細胞の除去効率をさらに向上することができる。Ｙ／Ｚ≦１００の関係を満たすことで、第二の細胞分離処理における生細胞と死細胞との分離効率が低下することをより効果的に防ぐことができる。 The flow rate of the cell suspension extracted from the culture vessel and the flow rate of the first to fourth solutions are appropriately determined according to the amount of the cell suspension stored in the culture vessel, the desired dead cell removal rate, etc. Can be set.
For example, let X be the amount of cell suspension in the culture vessel expressed in liters, Y be the flow rate expressed in liters / minute of the second solution subjected to the second cell separation process, It is preferable that both the following inequality A and inequality B are satisfied, where Z is the flow rate of the fourth solution generated by the cell separation treatment expressed in liters / minute.
Inequality A 0.3 ≦ 1440 × Z / X ≦ 5.0
Inequality B 2 ≦ Y / Z ≦ 100
By satisfying the relationship of 0.3 ≦ 1440 × Z / X, dead cell removal efficiency can be further improved. By satisfying 1440 × Z / X ≦ 5.0, the amount of the medium to be removed can be reduced, and the cost can be further reduced. By satisfying the relationship of 2 ≦ Y / Z, the dead cell removal efficiency can be further improved. By satisfy | filling the relationship of Y / Z <= 100, it can prevent more effectively that the isolation | separation efficiency of the living cell and dead cell in a 2nd cell separation process falls.
　Ｚ及びＸは、以下の不等式Ａ２を満たすことがより好ましく、以下の不等式Ａ３を満たすことがさらに好ましく、以下の不等式Ａ４を満たすことが一層好ましい。
　不等式Ａ２　　０．６≦１４４０×Ｚ／Ｘ≦４．０
　不等式Ａ３　　０．８≦１４４０×Ｚ／Ｘ≦３．０
　不等式Ａ４　　１．０≦１４４０×Ｚ／Ｘ≦２．５ Z and X preferably satisfy the following inequality A2, more preferably satisfy the following inequality A3, and more preferably satisfy the following inequality A4.
Inequality A2 0.6 ≦ 1440 × Z / X ≦ 4.0
Inequality A3 0.8 ≦ 1440 × Z / X ≦ 3.0
Inequality A4 1.0 ≦ 1440 × Z / X ≦ 2.5
　Ｙ及びＺは、以下の不等式Ｂ２を満たすことがより好ましく、以下の不等式Ｂ３を満たすことがさらに好ましく、以下の不等式Ｂ４を満たすことが一層好ましい。
　不等式Ｂ２　　４≦Ｙ／Ｚ≦５０
　不等式Ｂ３　　６≦Ｙ／Ｚ≦３０
　不等式Ｂ４　　８≦Ｙ／Ｚ≦２５ Y and Z preferably satisfy the following inequality B2, more preferably satisfy the following inequality B3, and more preferably satisfy the following inequality B4.
Inequality B2 4 ≦ Y / Z ≦ 50
Inequality B3 6 ≦ Y / Z ≦ 30
Inequality B4 8 ≦ Y / Z ≦ 25
　本開示に係る細胞培養方法により、細胞懸濁液から効率的に死細胞を除去することができる。このため、細胞懸濁液中の生細胞の成長に対する阻害が低減され、また生細胞が生産物を生産する細胞である場合には、生産物の品質を向上することができる。
　培養容器内の細胞懸濁液における生細胞濃度は、培養容器内の細胞懸濁液における生細胞及び死細胞の合計細胞濃度に対し、８０％以上９９．９％以下の濃度であることが好ましい。培養容器内の細胞懸濁液における生細胞濃度が、培養容器内の細胞懸濁液における生細胞及び死細胞の合計細胞濃度に対し、８０％以上であることで、培養容器内の生細胞の成長の阻害及び生産物の生産の阻害をより効果的に抑制できる。９９．９％は、培養技術上到達可能な上限値である。培養容器内の細胞懸濁液における生細胞濃度は、培養容器内の細胞懸濁液における生細胞及び死細胞の合計細胞濃度に対し、８５％以上９９％以下であることがより好ましく、９０％以上９９％以下であることがさらに好ましく、９２％以上９９％以下であることが一層好ましい。 By the cell culture method according to the present disclosure, dead cells can be efficiently removed from the cell suspension. For this reason, the inhibition with respect to the growth of the living cell in a cell suspension is reduced, and when the living cell is a cell which produces a product, the quality of a product can be improved.
The viable cell concentration in the cell suspension in the culture container is preferably 80% or more and 99.9% or less with respect to the total cell concentration of the living cells and dead cells in the cell suspension in the culture container. . The viable cell concentration in the cell suspension in the culture vessel is 80% or more with respect to the total cell concentration of the living cells and dead cells in the cell suspension in the culture vessel. Growth inhibition and production production inhibition can be more effectively suppressed. 99.9% is the upper limit that can be reached in the culture technique. The living cell concentration in the cell suspension in the culture vessel is more preferably 85% or more and 99% or less, with respect to the total cell concentration of the living cells and dead cells in the cell suspension in the culture vessel, 90% It is more preferably 99% or less and more preferably 92% or more and 99% or less.
　なお、上記の説明に記載の各部分での生細胞の濃度及び死細胞の濃度、並びに生細胞及び死細胞の合計細胞濃度は、測定対象となる各部分において細胞懸濁液又は溶液をサンプルとして採取して、ＢＥＣＫＭＡＮ　ＣＯＵＬＴＥＲ社のＶｉ－ＣＥＬＬ　ＸＲを用いて測定することで求められる。Ｖｉ－ＣＥＬＬ　ＸＲは、トリパンブルー染色を用いているため生細胞と死細胞とをそれぞれ計数することが可能である。Ｖｉ－ＣＥＬＬ　ＸＲによれば、生細胞の濃度及び死細胞の濃度、並びに生細胞及び死細胞の合計細胞濃度だけでなく、サンプル内における生細胞数、死細胞数、並びに生細胞及び死細胞の合計数も求めることができる。 The concentration of living cells and the concentration of dead cells in each part described in the above description, and the total cell concentration of living cells and dead cells are measured using a cell suspension or solution as a sample in each part to be measured. It is obtained by collecting and measuring using BECKMAN COULTER Vi-CELL XR. Since Vi-CELL XR uses trypan blue staining, it is possible to count live and dead cells. According to Vi-CELL XR, not only the concentration of live cells and dead cells, and the total cell concentration of live cells and dead cells, but also the number of live cells, dead cells, and live and dead cells in the sample. The total number can also be determined.
　本開示に係る生産物の製造方法は、
　生産物を生産する細胞を本開示に係る細胞培養方法を用いて培養することと、
　上記第四の溶液から生産物を回収することと、
　を含む、生産物の製造方法である。
　本開示に係る生産物の製造方法においては、本開示に係る細胞培養方法を用いて培養を行うため、培養容器内の細胞懸濁液からは死細胞が効率的に除去されており、生細胞の状態が良好であるため生産物の生産性を高くでき、また、生産物の品質を向上することができる。 A method for producing a product according to the present disclosure is as follows:
Culturing cells producing the product using the cell culture method according to the present disclosure;
Recovering the product from the fourth solution;
Is a method for producing a product.
In the method for producing a product according to the present disclosure, since culturing is performed using the cell culture method according to the present disclosure, dead cells are efficiently removed from the cell suspension in the culture container, and living cells Therefore, the productivity of the product can be increased, and the quality of the product can be improved.
　生産物を生産する細胞としては、先に例示した生産物を生産する細胞を用いることができ、また生産物としては先に例示した生産物とすることができる。生産物は抗体であることが好ましい。 The cells that produce the product exemplified above can be used as the cells that produce the product, and the products exemplified above can be used as the product. The product is preferably an antibody.
　生産物は、一般に、細胞に比してサイズが大幅に小さいため、本開示に係る細胞培養方法においては以下のような挙動を示す。すなわち、培養容器から細胞懸濁液を抜き出す際には、細胞懸濁液に含まれる生産物も共に抜き出される。そして第一の細胞分離処理においては、生産物は細胞に比して小さいサイズを有しているため、大きく濃度減少することなくそのまま第二の溶液に含まれる。第二の液体中に含まれる生産物は、第二の細胞分離処理においては強電界の影響を受けにくく、電場による遮蔽の影響を大きく受けずに、一部が第四の溶液に移行する。このため、第四の溶液から生産物を回収することができる。 Since the product is generally much smaller in size than the cell, the product exhibits the following behavior in the cell culture method according to the present disclosure. That is, when the cell suspension is extracted from the culture vessel, the product contained in the cell suspension is also extracted. In the first cell separation treatment, since the product has a smaller size than the cells, it is directly contained in the second solution without a significant decrease in concentration. The product contained in the second liquid is not easily affected by the strong electric field in the second cell separation process, and part of the product is transferred to the fourth solution without being greatly affected by the shielding by the electric field. For this reason, the product can be recovered from the fourth solution.
　生産物の回収は、単なる第四の溶液の回収、例えば第四の溶液をタンク中に回収することであってもかまわない。回収した第四の溶液に対しては生産物を死細胞から分離するための処理を行うことが好ましく、例えばフィルタ又は遠心分離等により死細胞を溶液から除去することができる。生産物の純度を向上したり、溶媒を変更したり、例えば粉末状にするなど形態を変更したい場合には、死細胞を除去した溶液をさらなる処理に供することができる。 The product may be recovered simply by recovering the fourth solution, for example, recovering the fourth solution in the tank. The recovered fourth solution is preferably subjected to a treatment for separating the product from dead cells. For example, dead cells can be removed from the solution by a filter or centrifugation. When it is desired to improve the purity of the product, change the solvent, or change the form such as powder, the solution from which dead cells have been removed can be subjected to further processing.
　例えば、第四の溶液に含まれる生産物は、精製処理により精製することができる。得られた生産物は、高い純度にまで精製することができる。生産物が抗体又はその断片などのポリペプチドである場合、生産物の分離及び精製は通常のポリペプチドで使用されている分離及び精製方法を使用すればよい。例えば、アフィニティークロマトグラフィー等のクロマトグラフィーカラム、フィルタ、限外濾過、塩析、透析、SDSポリアクリルアミドゲル電気泳動及び等電点電気泳動等を適宜選択、組み合わせれば、ポリペプチドを分離及び精製することができるが、分離及び生成に用いることのできる方法はこれらに限定されるものではない。得られたポリペプチドの濃度測定は吸光度の測定又は酵素結合免疫吸着検定法(Enzyme-linked immunosorbent assay；ELISA）等により行うことができる。　 For example, the product contained in the fourth solution can be purified by a purification treatment. The resulting product can be purified to high purity. When the product is a polypeptide such as an antibody or a fragment thereof, the separation and purification of the product may be performed using the separation and purification methods used in ordinary polypeptides. For example, if a chromatography column such as affinity chromatography, filter, ultrafiltration, salting out, dialysis, SDS polyacrylamide gel electrophoresis, isoelectric focusing etc. is appropriately selected and combined, the polypeptide is separated and purified. However, the methods that can be used for separation and production are not limited to these. The concentration of the obtained polypeptide can be measured by measuring absorbance, enzyme-linked immunosorbent assay (ELISA), or the like. *
　本開示に係る細胞培養装置は、
　細胞懸濁液を収容する培養容器と、
　上記培養容器から抜き出された上記細胞懸濁液を、上記細胞懸濁液中における細胞濃度よりも高い細胞濃度を有する第一の溶液と、上記細胞懸濁液中における細胞濃度よりも低い細胞濃度を有する第二の溶液と、に分離する第一の細胞分離処理を行う第一の細胞分離処理部と、
　上記第一の溶液を上記培養容器に戻す第一の循環流路と、
　上記第二の溶液に対して、誘電泳動により生細胞と死細胞とを分離する第二の細胞分離処理を施して、上記第二の溶液中における生細胞の個数の死細胞の個数に対する比である生細胞／死細胞比よりも高い生細胞／死細胞比を有する第三の溶液と、上記第二の溶液中における生細胞／死細胞比よりも低い生細胞／死細胞比を有する第四の溶液と、に上記第二の溶液を分離する第二の細胞分離処理部と、
　上記第三の溶液を上記培養容器に戻す第二の循環流路と、
　上記第四の溶液を回収するための、回収流路と、
　を含む、細胞培養装置である。本開示に係る細胞培養装置を用いることで、本開示に係る細胞培養方法を実施することができ、培養容器内の細胞懸濁液に含まれる死細胞を効率的に除去することができる。 The cell culture device according to the present disclosure is:
A culture vessel containing the cell suspension;
The cell suspension extracted from the culture vessel is divided into a first solution having a cell concentration higher than the cell concentration in the cell suspension, and cells lower than the cell concentration in the cell suspension. A second solution having a concentration, a first cell separation treatment unit for performing a first cell separation treatment to be separated,
A first circulation channel for returning the first solution to the culture vessel;
The second solution is subjected to a second cell separation process for separating live cells and dead cells by dielectrophoresis, and the ratio of the number of live cells to the number of dead cells in the second solution is as follows. A third solution having a higher ratio of live cells / dead cells than a ratio of live cells / dead cells, and a fourth solution having a ratio of live cells / dead cells lower than the ratio of live cells / dead cells in the second solution. A second cell separation processing unit for separating the second solution into
A second circulation channel for returning the third solution to the culture vessel;
A recovery channel for recovering the fourth solution;
Is a cell culture device. By using the cell culture device according to the present disclosure, the cell culture method according to the present disclosure can be performed, and dead cells included in the cell suspension in the culture container can be efficiently removed.
　第一の細胞分離処理部は、第一の細胞分離処理を行う。上述のとおり、第一の細胞分離処理として、遠心分離処理、定在波の音波による細胞分離処理などが挙げられるため、これに対応して、第一の細胞分離処理部は、遠心分離機、定在波の音波による細胞分離処理部などとすることができる。第一の細胞分離処理部は、定在波の音波による細胞分離処理部であることが好ましい。 The first cell separation processing unit performs the first cell separation processing. As described above, the first cell separation process includes a centrifugal separation process, a cell separation process using standing wave sound waves, and the like, and correspondingly, the first cell separation processing unit includes a centrifuge, A cell separation processing unit using standing wave sound waves can be used. The first cell separation processing unit is preferably a cell separation processing unit using standing wave sound waves.
　上述のとおり、培養容器と第一の細胞分離処理部とは直結していてもよいし、流路で連絡していてもよい。流路で連絡している場合には、培養容器から第一の細胞分離処理部の間の流路には送液装置を設けることができ、特に、磁気浮上型ポンプを設けることが好ましい。また、第一の細胞分離処理部と第二の細胞分離処理部とは直結していてもよいし、流路で連絡していてもよい。流路で連絡している場合には、第一の細胞分離処理部から第二の細胞分離処理部の間の流路には送液装置を設けることができ、特に、磁気浮上型ポンプを設けることが好ましい。 As described above, the culture vessel and the first cell separation processing unit may be directly connected or may be communicated with each other through a flow path. When communicating with the flow channel, a liquid feeding device can be provided in the flow channel between the culture vessel and the first cell separation processing unit, and it is particularly preferable to provide a magnetic levitation pump. Further, the first cell separation processing unit and the second cell separation processing unit may be directly connected or may be communicated with each other through a flow path. When communicating with the flow path, a liquid feeding device can be provided in the flow path between the first cell separation processing section and the second cell separation processing section, and in particular, a magnetic levitation pump is provided. It is preferable.
　第二の細胞分離処理部は、第二の溶液に対して誘電泳動による第二の細胞分離処理を行う。第二の細胞分離処理部は、交流電源に電気的に連絡した電極を含んで構成することができる。第二の細胞分離処理部は、生細胞を電極から引き離す方向に泳動させるものであることが好ましい。 The second cell separation processing unit performs a second cell separation process by dielectrophoresis on the second solution. The second cell separation processing unit can be configured to include an electrode electrically connected to an AC power source. It is preferable that the second cell separation processing unit migrates the living cells in a direction to separate them from the electrodes.
　本開示に係る細胞培養方法、生産物の製造方法、及び細胞培養装置の一例について、図１においてまとめて説明する。図１は、本開示に係る生産物の製造方法の実施に適用可能な細胞培養装置１００の構成の一例を示す図である。 An example of a cell culture method, a product production method, and a cell culture apparatus according to the present disclosure will be described together in FIG. FIG. 1 is a diagram illustrating an example of a configuration of a cell culture device 100 that can be applied to the production method for a product according to the present disclosure.
　細胞培養装置１００は、生産物の一例としての抗体を生産する細胞を培地とともに収容し培養する培養容器１０と、培養容器１０内に新鮮培地を供給する流路５６と、培養容器１０に収容されている細胞懸濁液を抜き出して、音波定在波型細胞分離処理部３０に送る流路５１と、培養容器１０から抜き出された細胞懸濁液に対して第一の細胞分離処理を施す第一の細胞分離処理部としての音波定在波型細胞分離処理部３０と、音波定在波型細胞分離処理部３０から排出される細胞濃度が増加した第一の溶液を培養容器１０に戻す循環流路５２と、音波定在波型細胞分離処理部３０から排出される細胞濃度が減少した第二の溶液を第二の細胞分離処理部４０に送る流路５３と、第二の溶液に対して誘電泳動による第二の細胞分離処理を施す第二の細胞分離処理部４０と、第二の細胞分離処理部４０から排出される生細胞／死細胞比が上昇した第三の溶液を培養容器に戻す循環流路５４と、第二の細胞分離処理部４０から排出される生細胞／死細胞比が低下した第四の溶液を回収のために送液する回収流路５５とを有する。矢印Ａ１は流路５６における新鮮培地の流れの方向を示し、矢印Ａ２は流路５１における細胞懸濁液の流れの方向を示し、矢印Ａ３は循環流路５２における第一の溶液の流れの方向を示し、矢印Ａ４は流路５３における第二の溶液の流れの方向を示し、矢印Ａ５は循環流路５４における第三の溶液の流れの方向を示し、矢印Ａ６は回収流路５５における第四の溶液の流れの方向を示す。 The cell culture device 100 is accommodated in the culture vessel 10 that accommodates and cultures cells that produce antibodies as an example of the product together with the culture medium, the flow channel 56 that supplies the fresh culture medium into the culture vessel 10, and the culture vessel 10. The first cell separation process is performed on the cell suspension extracted from the culture vessel 10 and the flow path 51 that is extracted from the cell suspension and sent to the sonic standing wave cell separation processing unit 30. Return the sonic standing wave cell separation processing unit 30 as the first cell separation processing unit and the first solution discharged from the sonic standing wave cell separation processing unit 30 to the culture vessel 10. A circulation channel 52, a channel 53 for sending the second solution with reduced cell concentration discharged from the sonic standing wave cell separation processing unit 30 to the second cell separation processing unit 40, and a second solution The second cell separation process by dielectrophoresis Cell separation processing unit 40, a circulation channel 54 for returning the third solution with an increased ratio of living cells / dead cells discharged from the second cell separation processing unit 40 to the culture vessel, and a second cell separation process And a recovery channel 55 for sending the fourth solution having a reduced ratio of living cells / dead cells discharged from the section 40 for recovery. Arrow A1 indicates the direction of the fresh medium flow in the flow path 56, arrow A2 indicates the direction of the cell suspension flow in the flow path 51, and arrow A3 indicates the direction of the first solution flow in the circulation flow path 52. The arrow A4 indicates the direction of the second solution flow in the flow path 53, the arrow A5 indicates the direction of the third solution flow in the circulation flow path 54, and the arrow A6 indicates the fourth direction in the recovery flow path 55. Shows the direction of the solution flow.
　培養容器１０の内部には、撹拌翼１１を有する撹拌装置が設けられている。撹拌翼１１を回転させることで、培養容器１０の内部に収容された培地が撹拌され、培地の均質性が保たれる。 A stirring device having a stirring blade 11 is provided inside the culture vessel 10. By rotating the stirring blade 11, the medium contained in the culture vessel 10 is stirred, and the uniformity of the medium is maintained.
　流路５１には、ポンプＰ１が設けられ、培養容器から細胞懸濁液を抜き出して音波定在波型細胞分離処理部３０へと送液する。音波定在波型細胞分離処理部３０に導入された細胞懸濁液は、トランスデューサーＴと音波反射板Ｍにより形成された音波の定在波Ｓに曝され、定在波Ｓの節又は腹に細胞が捕捉される。これにより培養容器から抜き出された細胞懸濁液よりも細胞濃度が減少した第二の溶液が得られ、流路５３に設けられたポンプＰ２により第二の細胞分離処理部４０へと送液される。一方、定在波Ｓの節又は腹に捕捉された細胞は凝集して沈降し、培養容器から抜き出された細胞懸濁液よりも細胞濃度が増加した第一の溶液として循環流路５２へと排出され、培養容器１０に戻される。第二の細胞分離処理部４０へと供給された第二の溶液においては、電極４１が誘電泳動力を及ぼす中、死細胞及び生産物としての抗体の一部が電極４１に印加される電圧により形成される強電界領域を通過し、第二の溶液中における生細胞／死細胞比よりも低い生細胞／死細胞比を有する第四の溶液としてポンプＰ３により回収流路５５へと排出される。死細胞及び生産物としての抗体の残りの部分及び生細胞は、第二の溶液中における生細胞／死細胞比よりも高い生細胞／死細胞比を有する第三の溶液として循環流路５４を通って培養容器１０へと戻される。この結果、回収流路５５へと排出される第四の溶液の量の分、培養容器１０内の細胞懸濁液の量が減少することになるため、ポンプＰ４を駆動して流路５６を通して新鮮培地を培養容器１０内に補充する。 In the flow path 51, a pump P1 is provided, and a cell suspension is extracted from the culture vessel and fed to the sonic standing wave cell separation processing unit 30. The cell suspension introduced into the sonic standing wave type cell separation processing unit 30 is exposed to the standing wave S of the sonic wave formed by the transducer T and the sonic wave reflector M, and the node or belly of the standing wave S is exposed. The cells are trapped. As a result, a second solution having a cell concentration reduced from that of the cell suspension extracted from the culture container is obtained, and the solution is fed to the second cell separation processing unit 40 by the pump P2 provided in the flow path 53. Is done. On the other hand, the cells trapped in the nodes or the abdomen of the standing wave S aggregate and settle, and enter the circulation channel 52 as a first solution having a higher cell concentration than the cell suspension extracted from the culture vessel. Is discharged and returned to the culture vessel 10. In the second solution supplied to the second cell separation processing unit 40, while the electrode 41 exerts a dielectrophoretic force, a dead cell and a part of the antibody as a product are generated by a voltage applied to the electrode 41. It passes through the formed strong electric field region, and is discharged to the recovery channel 55 by the pump P3 as a fourth solution having a living cell / dead cell ratio lower than the living cell / dead cell ratio in the second solution. . Dead cells and the remainder of the antibody as product and living cells are passed through the circulation channel 54 as a third solution having a higher ratio of living cells / dead cells than the ratio of living cells / dead cells in the second solution. It returns to the culture vessel 10 through. As a result, since the amount of the cell suspension in the culture vessel 10 is reduced by the amount of the fourth solution discharged to the recovery channel 55, the pump P4 is driven to pass through the channel 56. The culture medium 10 is replenished with fresh medium.
　生産物としての抗体の製造に用いる場合には、回収流路５５へと排出された第四の溶液は、死細胞は通過させないが生産物としての抗体は通過させる孔径を有するフィルタ５７を通り、フィルタ５７に死細胞が回収される。フィルタ５７を通過した生産物としての抗体を含む溶液は、さらなる精製処理部（図示せず）に供される。 When used for production of an antibody as a product, the fourth solution discharged to the recovery channel 55 passes through a filter 57 having a pore size that does not allow dead cells to pass but allows the antibody as a product to pass through. Dead cells are collected in the filter 57. The solution containing the antibody as a product that has passed through the filter 57 is supplied to a further purification processing section (not shown).
　培養容器１０、流路５１、音波定在波型細胞分離処理部３０、循環流路５２、流路５３、第二の細胞分離処理部４０、及び循環流路５４は、循環系を形成している。このため、これらの部分には、生細胞が繰り返し通過することになる。このことから、ポンプＰ１及びＰ２は生細胞へのダメージが少ないポンプを使用することが好ましく、特に、磁気浮上型ポンプを用いることが好ましい。 The culture vessel 10, the channel 51, the sonic standing wave cell separation processing unit 30, the circulation channel 52, the channel 53, the second cell separation processing unit 40, and the circulation channel 54 form a circulation system. Yes. For this reason, live cells repeatedly pass through these portions. Therefore, it is preferable to use pumps P1 and P2 that are less damaging to living cells, and it is particularly preferable to use a magnetic levitation pump.
　以上説明したように、本開示によれば、高濃度で細胞を培養しながら培養容器内の死細胞を除去する細胞培養方法及び細胞培養装置、並びに高濃度で細胞を培養しながら培養容器内の死細胞を除去する細胞培養方法を用いた生産物の製造方法を提供することができる。生産物の製造方法の場合、製造された生産物は、例えばバイオ医薬品及び再生医療等において用いることができる。 As described above, according to the present disclosure, the cell culture method and the cell culture apparatus for removing dead cells in the culture container while culturing the cells at a high concentration, and the cells in the culture container while culturing the cells at a high concentration A method for producing a product using a cell culture method for removing dead cells can be provided. In the case of the production method of the product, the produced product can be used, for example, in biopharmaceuticals and regenerative medicine.
　各図面における各符号が表す対象については、上記の説明のとおりであるが、符号と、符号が表す対象とを以下にまとめて記載する。
（符号の説明） About the object which each code | symbol in each drawing represents, it is as said description, However, A code | symbol and the object which a code | symbol represent are collectively described below.
(Explanation of symbols)
１０　培養容器
１１　撹拌翼
３０　音波定在波型細胞分離処理部
４０　細胞分離処理部
４１　電極
５１、５３、５４、５６　流路
５２、５４　循環流路
５５　回収流路
５７　フィルタ
１００　細胞培養装置
２００　磁気浮上型ポンプ
２１０　ケース
２１１　吸入口
２１２　排出口
２２０　回転羽根
２２１　ロータ磁石
２３０　ステータ
２４０　コイル
３００　音波定在波型細胞分離処理部
３１０、３３０、３４０　ポート
３２０　導入部
３５０　チャンバー
４１０ａ、４１０ｂ　棒状電極
４２０　交流電源
４３０　電圧制御部
４４０　生細胞
４５０　死細胞
４６０　生産物
Ａ１～Ａ１５　矢印
Ｐ１～Ｐ４　ポンプ
Ｄ：回転羽根の径
Ｅ：強電界領域
Ｌ：回転羽根の先端と回転羽根の周囲を囲うケースの内壁との距離
Ｍ：音波反射板
Ｓ：定在波
Ｔ：トランスデューサー DESCRIPTION OF SYMBOLS 10 Cultivation container 11 Stirring blade 30 Sonic standing wave type cell separation processing part 40 Cell separation processing part 41 Electrode 51, 53, 54, 56 Channel 52, 54 Circulation channel 55 Recovery channel 57 Filter 100 Cell culture device 200 Magnetic Floating pump 210 Case 211 Suction port 212 Discharge port 220 Rotating blade 221 Rotor magnet 230 Stator 240 Coil 300 Sonic standing wave type cell separation processing unit 310, 330, 340 Port 320 Introducing unit 350 Chamber 410a, 410b Rod electrode 420 AC power source 430 Voltage control unit 440 Live cell 450 Dead cell 460 Product A1 to A15 Arrows P1 to P4 Pump D: Diameter of rotating blade E: Strong electric field region L: Between the tip of the rotating blade and the inner wall of the case surrounding the rotating blade Distance M: Sound wave reflector S: Standing wave T: Transducer
　２０１８年６月１５日に出願された日本国特許出願２０１８－１１４４１０号の開示は、その全体が参照により本明細書に取り込まれる。
　本明細書に記載された全ての文献、特許出願、および技術規格は、個々の文献、特許出願、および技術規格が参照により取り込まれることが具体的かつ個々に記された場合と同程度に、本明細書中に参照により取り込まれる。 The disclosure of Japanese Patent Application No. 2018-114410 filed on Jun. 15, 2018 is incorporated herein by reference in its entirety.
All documents, patent applications, and technical standards mentioned in this specification are to the same extent as if each individual document, patent application, and technical standard were specifically and individually described to be incorporated by reference, Incorporated herein by reference.

Claims (21)

  1. 　培養容器内に収容された細胞懸濁液中で細胞を培養することと、
    　前記培養容器から前記細胞懸濁液を抜き出して、抜き出された前記細胞懸濁液を、前記細胞懸濁液中における細胞濃度よりも高い細胞濃度を有する第一の溶液と、前記細胞懸濁液中における細胞濃度よりも低い細胞濃度を有する第二の溶液と、に分離する第一の細胞分離処理を行うことと、
    　前記第一の溶液を前記培養容器に戻すことと、
    　前記第二の溶液に対して、誘電泳動により生細胞と死細胞とを分離する第二の細胞分離処理を施して、前記第二の溶液中における生細胞の個数の死細胞の個数に対する比である生細胞／死細胞比よりも高い生細胞／死細胞比を有する第三の溶液と、前記第二の溶液中における生細胞／死細胞比よりも低い生細胞／死細胞比を有する第四の溶液と、に前記第二の溶液を分離することと、
    　前記第三の溶液を前記培養容器に戻すことと、
    　前記第四の溶液を回収することと、
    　を含む細胞培養方法。     Culturing cells in a cell suspension contained in a culture vessel;
    The cell suspension is extracted from the culture vessel, and the extracted cell suspension is divided into a first solution having a cell concentration higher than the cell concentration in the cell suspension, and the cell suspension. Performing a first cell separation treatment to separate into a second solution having a cell concentration lower than the cell concentration in the liquid;
    Returning the first solution to the culture vessel;
    The second solution is subjected to a second cell separation process for separating live cells and dead cells by dielectrophoresis, and the ratio of the number of live cells to the number of dead cells in the second solution is A third solution having a higher ratio of live cells / dead cells than a ratio of live cells / dead cells, and a fourth solution having a ratio of live cells / dead cells lower than the ratio of live cells / dead cells in the second solution. Separating the second solution into a solution of
    Returning the third solution to the culture vessel;
    Recovering the fourth solution;
    A cell culture method comprising:
  2. 　前記第一の細胞分離処理が、定在波の音波を用いて定在波の腹又は節に細胞を捕捉する細胞分離処理である、請求項１に記載の細胞培養方法。 The cell culturing method according to claim 1, wherein the first cell separation treatment is a cell separation treatment in which cells are trapped in the antinodes or nodes of a standing wave using standing wave sound waves.
  3. 　前記第二の細胞分離処理が、電極を用いて、生細胞を前記電極から引き離す方向に泳動させることを含む、請求項１又は請求項２に記載の細胞培養方法。 The cell culturing method according to claim 1 or 2, wherein the second cell separation treatment includes using an electrode to migrate a living cell in a direction separating the electrode from the electrode.
  4. 　前記培養容器から抜き出された前記細胞懸濁液が、前記細胞懸濁液中に浮遊し且つ磁石を有する回転羽根を磁力により回転させることで前記細胞懸濁液を送液するポンプにより送液される、請求項１～請求項３のうちいずれか一項に記載の細胞培養方法。 The cell suspension extracted from the culture vessel is floated in the cell suspension and fed by a pump that feeds the cell suspension by rotating a rotating blade having a magnet by a magnetic force. The cell culture method according to any one of claims 1 to 3, wherein the cell culture method is performed.
  5. 　前記第二の溶液が、前記第二の溶液中に浮遊し且つ磁石を有する回転羽根を磁力により回転させることで前記第二の溶液を送液するポンプにより送液される、請求項１～請求項４のうちいずれか一項に記載の細胞培養方法。 The second solution is fed by a pump that feeds the second solution by rotating a rotating blade floating in the second solution and having a magnet by a magnetic force. Item 5. The cell culture method according to any one of Items4.
  6. 　前記第二の溶液における生細胞及び死細胞の合計細胞濃度が、前記培養容器内の前記細胞懸濁液における生細胞及び死細胞の合計細胞濃度に対し、０．１％以上５０％以下の濃度である、請求項１～請求項５のうちいずれか一項に記載の細胞培養方法。 The total cell concentration of live cells and dead cells in the second solution is a concentration of 0.1% or more and 50% or less with respect to the total cell concentration of live cells and dead cells in the cell suspension in the culture container. The cell culture method according to any one of claims 1 to 5, wherein
  7. 　前記第四の溶液における生細胞濃度が、前記第二の溶液における生細胞濃度の５０％以下の濃度である、請求項１～請求項６のうちいずれか一項に記載の細胞培養方法。 The cell culture method according to any one of claims 1 to 6, wherein the viable cell concentration in the fourth solution is 50% or less of the viable cell concentration in the second solution.
  8. 　前記培養容器内の前記細胞懸濁液における生細胞濃度が２０×１０６ｃｅｌｌｓ／ｍｌ以上１５０×１０６ｃｅｌｌｓ／ｍｌ以下である、請求項１～請求項７のうちいずれか一項に記載の細胞培養方法。 The viable cell concentration in the cell suspension in the culture vessel is 20 × 10 6 cells / ml or more and 150 × 10 6 cells / ml or less, according to any one of claims 1 to 7. Cell culture method.
  9. 　前記培養容器内の前記細胞懸濁液における生細胞濃度が、前記培養容器内の前記細胞懸濁液における生細胞及び死細胞の合計細胞濃度に対し、８０％以上９９．９％以下の濃度である、請求項１～請求項８のうちいずれか一項に記載の細胞培養方法。 The concentration of viable cells in the cell suspension in the culture vessel is 80% or more and 99.9% or less of the total cell concentration of live cells and dead cells in the cell suspension in the culture vessel. The cell culture method according to any one of claims 1 to 8, wherein
  10. 　前記第二の溶液における生細胞及び死細胞の合計細胞濃度が、０．５×１０６ｃｅｌｌｓ／ｍｌ以上５０×１０６ｃｅｌｌｓ／ｍｌ以下である、請求項１～請求項９のうちいずれか一項に記載の細胞培養方法。 10. The total cell concentration of live cells and dead cells in the second solution is 0.5 × 10 6 cells / ml or more and 50 × 10 6 cells / ml or less. The cell culture method according to Item.
  11. 　前記第四の溶液における死細胞濃度が、０．０１×１０６ｃｅｌｌｓ／ｍｌ以上１０×１０６ｃｅｌｌｓ／ｍｌ以下である、請求項１～請求項１０のうちいずれか一項に記載の細胞培養方法。 The cell culture according to any one of claims 1 to 10, wherein a concentration of dead cells in the fourth solution is 0.01 x 10 6 cells / ml or more and 10 x 10 6 cells / ml or less. Method.
  12. 　前記第四の溶液における生細胞濃度が、前記第四の溶液における生細胞及び死細胞の合計細胞濃度に対して２０％以下である、請求項１～請求項１１のうちいずれか一項に記載の細胞培養方法。 The living cell concentration in the fourth solution is 20% or less with respect to the total cell concentration of living cells and dead cells in the fourth solution. Cell culture method.
  13. 　前記培養容器内の前記細胞懸濁液のリットルで表した液量をＸとし、前記第二の細胞分離処理に供される前記第二の溶液のリットル／分で表した流量をＹとし、前記第二の細胞分離処理により生じる前記第四の溶液のリットル／分で表した流量をＺとしたときに、以下の不等式Ａ及び不等式Ｂの両方が満たされる、請求項１～請求項１２のうちいずれか一項に記載の細胞培養方法。
    　不等式Ａ　　０．３≦１４４０×Ｚ／Ｘ≦５．０
    　不等式Ｂ　　２≦Ｙ／Ｚ≦１００     The amount of liquid expressed in liters of the cell suspension in the culture vessel is set as X, the flow rate expressed in liters / minute of the second solution subjected to the second cell separation process is set as Y, and The following inequality A and inequality B are both satisfied, where Z is a flow rate expressed in liters / minute of the fourth solution generated by the second cell separation treatment: The cell culture method according to any one of the above.
    Inequality A 0.3 ≦ 1440 × Z / X ≦ 5.0
    Inequality B 2 ≦ Y / Z ≦ 100
  14. 　前記細胞懸濁液に含まれる細胞がＣＨＯ細胞である、請求項１～請求項１３のうちいずれか一項に記載の細胞培養方法。 The cell culture method according to any one of claims 1 to 13, wherein the cells contained in the cell suspension are CHO cells.
  15. 　生産物を生産する細胞を請求項１～請求項１４のうちいずれか一項に記載の細胞培養方法を用いて培養することと、
    　前記第四の溶液から生産物を回収することと、
    　を含む、生産物の製造方法。     Culturing cells producing the product using the cell culture method according to any one of claims 1 to 14;
    Recovering the product from the fourth solution;
    A method for producing a product, comprising:
  16. 　前記生産物が抗体である、請求項１５に記載の生産物の製造方法。 The method for producing a product according to claim 15, wherein the product is an antibody.
  17. 　細胞懸濁液を収容する培養容器と、
    　前記培養容器から抜き出された前記細胞懸濁液を、前記細胞懸濁液中における細胞濃度よりも高い細胞濃度を有する第一の溶液と、前記細胞懸濁液中における細胞濃度よりも低い細胞濃度を有する第二の溶液と、に分離する第一の細胞分離処理を行う第一の細胞分離処理部と、
    　前記第一の溶液を前記培養容器に戻す第一の循環流路と、
    　前記第二の溶液に対して、誘電泳動により生細胞と死細胞とを分離する第二の細胞分離処理を施して、前記第二の溶液中における生細胞の個数の死細胞の個数に対する比である生細胞／死細胞比よりも高い生細胞／死細胞比を有する第三の溶液と、前記第二の溶液中における生細胞／死細胞比よりも低い生細胞／死細胞比を有する第四の溶液と、に前記第二の溶液を分離する第二の細胞分離処理部と、
    　前記第三の溶液を前記培養容器に戻す第二の循環流路と、
    　前記第四の溶液を回収するための、回収流路と、
    　を含む、細胞培養装置。     A culture vessel containing the cell suspension;
    The cell suspension extracted from the culture container is divided into a first solution having a cell concentration higher than the cell concentration in the cell suspension, and cells lower than the cell concentration in the cell suspension. A second solution having a concentration, a first cell separation treatment unit for performing a first cell separation treatment to be separated,
    A first circulation channel for returning the first solution to the culture vessel;
    The second solution is subjected to a second cell separation process for separating live cells and dead cells by dielectrophoresis, and the ratio of the number of live cells to the number of dead cells in the second solution is A third solution having a higher ratio of live cells / dead cells than a ratio of live cells / dead cells, and a fourth solution having a ratio of live cells / dead cells lower than the ratio of live cells / dead cells in the second solution. A second cell separation processing unit for separating the second solution into
    A second circulation channel for returning the third solution to the culture vessel;
    A recovery channel for recovering the fourth solution;
    A cell culture device.
  18. 　前記第一の細胞分離処理部が、定在波の音波を用いて定在波の腹又は節に細胞を捕捉する細胞分離処理部である、請求項１７に記載の細胞培養装置。 The cell culture device according to claim 17, wherein the first cell separation processing unit is a cell separation processing unit that captures cells in an antinode or node of a standing wave using a standing wave sound wave.
  19. 　前記第二の細胞分離処理部が電極を含み、前記第二の細胞分離処理部は、生細胞を前記電極から引き離す方向に泳動させる、請求項１７又は請求項１８に記載の細胞培養装置。 The cell culture device according to claim 17 or 18, wherein the second cell separation processing unit includes an electrode, and the second cell separation processing unit migrates a living cell in a direction to separate the living cell from the electrode.
  20. 　前記培養容器から前記第一の細胞分離処理部の間に設けられた、前記細胞懸濁液中に浮遊し且つ磁石を有する回転羽根を磁力により回転させることで前記細胞懸濁液を送液するポンプをさらに含む、請求項１７～請求項１９のうちいずれか一項に記載の細胞培養装置。 The cell suspension is fed from the culture vessel by rotating magnetically a rotating blade provided between the first cell separation processing unit and suspended in the cell suspension and having a magnet. The cell culture device according to any one of claims 17 to 19, further comprising a pump.
  21. 　前記第一の細胞分離処理部と前記第二の細胞分離処理部との間に設けられた、前記第二の溶液中に浮遊し且つ磁石を有する回転羽根を磁力により回転させることで前記第二の溶液を送液するポンプをさらに含む、請求項１７～請求項２０のうちいずれか一項に記載の細胞培養装置。 By rotating a rotating blade provided between the first cell separation processing unit and the second cell separation processing unit, which floats in the second solution and has a magnet, by the magnetic force, the second The cell culture device according to any one of claims 17 to 20, further comprising a pump for feeding the solution.
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