WO2020044984A1 - Cell culture method and cell culture device - Google Patents

Cell culture method and cell culture device Download PDF

Info

Publication number
WO2020044984A1
WO2020044984A1 PCT/JP2019/030859 JP2019030859W WO2020044984A1 WO 2020044984 A1 WO2020044984 A1 WO 2020044984A1 JP 2019030859 W JP2019030859 W JP 2019030859W WO 2020044984 A1 WO2020044984 A1 WO 2020044984A1
Authority
WO
WIPO (PCT)
Prior art keywords
cell
cell suspension
concentration
unit
membrane
Prior art date
Application number
PCT/JP2019/030859
Other languages
French (fr)
Japanese (ja)
Inventor
淳史 稲田
英俊 高山
Original Assignee
富士フイルム株式会社
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 富士フイルム株式会社 filed Critical 富士フイルム株式会社
Publication of WO2020044984A1 publication Critical patent/WO2020044984A1/en

Links

Images

Classifications

    • 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
    • C12M1/33—Disintegrators
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M3/00—Tissue, human, animal or plant cell, or virus culture apparatus
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • 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

  • Japanese Patent Application Laid-Open No. 2016-131538 discloses a culture vessel, a storage vessel, a division processing section for performing division processing for dividing a cell aggregate, and a medium supply section for supplying a culture medium in a flow path.
  • a cell culture device comprising:
  • the cell aggregates In culturing stem cells such as iPS cells (induced pluripotent stem cells), if the size of cell aggregates (spheres) generated by culturing the cells becomes excessive, the cell aggregates adhere and fuse to each other, and the cells start to differentiate. Or cells in the center of the cell aggregate may be necrotic. Therefore, in order to prevent the size of the cell aggregate from becoming excessively large, the cell aggregate is divided (disrupted) into a plurality of smaller-sized cell aggregates at an appropriate time during the cell culture period. Division processing has been performed. As a method of dividing the cell aggregate, a method of mechanically dividing the cell aggregate by passing a cell suspension containing the cell aggregate through a mesh having a plurality of openings (mesh) has been proposed. . In a division process using a mesh, cell aggregates are damaged at the time of mesh collision, and dead cells are inevitably generated.
  • iPS cells induced pluripotent stem cells
  • the culture medium is altered by metabolites secreted from cells or dead cells. Therefore, at an appropriate time during the culture period, a medium exchange process for exchanging the used medium in the culture container with a fresh medium is required.
  • the medium exchange process includes a concentration separation process for separating the used medium from the cells, and a mixing process of mixing the separated cells with a fresh medium.
  • culture medium exchange processing was performed on a cell suspension contained in a culture vessel by culture medium exchange means. It is assumed that the configuration is such that the division processing is performed later by division means including a mesh as necessary.
  • FIG. 1 is a graph showing an example of the frequency distribution of the size of cell aggregates at each stage of cell culture using the cell culture device having the above configuration.
  • the solid line indicates the state before the medium exchange processing
  • the dashed line indicates the state after the medium exchange processing
  • the dotted line indicates the state after the division processing.
  • the cell culture apparatus having the above configuration when applied to a large-scale culture in which, for example, about 1 ⁇ 10 9 or more cells are to be cultured, the amount of debris generated by the division treatment increases.
  • the mesh constituting the dividing means when mass culture is performed, the mesh constituting the dividing means may be clogged, cells may stay on the mesh, and the state of the mesh may change every moment as the throughput increases. In such a situation where the state of the mesh changes every moment, it becomes difficult to predict the cell concentration in the cell suspension after the division, and as a result, the control of the cell concentration of the cell suspension recovered after the division is performed. Becomes difficult.
  • the cell concentration in the cell suspension is an important parameter related to cell growth and viability, and it is important to control the cell concentration in the cell suspension in an appropriate range.
  • the disclosed technology provides a cell culture method and a cell culture device capable of suppressing mixing of unnecessary components such as dead cells generated by the division processing into a cell suspension collected after the division processing.
  • the cell culture method includes a dividing step of dividing the cell aggregate, and separating the components of the cell suspension containing the cell aggregate divided in the dividing step to increase the concentration of cells in the cell suspension.
  • a concentration separation step of concentrating a mixing step of obtaining a mixed liquid obtained by mixing the cell suspension and the medium that have undergone the concentration separation step, a measurement step of measuring the concentration of cells in the mixture, and after the measurement step, the mixed liquid And recovering. This makes it possible to prevent unnecessary components such as dead cells generated by the dividing process from being mixed into the cell suspension collected after the dividing process.
  • the cell culture method according to the disclosed technology may further include a replenishing step of replenishing the mixed solution with a medium based on the concentration measured in the measuring step. Thereby, the concentration of the cells in the collected liquid mixture can be adjusted to an appropriate concentration.
  • the concentration separation step may include a membrane separation treatment for separating a cell suspension containing the cell aggregates divided in the division step.
  • the membrane separation process includes a first membrane separation process of membrane-separating the cell suspension containing the cell aggregates divided in the dividing step using the first filtration membrane, and a component blocked by the first filtration membrane. And a second membrane separation process of performing membrane separation using a second filtration membrane. As described above, by performing the membrane separation process in two stages, the separability in the membrane separation process can be improved.
  • the pore size of the first filtration membrane may be smaller than the pore size of the second filtration membrane.
  • a cell suspension from which debris such as dead cells has been removed can be subjected to membrane separation using the second filter membrane.
  • the component blocked by the second filtration membrane and the component permeated through the second filtration membrane may be separately recovered. Thereby, culturing can be continued in an optimal environment for the collected components.
  • a mixture of the component blocked by the first filtration membrane and the diluent may be subjected to membrane separation using the second filtration membrane.
  • the concentration of cells in the cell suspension to be subjected to membrane separation using the second filtration membrane can be adjusted to an appropriate concentration.
  • the cell culture method according to the disclosed technology may further include a dilution step of adding a diluent to a cell suspension containing the cell aggregate before the division in the division step.
  • a dilution step of adding a diluent to a cell suspension containing the cell aggregate before the division in the division step may be optimized.
  • mesh clogging over time due to a medium-containing component such as a thickener in the dividing step can be reduced.
  • the concentration / separation step may include a centrifugation treatment for separating components of the cell suspension containing the cell aggregates divided in the division step by centrifugation.
  • the centrifugation process includes a first centrifugation process of separating the components of the cell suspension containing the cell aggregates divided in the division step by centrifugation under the first condition, and a first centrifugation process.
  • a second centrifugation process of separating the component of the cell suspension thus performed by centrifugation under a second condition different from the first condition.
  • the cell culture device includes a dividing unit that divides a cell aggregate, a concentration separation unit that separates components of a cell suspension to concentrate the concentration of cells in the cell suspension, and contains a medium.
  • Medium container for monitoring for monitoring, a monitor unit for monitoring the cell suspension, a dividing unit, a concentration separation unit, a channel connected to the medium container and the monitor unit, and a cell suspension and a medium via the channel.
  • a controller for controlling the transfer of the data The control unit transfers the cell suspension containing the cell aggregates divided in the division unit to the concentration and separation unit, and the cell suspension concentrated in the concentration and separation unit and the medium contained in the medium containing container.
  • the mixed liquid mixture is transferred to the monitor, and the mixed liquid monitored by the monitor is transferred to a container connected to the flow path. This makes it possible to prevent unnecessary components such as dead cells generated by the dividing process from being mixed into the cell suspension collected after the dividing process.
  • the cell culture device may further include a culture container connected to the flow channel.
  • the control unit may transfer the cell suspension containing the cell aggregate contained in the culture container to the division unit, and may transfer the mixed solution monitored by the monitor unit to the culture container.
  • the cell culture device may further include a culture container connected to the flow channel and a collection container connected to the flow channel.
  • the control unit may transfer the cell suspension containing the cell aggregate contained in the culture container to the division unit, and may transfer the mixed solution monitored by the monitor unit to the collection container.
  • the control unit may replenish the mixed solution with the medium contained in the medium containing container based on the concentration of cells contained in the mixed solution derived based on the result of monitoring by the monitor unit. Thereby, the concentration of the cells in the collected liquid mixture can be adjusted to an appropriate concentration.
  • the concentration and separation unit may include a filtration unit provided with a filtration membrane for membrane-separating the cell suspension.
  • the concentration / separation unit was provided with a first filtration unit provided with a first filtration membrane for separating a cell suspension by membrane, and a second filtration membrane provided for separating components blocked by the first filtration membrane. And a second filtration unit. As described above, by performing the membrane separation process in two stages, the separability in the membrane separation process can be improved.
  • the pore size of the first filtration membrane may be smaller than the pore size of the second filtration membrane.
  • a cell suspension from which debris such as dead cells has been removed can be subjected to membrane separation using the second filter membrane.
  • the cell culture device includes a first collection container that stores components blocked by the second filtration membrane, and a second collection container that stores components that have passed through the second filtration membrane. May be included. Thereby, culturing can be continued in an optimal environment for the collected components.
  • the concentration and separation unit may include a centrifugal separation mechanism that separates the cell suspension by centrifugation.
  • the concentration / separation unit includes a first centrifugal separation mechanism that separates the components of the cell suspension by centrifugation under the first condition, and a component of the cell suspension processed by the first centrifugal separation mechanism.
  • FIG. 1 is a diagram illustrating an example of a configuration of a cell culture device according to an embodiment of the disclosed technology.
  • FIG. 6 is a cross-sectional view illustrating an example of a configuration of a division unit according to an embodiment of the disclosed technology.
  • FIG. 4B is a plan view of the mesh.
  • FIG. 4B is an enlarged view of a portion surrounded by a broken line in FIG. 4B according to the embodiment of the disclosed technology.
  • 11 is a flowchart illustrating an example of a flow of a process performed by a control unit according to an embodiment of the disclosed technology.
  • 1 is a diagram illustrating an example of a configuration of a cell culture device according to an embodiment of the disclosed technology.
  • 1 is a diagram illustrating an example of a configuration of a cell culture device according to an embodiment of the disclosed technology.
  • 11 is a flowchart illustrating an example of a flow of a process performed by a control unit according to an embodiment of the disclosed technology.
  • 1 is a diagram illustrating an example of a configuration of a cell culture device according to an embodiment of the disclosed technology.
  • 1 is a diagram illustrating an example of a configuration of a cell culture device according to an embodiment of the disclosed technology.
  • FIG. 2 is a process flowchart illustrating an example of a processing flow in the cell culture method according to the embodiment of the disclosed technology.
  • proliferating stem cells such as iPS cells, mesenchymal germ cells, and ES cells are to be cultured.
  • the cells form a substantially spherical aggregate (sphere) and are cultured in a state of being suspended in the medium.
  • the cell culture method according to the present embodiment includes a division step A1, a concentration separation step A2, a mixing step A3, a measurement step A4, a replenishment step A5, and a collection step A6.
  • dividing step A1 a dividing process is performed to divide the cell aggregates contained in the cell suspension transferred from the culture vessel into smaller aggregates.
  • the division treatment is performed by passing the cell aggregate through a mesh.
  • the dividing step A1 is performed, for example, when the average size of the cell aggregate contained in the culture container becomes larger than a predetermined size.
  • stem cells such as iPS cells
  • the size of cell aggregates generated by suspension culture of cells becomes excessive, the cell aggregates adhere and fuse with each other, and the cells start to differentiate, or the center of the cell aggregates Some cells may become necrotic. The above-mentioned occurrence is suppressed by dividing the cell aggregate that has grown to a predetermined size in the dividing step A1.
  • the concentration separation step A2 the components of the cell suspension containing the cell aggregates divided in the division step A1 are separated, and the concentration of cells in the cell suspension is concentrated. That is, in the concentration separation step A2, debris such as undersized cell aggregates, single cells that do not form cell aggregates, and dead cells generated in the division step A1 are separated from cell aggregates divided into appropriate sizes. Is done.
  • the concentration separation step A2 may include a filtration treatment for filtering the cell suspension containing the cell aggregates divided in the division step A1, and the filtration treatment includes, for example, a membrane separation treatment using a filtration membrane. You may go out.
  • a membrane separation method it is preferable to apply a tangential flow method in which damage to cells is smaller than a dead end method.
  • the concentration separation step A2 may include a centrifugal separation process of separating the components of the cell suspension containing the cell aggregates divided in the division step A1 by centrifugation.
  • centrifugation separation debris such as undersized cell aggregates, single cells that do not form cell aggregates, dead cells, etc. generated in the division step A1, and cell aggregates divided into appropriate sizes are separated. .
  • a mixing process of mixing the cell suspension containing the cell aggregates of an appropriate size separated in the concentration separation step A2 with a fresh medium is performed.
  • a mixed liquid obtained by mixing the cell suspension that has passed through the dividing step A1 and the concentration separation step A2 with a fresh medium is obtained. Since the cell concentration of the cell suspension is excessively increased by passing through the concentration separation step A2, the state where the cell concentration is excessively high is relaxed by mixing the cell suspension and the medium that have passed through the concentration separation step A2. Is done.
  • the cell concentration in the mixed solution obtained in the mixing step A3 is measured.
  • the cell concentration can be derived, for example, as a ratio of the volume of cells (cell aggregates) to the unit volume of the mixture.
  • the derivation of the cell concentration may be derived by analyzing an image obtained by imaging the mixed solution. In this case, the cell concentration may be derived from the ratio between the integrated value of the individual areas of the cell aggregates included in the image and the area of the imaging visual field.
  • the medium is replenished to the above-mentioned mixture according to the cell concentration measured in the measurement step A4. That is, when the amount of the medium mixed in the mixing step A3 is insufficient and the cell concentration of the mixture measured in the measurement step A4 is higher than a predetermined concentration, a fresh medium is added to the mixture. . On the other hand, when the amount of the medium mixed in the mixing step A3 is appropriate and the cell concentration of the mixed liquid measured in the measuring step A4 is within an appropriate range, the medium is not supplemented to the mixed liquid. In this case, the replenishment step A5 is skipped.
  • the cell suspension (mixture) that has passed through the measurement step A4 or the cell suspension (mixture) that has passed through the replenishment step A5 is recovered in the original culture vessel or another recovery vessel. .
  • FIG. 3 is a diagram illustrating an example of a configuration of a cell culture device 1 according to an embodiment of the disclosed technology for realizing the above-described cell culture method.
  • the cell culture device 1 includes a culture container 10, a collection container 11, a medium storage container 40, a filtration unit 30, which is an example of a concentration separation unit, a waste liquid container 31, a division unit 20, monitor units 60A and 60B, a control unit 50, and a pump P1. To P4 and valves V1 to V6.
  • the culture container 10, the collection container 11, the medium storage container 40, the filtration unit 30, the waste liquid container 31, the division unit 20, and the monitor units 60A and 60B are connected to the flow path 70, respectively.
  • the culture container 10 contains a plurality of cells to be cultured and a cell suspension containing a medium.
  • a plurality of cells are cultured in a state where they form spherical cell aggregates (spheres) and are suspended in the medium.
  • the culture vessel 10 has, for example, a volume that can accommodate 1 ⁇ 10 9 or more cells.
  • the form of the culture container 10 is not particularly limited, and for example, a glass container or a metal container can be used as the culture container 10.
  • the culture vessel 10 may have, for example, a form of a bag including a film having gas permeability.
  • the dividing unit 20 performs the dividing step A1. That is, the dividing unit 20 performs a dividing process of dividing the cell aggregates contained in the cell suspension transferred from the culture container 10 into smaller aggregates.
  • FIG. 4A is a cross-sectional view illustrating an example of the configuration of the dividing unit 20.
  • the dividing unit 20 includes a case 201 having an inlet 202 and an outlet 203, and a mesh 210 provided inside the case 201 between the inlet 202 and the outlet 203.
  • 4B is a plan view of the mesh 210
  • FIG. 4C is an enlarged view of a portion Y surrounded by a broken line in FIG. 4B.
  • the mesh 210 has a plurality of openings (mesh) 211 formed by, for example, plain weaving a plurality of fibrous members 212.
  • the weaving method of the fibrous member 212 is not limited to plain weaving.
  • the material of the fibrous member 212 is not particularly limited, but is preferably made of a material having high corrosion resistance. For example, nylon or stainless steel can be suitably used.
  • the mesh 210 is installed in the case 201 such that the main surface having the plurality of openings 211 extends in a direction intersecting with the flow direction FL of the cell suspension.
  • the pore diameter L of the mesh 210 included in the division unit 20 is, for example, smaller than the average diameter of the cell aggregate before the division processing, and is determined according to the target size of the cell aggregate after the division processing.
  • As the average diameter of the cell aggregate it is possible to apply the arithmetic average of the diameter of the spherical shape when each of the cell aggregates is approximated to a spherical shape.
  • the filtration unit 30, which is an example of the concentration separation unit, performs the concentration separation step A2. That is, the filtration unit 30 separates the components of the cell suspension and concentrates the concentration of the cells in the cell suspension. More specifically, the filtration unit 30 includes debris such as undersized cell aggregates, single cells that do not form cell aggregates, dead cells, and the like generated by the division processing in the division unit 20, and cell aggregates of appropriate size. And a filtration treatment for separating The filtration process in the filtration unit 30 may be performed by, for example, membrane separation using a filtration membrane. In this case, the membrane separation method is preferably a tangential flow method in which damage to cells is smaller than the dead end method. Debris such as undersized cell aggregates, single cells that do not form cell aggregates, and dead cells separated in the filtration unit 30 are stored in the waste liquid container 31.
  • the culture medium container 40 stores a fresh culture medium.
  • the mixing step A3 is performed by mixing the cell suspension from which debris has been removed in the filtration unit 30 with the medium contained in the medium containing container 40.
  • the replenishment step A5 further includes adding the medium contained in the medium container 40 to a mixed solution obtained by mixing the cell suspension from which debris has been removed in the filtration unit 30 and the medium contained in the medium container 40. It is done by adding.
  • the monitor section 60A is provided in a section X1 between the culture vessel 10 and the division section 20 in the flow path 70, and the monitor section 60B is provided in a section X2 between the filtration section 30 and the collection vessel 11 in the flow path 70. It is provided in.
  • the monitoring units 60A and 60B monitor the cell suspension passing through the sections X1 and X2, respectively.
  • Each of the monitor units 60A and 60B includes a flow cell 61 and an imaging device 62.
  • the entire flow cell 61 is made of a light-transmitting material such as glass or plastic.
  • the flow cell 61 has a first distribution port 61a and a second distribution port 61b communicating with the first distribution port 61a.
  • the imaging device 62 has an imaging field of view set in a region between the first distribution port 61a and the second distribution port 61b of the flow cell 61, and includes cells included in a cell suspension flowing inside the flow cell 61 ( (Cell aggregate) is continuously imaged through the flow cell 61.
  • the plurality of images captured by the imaging device 62 are transmitted to the control unit 50.
  • the measurement step A4 is realized using the monitor unit 60B.
  • the collection container 11 contains a mixed liquid obtained by mixing the cell suspension from which debris has been removed in the filtration unit 30 and the fresh medium stored in the medium storage container 40.
  • the form of the collection container 11 is not particularly limited.
  • a glass container or a metal container can be used as the collection container 11.
  • the collection container 11 may have, for example, a form of a bag including a film having gas permeability.
  • the flow path 70 transfers the cell suspension from the culture container 10 to the dividing unit 20, transfers the cell suspension from the dividing unit 20 to the filtering unit 30, and transfers the cell suspension from the filtering unit 30 to the collecting container 11 and the waste liquid container 31. It is configured to allow transfer of the cell suspension.
  • the flow path 70 allows the medium extracted from the medium storage container 40 to join the cell suspension from which debris has been removed in the filtration unit 30 and the cell suspension that has passed through the monitor unit 60B. It is configured.
  • the flow path 70 is configured such that the cell suspension that has passed through the monitor unit 60 ⁇ / b> A reaches the filtration unit 30 via the division unit 20 and the filtration unit 30 without passing through the division unit 20. It is configured to form a second path.
  • the pumps P1 to P4 and the valves V1 to V6 are appropriately disposed at various points in the flow path 70.
  • the pumps P1 to P4 are driven according to a control signal supplied from the control unit 50, and the valves V1 to V6 are opened and closed according to the control signal supplied from the control unit 50.
  • the configuration of the flow path 70 and the arrangement of the pumps P1 to P4 and the valves V1 to V6 are not limited to those illustrated in FIG. 3, but may be configured so that the cell culture method according to the present embodiment can be performed. I just need.
  • the controller 50 controls the transfer of the cell suspension through the flow path 70 by controlling the driving of the pumps P1 to P4 and controlling the opening and closing of the valves V1 to V3 using the control signal.
  • FIG. 5 is a flowchart illustrating an example of a flow of processing performed in the control unit 50 when performing cell culture in the cell culture device 1.
  • step S1 the control unit 50 drives the pump P1 to transfer the cell suspension contained in the culture container 10 to the monitor unit 60A.
  • the monitoring unit 60A monitors the cell suspension passing through the section X1 of the channel 70. That is, the imaging device 62 continuously images cells (cell aggregates) contained in the cell suspension passing through the flow cell 61.
  • the imaging device 62 of the monitor unit 60A captures, for example, all the cells (cell aggregates) included in the cell suspension passing through the section X1 of the flow channel 70 at intervals at which the imaging can be performed. Note that the imaging device 62 may image a part of cells (cell aggregates) included in the cell suspension passing through the section X1 of the flow channel 70.
  • step S2 the control unit 50 acquires an image of the cell captured by the imaging device 62 of the monitor unit 60A.
  • step S3 the control unit 50 determines whether or not the division processing is necessary based on the image acquired in step S2.
  • the control unit 50 determines whether or not the division processing is necessary, for example, as follows.
  • the control unit 50 derives, for example, the average size of the cell aggregates included in the image acquired from the monitor unit 60A in step S2, and when the derived average size is larger than a predetermined size, a division process is necessary. It is determined that there is, and if the derived average size is smaller than the predetermined size, it is determined that the dividing process is unnecessary.
  • As the average size of the cell aggregate it is possible to apply the arithmetic mean of the diameter of the spherical shape when each of the cell aggregates is approximated to a spherical shape.
  • step S4 the control unit 50 controls the valves V1 and V2 to be open and the valves V3 and V4 to be closed so that the cell suspension is transmitted to the division unit 20.
  • the cell aggregates contained in the cell suspension transferred to the dividing unit 20 pass through the mesh 210 of the dividing unit 20 (see FIGS. 4A, 4B, and 4C) to form smaller cell aggregates.
  • the cell suspension containing the divided cell aggregates is transferred to the filtration unit 30 in step S5.
  • step S5 the valves V3 and V4 are opened and the valves V1 and V2 are closed, thereby dividing the cell suspension. It is transferred to the filtration unit 30 without passing through the unit 20.
  • the filtration unit 30 performs a filtration process on the transferred cell suspension. That is, the filtration unit 30 separates debris such as undersized cell aggregates, single cells that do not form cell aggregates, dead cells, etc., contained in the transferred cell suspension, and cell aggregates of appropriate size. To separate. When the cell suspension is transferred to the filtration unit 30 via the division unit 20, debris such as undersized cell aggregates, single cells, and dead cells generated by the division processing in the division unit 20, It is removed in the filtration unit 30.
  • step S ⁇ b> 6 the control unit 50 drives the pump P ⁇ b> 2 to transfer the debris such as the undersized cell aggregates, single cells, and dead cells separated by the filtration unit 30 to the waste liquid container 31.
  • step S7 the control unit 50 drives the pump P3 to transfer the cell suspension from which debris has been removed to the monitor unit 60B.
  • step S8 which is performed in parallel with step S7, the control unit 50 drives the pump P4 to control the valve V5 to open and the valve V6 to close, so that the control unit 50 is stored in the culture medium storage container 40.
  • a fresh culture medium is extracted from the culture medium container 40, and the culture medium and the cell suspension from which the debris flows from the filtration unit 30 to the monitor unit 60B are removed on the flow path 70 and are mixed.
  • the monitor unit 60 ⁇ / b> B monitors a liquid mixture that passes through the section X ⁇ b> 2 of the channel 70 and is a mixture of the cell suspension from which debris has been removed and the fresh medium extracted from the medium container 40. That is, the imaging device 62 of the monitor unit 60B continuously captures images of cells (cell aggregates) contained in the mixed solution passing through the flow cell 61. The imaging device 62 images all cells (cell aggregates) included in the cell suspension passing through the section X2 of the flow path 70 at intervals at which imaging can be performed, for example. Note that the imaging device 62 may image a part of cells (cell aggregates) included in the cell suspension passing through the section X2 of the flow path 70.
  • step S9 the control unit 50 acquires an image of the cell captured by the imaging device 62 of the monitor unit 60B.
  • step S10 the control unit 50 determines whether or not it is necessary to replenish the mixture with the medium based on the image obtained by imaging the cells contained in the mixture obtained in step S9.
  • the control unit 50 determines whether or not the medium needs to be replenished, for example, as follows. For example, the control unit 50 derives the cell concentration in the mixed solution from the image acquired from the monitor unit 60B in step S9, and when the derived cell concentration is higher than a predetermined value, the medium needs to be replenished. If the derived cell concentration is smaller than the predetermined value, it is determined that the medium need not be replenished.
  • the cell concentration in the mixed solution can be derived, for example, from the ratio of the integrated value of the individual size of the cell aggregate included in the image acquired in step S9 and the area of the imaging visual field of the imaging device 62.
  • Control unit 50 shifts the processing to step S11 when judging that medium replenishment is necessary, and ends processing when judging that medium replenishment is not necessary.
  • control unit 50 determines that supplementation of the culture medium is necessary, in step S11, based on the cell concentration of the mixture liquid derived in step S10, the control unit 50 adjusts the cell concentration of the mixture liquid to a predetermined concentration.
  • the replenishment amount is derived.
  • step S12 the control unit 50 drives the pump P4 and controls the valve V6 to the open state, so that the fresh medium stored in the medium storage container 40 is dispensed in an amount corresponding to the replenishment amount derived in step S11. Then, the extracted medium and the mixed solution that has passed through the monitor unit 60B are joined on the flow path 70. Thus, the mixture is supplemented with fresh medium, and the cell concentration in the mixture is adjusted to an appropriate concentration. At this time, the valve V5 is kept open.
  • Concentration adjustment processing for adjusting the cell concentration by replenishing the mixture is performed in parallel. Adjustment of the cell concentration can be realized, for example, by controlling the degree of opening of the valves V5 and V6.
  • the mixed solution adjusted to an appropriate concentration is transferred to the collection container 11.
  • the cells transferred to the collection container 11 are continuously cultured in the collection container 11. Note that, in the present embodiment, the case where the feeding of the culture medium passing through the valves V5 and V6 is performed using the common culture medium storage container 40 and the common pump P4 is described. It is also possible to use a pump.
  • the concentration separation step A2 is performed after the division step A1.
  • Debris such as undersized cell aggregates, single cells, dead cells and the like generated in the dividing step A1 are removed in the concentration separation step A2. Therefore, mixing of unnecessary components (debris) into the cell suspension collected in the collection container 11 is suppressed. Thereby, the growth rate and the survival rate of the cells cultured in the collection container 11 can be increased.
  • the cell culture method and the cell culture device according to the embodiment of the disclosed technology, it is ensured that the cell suspension having an appropriate cell concentration is collected in the collection container 11 by the measurement step A4 and the replenishment step A5. Is done. Further, even in a situation where it is difficult to predict the cell concentration in the cell suspension after the division process, the measurement step A4 and the replenishment step A5 are performed immediately before the collection step A6, so that the cells collected in the collection container 11 are obtained. It becomes possible to control the cell concentration in the suspension within an appropriate range.
  • the case where the replenishing medium and the mixed solution before the concentration adjustment are combined on the flow path 70 and then transferred to the collection container 11 has been described as an example. May be transferred to the collection container 11 after transferring to the collection container 11.
  • FIG. 3 illustrates an example of a configuration in which the cell suspension that has passed through each step is collected in the collection container 11. As illustrated in FIG. 6, the cell culture device 1 has passed through each step.
  • the cell suspension may be configured to be collected in the culture container 10.
  • the filtration unit 30 is illustrated as an example of the concentration separation unit that performs the concentration separation step A1, but the present invention is not limited to this mode.
  • the concentration separation unit that performs the concentration separation step A1 may include, for example, a centrifugal separation mechanism that separates components of the cell suspension by centrifugation.
  • the centrifugation mechanism separates the undersized cell aggregates generated by the division process in the division unit 20, single cells that do not form the cell aggregates, debris such as dead cells, and the appropriate size cell aggregates by centrifugation. To separate.
  • FIG. 7 is a diagram illustrating an example of a configuration of a cell culture device 1A according to the second embodiment of the disclosed technology.
  • the cell culture device 1A includes the diluent storage container 41, two filtration units 30A and 30B, which are examples of a concentration separation unit, three monitoring units 60A, 60B and 60C, and two collection containers 11A and 11B is described above. This is different from the cell culture device 1 according to the first embodiment described above.
  • the diluent storage container 41 is a container for storing the diluent.
  • the basic components of the diluent may be the same as the basic components of the medium used for the cell culture performed in the culture vessel 10.
  • the culture medium used for cell culture performed in the culture vessel 10 may be supplemented with a supplement for supplying cells with nutrients, and an additive such as a thickener for increasing the viscosity of the culture medium. It is preferable not to include such additives. That is, the viscosity of the diluent is preferably lower than the viscosity of the culture medium contained in the culture vessel 10.
  • the filtration unit 30B is provided downstream of the filtration unit 30A in the flow direction of the cell suspension.
  • the filtration process in the filtration units 30A and 30B is performed by a membrane separation process using a filtration membrane.
  • the method of membrane separation in the filtration units 30A and 30B is preferably a tangential flow method.
  • the pore size of the filtration membrane of the filtration unit 30A is smaller than the pore size of the filtration membrane of the filtration unit 30B.
  • the filtration unit 30A separates debris, such as single cells and dead cells, generated by the division processing in the division unit 20, and cell aggregates larger in size than the debris.
  • the debris that has passed through the filtration membrane of the filtration unit 30A is stored in the waste liquid container 31.
  • the cell aggregate blocked by the filtration membrane of the filtration unit 30A is transferred to the filtration unit 30B.
  • the filtration unit 30B separates the cell aggregate transferred from the filtration unit 30A into a cell aggregate having a relatively large size and a cell aggregate having a relatively small size.
  • the cell aggregate having a relatively small size that has passed through the filtration membrane of the filtration unit 30B is transferred to the collection container 11B via the monitor unit 60C.
  • the relatively large cell aggregate blocked by the filtration membrane of the filtration unit 30B is transferred to the collection container 11A via the monitor unit 60B.
  • the membrane separation process performed in the filtration unit 30A is an example of the first membrane separation process in the disclosed technology
  • the membrane separation process performed in the filtration unit 30B is the second membrane separation process in the disclosed technology. It is an example of processing.
  • the filtration membrane of the filtration unit 30A is an example of a first filtration membrane in the disclosed technology
  • the filtration membrane of the filtration unit 30B is an example of a second filtration membrane in the disclosed technology.
  • the monitor unit 60 ⁇ / b> A is provided in a section X ⁇ b> 1 of the flow channel 70 between the culture vessel 10 and the division unit 20.
  • the monitor unit 60B is provided in a section X2 of the flow path 70 between the filtration unit 30B and the collection container 11A.
  • the monitor unit 60C is provided in a section X3 of the flow path 70 between the filtration unit 30B and the collection container 11B.
  • the monitoring units 60A, 60B and 60C monitor the cell suspension passing through the sections X1, X2 and X3, respectively.
  • Each of the monitor units 60A, 60B, and 60C includes a flow cell 61 and an imaging device 62.
  • the channel 70 is configured such that the diluent stored in the diluent storage container 41 can join the cell suspension that has passed through the monitor unit 60A.
  • the flow path 70 transfers the cell suspension from the dividing section 20 to the filtering section 30A, transfers the cell suspension from the filtering section 30A to the filtering section 30B and the waste liquid container 31, and transfers the cell suspension from the filtering section 30B to the collecting vessel 11A. And 11B to allow the transfer of the cell suspension.
  • the flow path 70 is configured so that the medium contained in the medium container 40 can be combined with the components separated by the filtration unit 30B.
  • FIG. 8 is a flowchart illustrating an example of a flow of processing performed by the control unit 50 when performing cell culture in the cell culture device 1A.
  • step S21 the control unit 50 drives the pump P1 to transfer the cell suspension contained in the culture container 10 to the monitor unit 60A.
  • the monitoring unit 60A monitors the cell suspension passing through the section X1 of the channel 70. That is, the imaging device 62 continuously images cells (cell aggregates) contained in the cell suspension passing through the flow cell 61.
  • step S22 the control unit 50 acquires an image of the cell captured by the imaging device 62 of the monitor unit 60A.
  • step S23 the control unit 50 determines whether the division processing is necessary based on the image acquired in step S22.
  • the process proceeds to step S24, and when it determines that the division process is not necessary, the process proceeds to step S25.
  • step S24 the valves V1 and V2 are opened and the valves V3 and V4 are closed so that the cell suspension is transferred to the dividing unit 20. Transfer.
  • step S24a performed in parallel with step S24, the control unit 50 drives the pump P5 to extract the diluent stored in the diluent storage container 41 from the diluent storage container 41, and The cell suspension flowing toward the dividing section 20 and the cell suspension are combined on the flow path 70, and they are mixed. Since the cell suspension before the splitting treatment contains many relatively large cell aggregates, it is assumed that the cell suspension before the splitting treatment has an excessively high cell concentration.
  • the cell concentration in the cell suspension before the division treatment can be adjusted to an appropriate concentration.
  • the viscosity of the cell suspension before the division processing can be reduced, thereby causing clogging of the mesh of the division unit 20. Can be suppressed.
  • the cell aggregates contained in the cell suspension transferred to the dividing unit 20 pass through the mesh 210 of the dividing unit 20 (see FIGS. 4A, 4B, and 4C) to form smaller cell aggregates. Divided.
  • the cell suspension containing the divided cell aggregates is transferred to the filtration unit 30A in step S25.
  • the valves V3 and V4 are opened and the valves V1 and V2 are closed so that the cell suspension is divided. It is transferred to the filtration unit 30A without passing through the filter unit 20.
  • the filtration unit 30A separates debris, such as single cells and dead cells, contained in the transferred cell suspension, and cell aggregates larger in size than the debris.
  • step S26 the control unit 50 drives the pump P2 to transfer the debris that has passed through the filtration membrane of the filtration unit 30A to the waste liquid container 31.
  • the cell suspension containing the cell aggregate blocked by the filtration membrane of the filtration unit 30A is transferred to the filtration unit 30B.
  • the filtration unit 30B separates the cell aggregate transferred from the filtration unit 30A into a relatively large cell aggregate and a relatively small cell aggregate.
  • step S27 the control unit 50 drives the pumps P3a and P3b to transfer the cell suspension containing the relatively large cell aggregate blocked by the filtration membrane of the filtration unit 30B to the monitoring unit 60B. Then, the cell suspension containing the cell aggregate having a relatively small size that has passed through the filtration membrane of the filtration unit 30B is transferred to the monitor unit 60C.
  • step S28 which is performed in parallel with step S27, the control unit 50 drives the pumps P4a and P4b to control the valves V5 and V7 to open and the valves V6 and V8 to close, so that the culture medium container
  • the fresh medium contained in 40 is extracted from the medium containing container 40, and this medium and the cell suspension flowing from the filtration unit 30B to each of the monitor units 60B and 60C are merged on the channel 70, Mix these.
  • the first mixed solution obtained by mixing the cell suspension containing the cell aggregate having a relatively large size and the medium transferred from the medium storage container 40 is mixed with the cell mixture having a relatively small size.
  • a second liquid mixture in which the cell suspension containing the aggregate and the medium transferred from the medium storage container 40 are mixed is generated.
  • the monitor unit 60B monitors the first mixed liquid passing through the section X2 of the flow path 70, and the monitor unit 60C monitors the second mixed liquid passing through the section X3 of the flow path 70.
  • step S29 the control unit 50 acquires an image of a cell captured by the imaging device 62 of the monitor units 60B and 60C.
  • step S30 the control unit 50 determines whether the replenishment of the culture medium is necessary for each of the first mixed liquid and the second mixed liquid based on the image acquired in step S29.
  • the control unit 50 shifts the processing to step S31 and determines that the replenishment of the medium is not necessary. If so, the process ends.
  • step S31 the controller 50 sets the cell concentration of the mixture to a predetermined concentration. Deriving the replenishment amount of the medium.
  • step S32 the control unit 50 drives the pump P4a and controls the valve V6 to be in the open state, whereby the fresh culture medium stored in the culture medium storage container 40 is controlled. Is extracted by an amount corresponding to the replenishment amount derived in step S31, and the extracted medium and the first mixed solution that has passed through the monitor unit 60B are combined on the flow path 70.
  • step S32 the control unit 50 drives the pump P4b and controls the valve V8 to the open state, so that the fresh culture medium stored in the culture medium storage container 40 is opened.
  • the adjustment of the cell concentration can be realized, for example, by controlling the degree of opening of the valves V5 to V8.
  • the first mixed solution adjusted to the appropriate concentration is transferred to the collection container 11A, and the second mixed solution adjusted to the appropriate concentration is transferred to the collection container 11B.
  • the case where the supply of the culture medium passing through the valves V5 and V6 is performed using the common culture medium storage container 40 and the common pump P4a is illustrated. It is also possible to use a pump.
  • the case where the feeding of the culture medium passing through the valves V7 and V8 is performed using the common culture medium storage container 40 and the common pump P4b is illustrated, but it is also possible to use separate culture medium storage containers and pumps. It is possible.
  • the cell culture device 1A according to the second embodiment of the disclosed technology similarly to the cell culture device 1 according to the first embodiment, unnecessary components in the cell suspension collected in the collection containers 11A and 11B. (Debris) is suppressed. Thereby, the growth rate and the survival rate of the cells cultured in the collection containers 11A and 11B, respectively, can be increased.
  • the two-stage filtration is performed by the two filtration units 30A and 30B having the filtration membranes having different pore sizes from each other, thereby improving the separation property in the filtration. be able to.
  • the components that have passed through the filtration membrane of the subsequent filtration unit 30B may contain many cells that can be expected to proliferate in the subsequent culture, if only the components blocked by the filtration membrane of the filtration unit 30B are used, In addition, by recovering the components that have passed through the filtration membrane of the filtration unit 30B, cell loss can be reduced.
  • Separate collection vessels 11A and 11B separate the cell aggregate having a relatively large size blocked by the filtration membrane of the filtration unit 30B and the cell aggregate having a relatively small size transmitted through the filtration membrane of the filtration unit 30B. By collecting the cells in a suitable size, the cells can be cultured in an environment suitable for the cell aggregate of each size, and the survival rate and proliferation rate of the cells can be improved.
  • the replenishment medium and the first and second mixed liquids are merged on the flow path 70, they are transferred to the collection containers 11A and 11B, respectively.
  • the supplemental medium may be transferred to the collection containers 11A and 11B.
  • FIG. 7 illustrates a configuration in which components blocked by the filtration membrane of the filtration unit 30B are collected in the collection container 11A.
  • the cell culture device 1A includes a filtration unit.
  • the components blocked by the 30B filtration membrane may be collected in the culture vessel 10.
  • the cell culture device 1A may include a diluent container 42 connected to a flow path disposed between the filtration unit 30A and the filtration unit 30B.
  • the cell suspension blocked by the filtration membrane of the filtration unit 30A is mixed with the diluent stored in the diluent storage container 42 and transferred to the filtration unit 30B. It is assumed that the cell concentration of the cell suspension blocked by the filtration membrane of the filtration unit 30A is excessively high.
  • the cell concentration in the cell suspension transferred to the filtration unit 30B can be adjusted appropriately. High concentration.
  • the concentration and separation unit may include two centrifugal separation mechanisms. That is, the concentration / separation unit separates the components of the cell suspension by a first centrifugation mechanism that separates the components of the cell suspension by centrifugation under the first condition, and the components of the cell suspension processed by the first centrifugation mechanism. And a second centrifugal separation mechanism for separating by centrifugation under a second condition different from the first condition.
  • the conditions for centrifugation include, for example, the centrifugal force generated by the centrifugal separation mechanism and the processing time of centrifugation.

Abstract

A cell culture method which comprises: a division step for dividing a cell aggregate; a concentration/separation step for separating components of a cell suspension, which contains the cell aggregate divided in the division step, to thereby concentrate the concentration of the cells in the cell suspension; a mixing step for mixing the cell suspension having been treated in the concentration/separation step with a medium to give a liquid mixture; a measurement step for measuring the concentration of the cells in the liquid mixture; and a recovery step for, after the measurement step, recovering the liquid mixture.

Description

細胞培養方法及び細胞培養装置Cell culture method and cell culture device
　本願は２０１８年８月２７日出願の日本出願第２０１８－１５８５９８号の優先権を主張すると共に、その全文を参照により本明細書に援用する。
　開示の技術は、細胞培養方法及び細胞培養装置に関する。 This application claims the priority of Japanese Patent Application No. 2018-158598 filed on Aug. 27, 2018, which is incorporated herein by reference in its entirety.
The disclosed technology relates to a cell culture method and a cell culture device.
細胞培養装置に関し、例えば、特開２０１６－１３１５３８号公報には、培養容器、貯留容器、細胞凝集体を分割する分割処理を行う分割処理部と、流路内に培地を供給する培地供給部と、を含む細胞培養装置が記載されている。 Regarding the cell culture apparatus, for example, Japanese Patent Application Laid-Open No. 2016-131538 discloses a culture vessel, a storage vessel, a division processing section for performing division processing for dividing a cell aggregate, and a medium supply section for supplying a culture medium in a flow path. And a cell culture device comprising:
　ｉＰＳ細胞（induced pluripotent stem cells）等の幹細胞の培養においては、細胞を培養することによって生じる細胞凝集体（スフェア）のサイズが過大となると、細胞凝集体同士が接着融合し、細胞が分化を開始したり、細胞凝集体の中心部の細胞が壊死したりする場合がある。従って、細胞凝集体のサイズが過大となることを防止するために、細胞の培養期間中の適切な時期に、細胞凝集体を、より小さいサイズの複数の細胞凝集体に分割（解砕）する分割処理が行われている。細胞凝集体を分割する手法として、細胞凝集体を含む細胞懸濁液を、複数の開口部（網目）を有するメッシュに通過させることで細胞凝集体を機械的に分割する手法が提案されている。メッシュを用いた分割処理においては、メッシュ衝突時に細胞凝集体がダメージを受け、死細胞が不可避的に発生する。 In culturing stem cells such as iPS cells (induced pluripotent stem cells), if the size of cell aggregates (spheres) generated by culturing the cells becomes excessive, the cell aggregates adhere and fuse to each other, and the cells start to differentiate. Or cells in the center of the cell aggregate may be necrotic. Therefore, in order to prevent the size of the cell aggregate from becoming excessively large, the cell aggregate is divided (disrupted) into a plurality of smaller-sized cell aggregates at an appropriate time during the cell culture period. Division processing has been performed. As a method of dividing the cell aggregate, a method of mechanically dividing the cell aggregate by passing a cell suspension containing the cell aggregate through a mesh having a plurality of openings (mesh) has been proposed. . In a division process using a mesh, cell aggregates are damaged at the time of mesh collision, and dead cells are inevitably generated.
　また、細胞培養においては、細胞から分泌される代謝物や死細胞などによって培地が変質する。そのため、培養期間中における適切な時期に、培養容器内における使用済みの培地を新鮮な培地に交換する培地交換処理が必要となる。培地交換処理は、使用済みの培地と細胞とを分離するための濃縮分離処理と、分離された細胞と新鮮な培地とを混合する混合処理とを含む。ここで、上記の分割処理及び培地交換処理を含む一連の処理を自動化する細胞処理装置としては、培養容器に収容されている細胞懸濁液に対して、培地交換手段によって培地交換処理を行った後に、必要に応じてメッシュを含む分割手段によって分割処理を実施するように構成されたものが想定される。 培 地 In cell culture, the culture medium is altered by metabolites secreted from cells or dead cells. Therefore, at an appropriate time during the culture period, a medium exchange process for exchanging the used medium in the culture container with a fresh medium is required. The medium exchange process includes a concentration separation process for separating the used medium from the cells, and a mixing process of mixing the separated cells with a fresh medium. Here, as a cell processing apparatus for automating a series of processing including the above-mentioned division processing and culture medium exchange processing, culture medium exchange processing was performed on a cell suspension contained in a culture vessel by culture medium exchange means. It is assumed that the configuration is such that the division processing is performed later by division means including a mesh as necessary.
　図１は、上記の構成を有する細胞培養装置を用いた細胞培養の各段階における細胞凝集体のサイズの頻度分布の一例を示すグラフである。図１に示すグラフにおいて、実線は、培地交換処理前の状態を示し、一点鎖線は培地交換処理後の状態を示し、点線は分割処理後の状態を示す。培地交換処理を実施することで、過小サイズの細胞凝集体、細胞凝集体を形成しない単一細胞、死細胞等のデブリスが除去される。しかしながら、このようなデブリスは、メッシュを含む分割手段による分割処理によっても発生する。従って、培地交換処理を実施したとしても、その後に分割処理を実施した場合には、分割処理後に回収される細胞懸濁液には、多量のデブリスが混入する。過小サイズの細胞凝集体、細胞凝集体を形成しない単一細胞、死細胞等のデブリスは、その後の培養で増殖を見込めない上、細胞の増殖性及び生存率に悪影響を与えるおそれがある。 FIG. 1 is a graph showing an example of the frequency distribution of the size of cell aggregates at each stage of cell culture using the cell culture device having the above configuration. In the graph shown in FIG. 1, the solid line indicates the state before the medium exchange processing, the dashed line indicates the state after the medium exchange processing, and the dotted line indicates the state after the division processing. By performing the medium exchange treatment, debris such as undersized cell aggregates, single cells that do not form cell aggregates, and dead cells are removed. However, such debris is also generated by division processing by division means including a mesh. Therefore, even if the culture medium exchange processing is performed, when the division processing is performed thereafter, a large amount of debris is mixed in the cell suspension recovered after the division processing. Debris, such as undersized cell aggregates, single cells that do not form cell aggregates, and dead cells, may not be expected to grow in subsequent cultures and may adversely affect cell growth and viability.
　また、上記の構成の細胞培養装置を、例えば１×１０９個程度あるいはそれよりも多くの細胞を培養対象とする大量培養に適用した場合、分割処理によって発生するデブリスの量が増加する。また、大量培養を実施した場合には、分割手段を構成するメッシュが目詰まりを起こし、細胞がメッシュ上に滞留し、メッシュの状態が処理量の増加に伴って刻々と変化するおそれがある。このようにメッシュの状態が刻々と変化する状況においては、分割処理後の細胞懸濁液における細胞濃度の予測が困難となり、その結果、分割処理後に回収される細胞懸濁液の細胞濃度のコントロールが困難となる。細胞懸濁液における細胞濃度は、細胞の増殖性及び生存率に関わる重要なパラメータであり、細胞懸濁液の細胞濃度を適正範囲にコントロールすることは重要である。 In addition, when the cell culture apparatus having the above configuration is applied to a large-scale culture in which, for example, about 1 × 10 9 or more cells are to be cultured, the amount of debris generated by the division treatment increases. In addition, when mass culture is performed, the mesh constituting the dividing means may be clogged, cells may stay on the mesh, and the state of the mesh may change every moment as the throughput increases. In such a situation where the state of the mesh changes every moment, it becomes difficult to predict the cell concentration in the cell suspension after the division, and as a result, the control of the cell concentration of the cell suspension recovered after the division is performed. Becomes difficult. The cell concentration in the cell suspension is an important parameter related to cell growth and viability, and it is important to control the cell concentration in the cell suspension in an appropriate range.
　このように、培地交換処理後に分割処理を行い、分割処理後の細胞懸濁液をそのまま回収する細胞培養方法及び細胞培養装置によれば、回収される細胞懸濁液に培養に不要な成分が多く混入するおそれがある。また、回収される細胞懸濁液における細胞濃度を適正範囲にコントロールすることが困難となる。 As described above, according to the cell culture method and the cell culture apparatus in which the division treatment is performed after the medium exchange treatment and the cell suspension after the division treatment is directly collected, components unnecessary for culture are contained in the collected cell suspension. There is a possibility that a large amount will be mixed. In addition, it becomes difficult to control the cell concentration in the recovered cell suspension within an appropriate range.
　開示の技術は、分割処理によって発生する死細胞等の不要成分の、分割処理後に回収される細胞懸濁液への混入を抑制できる細胞培養方法及び細胞培養装置を提供する。 (4) The disclosed technology provides a cell culture method and a cell culture device capable of suppressing mixing of unnecessary components such as dead cells generated by the division processing into a cell suspension collected after the division processing.
　開示の技術に係る細胞培養方法は、細胞凝集体を分割する分割工程と、分割工程において分割された細胞凝集体を含む細胞懸濁液の成分を分離して細胞懸濁液における細胞の濃度を濃縮する濃縮分離工程と、濃縮分離工程を経た細胞懸濁液と培地とを混合した混合液を得る混合工程と、混合液における細胞の濃度を測定する測定工程と、測定工程の後に、混合液を回収する回収工程と、を含む。これにより、分割処理によって発生する死細胞等の不要成分の、分割処理後に回収される細胞懸濁液への混入を抑制することが可能となる。 The cell culture method according to the disclosed technology includes a dividing step of dividing the cell aggregate, and separating the components of the cell suspension containing the cell aggregate divided in the dividing step to increase the concentration of cells in the cell suspension. A concentration separation step of concentrating, a mixing step of obtaining a mixed liquid obtained by mixing the cell suspension and the medium that have undergone the concentration separation step, a measurement step of measuring the concentration of cells in the mixture, and after the measurement step, the mixed liquid And recovering. This makes it possible to prevent unnecessary components such as dead cells generated by the dividing process from being mixed into the cell suspension collected after the dividing process.
　開示の技術に係る細胞培養方法は、測定工程において測定された濃度に基づいて、混合液に培地を補充する補充工程を更に含んでいてもよい。これにより、回収される混合液における細胞の濃度を適正な濃度にすることができる。 細胞 The cell culture method according to the disclosed technology may further include a replenishing step of replenishing the mixed solution with a medium based on the concentration measured in the measuring step. Thereby, the concentration of the cells in the collected liquid mixture can be adjusted to an appropriate concentration.
　濃縮分離工程は、分割工程において分割された細胞凝集体を含む細胞懸濁液を膜分離する膜分離処理を含んでいてもよい。膜分離処理は、分割工程において分割された細胞凝集体を含む細胞懸濁液を第１の濾過膜を用いて膜分離する第１の膜分離処理と、第１の濾過膜によって阻止された成分を、第２の濾過膜を用いて膜分離する第２の膜分離処理とを含んでいてもよい。このように、膜分離処理を２段階で行うことで、膜分離処理における分離性を高めることができる。 (4) The concentration separation step may include a membrane separation treatment for separating a cell suspension containing the cell aggregates divided in the division step. The membrane separation process includes a first membrane separation process of membrane-separating the cell suspension containing the cell aggregates divided in the dividing step using the first filtration membrane, and a component blocked by the first filtration membrane. And a second membrane separation process of performing membrane separation using a second filtration membrane. As described above, by performing the membrane separation process in two stages, the separability in the membrane separation process can be improved.
　第１の濾過膜の孔径は、第２の濾過膜の孔径よりも小さくてもよい。これにより、例えば、死細胞等のデブリスが除去された細胞懸濁液を、第２の濾膜膜を用いた膜分離の対象とすることができる。 孔 The pore size of the first filtration membrane may be smaller than the pore size of the second filtration membrane. Thus, for example, a cell suspension from which debris such as dead cells has been removed can be subjected to membrane separation using the second filter membrane.
　回収において、第２の濾過膜によって阻止された成分と、第２の濾過膜を透過した成分とを別々に回収してもよい。これにより、回収された各成分について、最適な環境で培養を継続することができる。 In the recovery, the component blocked by the second filtration membrane and the component permeated through the second filtration membrane may be separately recovered. Thereby, culturing can be continued in an optimal environment for the collected components.
　第１の濾過膜によって阻止された成分と希釈液とを混合したものを第２の濾過膜を用いて膜分離してもよい。これにより、第２の濾過膜を用いた膜分離の対象となる細胞懸濁液における細胞の濃度を適切な濃度とすることができる。 混合 A mixture of the component blocked by the first filtration membrane and the diluent may be subjected to membrane separation using the second filtration membrane. Thereby, the concentration of cells in the cell suspension to be subjected to membrane separation using the second filtration membrane can be adjusted to an appropriate concentration.
　開示の技術に係る細胞培養方法は、分割工程において分割される前の細胞凝集体を含む細胞懸濁液に希釈液を添加する希釈工程を更に含んでいてもよい。これにより、分割処理の対象となる細胞凝集体を含む細胞懸濁液における細胞の濃度及び粘度を最適化できる。また、分割工程における増粘剤等の培地含有成分による経時のメッシュ目詰まりを緩和させることができる。 (4) The cell culture method according to the disclosed technology may further include a dilution step of adding a diluent to a cell suspension containing the cell aggregate before the division in the division step. Thereby, the concentration and the viscosity of the cells in the cell suspension containing the cell aggregate to be subjected to the division treatment can be optimized. In addition, mesh clogging over time due to a medium-containing component such as a thickener in the dividing step can be reduced.
　濃縮分離工程は、分割工程において分割された細胞凝集体を含む細胞懸濁液の成分を遠心分離により分離する遠心分離処理を含んでいてもよい。遠心分離処理は、分割工程において分割された細胞凝集体を含む細胞懸濁液の成分を、第１の条件による遠心分離により分離する第１の遠心分離処理と、第１の遠心分離処理が施された細胞懸濁液の成分を、第１の条件とは異なる第２の条件による遠心分離により分離する第２の遠心分離処理と、を含んでいてもよい。 (4) The concentration / separation step may include a centrifugation treatment for separating components of the cell suspension containing the cell aggregates divided in the division step by centrifugation. The centrifugation process includes a first centrifugation process of separating the components of the cell suspension containing the cell aggregates divided in the division step by centrifugation under the first condition, and a first centrifugation process. A second centrifugation process of separating the component of the cell suspension thus performed by centrifugation under a second condition different from the first condition.
　開示の技術に係る細胞培養装置は、細胞凝集体を分割する分割部と、細胞懸濁液の成分を分離して細胞懸濁液における細胞の濃度を濃縮する濃縮分離部と、培地を収容するための培地収容容器と、細胞懸濁液をモニタするモニタ部と、分割部、濃縮分離部、培地収容容器及びモニタ部に接続された流路と、流路を介した細胞懸濁液及び培地の移送を制御する制御部と、を含む。制御部は、分割部において分割された細胞凝集体を含む細胞懸濁液を濃縮分離部に移送し、濃縮分離部において濃縮された細胞懸濁液と培地収容容器に収容されている培地とを混合した混合液をモニタ部に移送し、モニタ部によりモニタされた混合液を流路に接続された容器に移送する。これにより、分割処理によって発生する死細胞等の不要成分の、分割処理後に回収される細胞懸濁液への混入を抑制することが可能となる。 The cell culture device according to the disclosed technology includes a dividing unit that divides a cell aggregate, a concentration separation unit that separates components of a cell suspension to concentrate the concentration of cells in the cell suspension, and contains a medium. Medium container for monitoring, a monitor unit for monitoring the cell suspension, a dividing unit, a concentration separation unit, a channel connected to the medium container and the monitor unit, and a cell suspension and a medium via the channel. And a controller for controlling the transfer of the data. The control unit transfers the cell suspension containing the cell aggregates divided in the division unit to the concentration and separation unit, and the cell suspension concentrated in the concentration and separation unit and the medium contained in the medium containing container. The mixed liquid mixture is transferred to the monitor, and the mixed liquid monitored by the monitor is transferred to a container connected to the flow path. This makes it possible to prevent unnecessary components such as dead cells generated by the dividing process from being mixed into the cell suspension collected after the dividing process.
　開示の技術に係る細胞培養装置は、流路に接続された培養容器を更に含んでいてもよい。制御部は、培養容器に収容されている細胞凝集体を含む細胞懸濁液を分割部に移送し、モニタ部によりモニタされた混合液を培養容器に移送してもよい。 細胞 The cell culture device according to the disclosed technology may further include a culture container connected to the flow channel. The control unit may transfer the cell suspension containing the cell aggregate contained in the culture container to the division unit, and may transfer the mixed solution monitored by the monitor unit to the culture container.
　開示の技術に係る細胞培養装置は、流路に接続された培養容器と、流路に接続された回収容器と、を更に含んでいてもよい。制御部は、培養容器に収容されている細胞凝集体を含む細胞懸濁液を分割部に移送し、モニタ部によりモニタされた混合液を回収容器に移送してもよい。 細胞 The cell culture device according to the disclosed technology may further include a culture container connected to the flow channel and a collection container connected to the flow channel. The control unit may transfer the cell suspension containing the cell aggregate contained in the culture container to the division unit, and may transfer the mixed solution monitored by the monitor unit to the collection container.
　制御部は、モニタ部によるモニタの結果に基づいて導出された混合液に含まれる細胞の濃度に基づいて、培地収容容器に収容されている培地を混合液に補充してもよい。これにより、回収される混合液における細胞の濃度を適正な濃度にすることができる。 The control unit may replenish the mixed solution with the medium contained in the medium containing container based on the concentration of cells contained in the mixed solution derived based on the result of monitoring by the monitor unit. Thereby, the concentration of the cells in the collected liquid mixture can be adjusted to an appropriate concentration.
　濃縮分離部は、細胞懸濁液を膜分離する濾過膜を備えた濾過部を含んでいてもよい。濃縮分離部は、細胞懸濁液を膜分離する第１の濾過膜を備えた第１の濾過部と、第１の濾過膜によって阻止された成分を膜分離する第２の濾過膜を備えた第２の濾過部と、を含んでいてもよい。このように、膜分離処理を２段階で行うことで、膜分離処理における分離性を高めることができる。 The concentration and separation unit may include a filtration unit provided with a filtration membrane for membrane-separating the cell suspension. The concentration / separation unit was provided with a first filtration unit provided with a first filtration membrane for separating a cell suspension by membrane, and a second filtration membrane provided for separating components blocked by the first filtration membrane. And a second filtration unit. As described above, by performing the membrane separation process in two stages, the separability in the membrane separation process can be improved.
　第１の濾過膜の孔径は、第２の濾過膜の孔径よりも小さくてもよい。これにより、例えば、死細胞等のデブリスが除去された細胞懸濁液を、第２の濾膜を用いた膜分離の対象とすることができる。 孔 The pore size of the first filtration membrane may be smaller than the pore size of the second filtration membrane. Thus, for example, a cell suspension from which debris such as dead cells has been removed can be subjected to membrane separation using the second filter membrane.
　開示の技術に係る細胞培養装置は、第２の濾過膜によって阻止された成分が収容される第１の回収容器と、第２の濾過膜を透過した成分が収容される第２の回収容器と、を含んでいてもよい。これにより、回収された各成分について、最適な環境で培養を継続することができる。 The cell culture device according to the disclosed technology includes a first collection container that stores components blocked by the second filtration membrane, and a second collection container that stores components that have passed through the second filtration membrane. May be included. Thereby, culturing can be continued in an optimal environment for the collected components.
濃縮分離部は、細胞懸濁液を遠心分離により分離する遠心分離機構を含んでいてもよい。濃縮分離部は、細胞懸濁液の成分を、第１の条件による遠心分離により分離する第１の遠心分離機構と、第１の遠心分離機構により処理された細胞懸濁液の成分を、第１の条件とは異なる第２の条件による遠心分離により分離する第２の遠心分離機構と、を含んでいてもよい。 The concentration and separation unit may include a centrifugal separation mechanism that separates the cell suspension by centrifugation. The concentration / separation unit includes a first centrifugal separation mechanism that separates the components of the cell suspension by centrifugation under the first condition, and a component of the cell suspension processed by the first centrifugal separation mechanism. A second centrifugal separation mechanism for separating by centrifugation under a second condition different from the first condition.
　開示の技術によれば、分割処理によって発生する死細胞等の不要成分の、分割処理後に回収される細胞懸濁液への混入を抑制することが可能となる。 According to the disclosed technology, it is possible to suppress mixing of unnecessary components such as dead cells generated by the division processing into the cell suspension collected after the division processing.
細胞培養の各段階における細胞凝集体のサイズの頻度分布の一例を示すグラフである。It is a graph which shows an example of the frequency distribution of the size of a cell aggregate in each stage of cell culture. 開示の技術の実施形態に係る細胞培養方法における処理の流れの一例を示す工程フロー図である。It is a process flow figure showing an example of a flow of processing in a cell culture method concerning an embodiment of an art of an indication. 開示の技術の実施形態に係る細胞培養装置の構成の一例を示す図である。1 is a diagram illustrating an example of a configuration of a cell culture device according to an embodiment of the disclosed technology. 開示の技術の実施形態に係る分割部の構成の一例を示す断面図である。FIG. 6 is a cross-sectional view illustrating an example of a configuration of a division unit according to an embodiment of the disclosed technology. 図４Ｂは、メッシュの平面図である。FIG. 4B is a plan view of the mesh. 開示の技術の実施形態に係る図４Ｂにおいて破線で囲んだ部分の拡大図である。FIG. 4B is an enlarged view of a portion surrounded by a broken line in FIG. 4B according to the embodiment of the disclosed technology. 開示の技術の実施形態に係る制御部において実施される処理の流れの一例を示すフローチャートである。11 is a flowchart illustrating an example of a flow of a process performed by a control unit according to an embodiment of the disclosed technology. 開示の技術の実施形態に係る細胞培養装置の構成の一例を示す図である。1 is a diagram illustrating an example of a configuration of a cell culture device according to an embodiment of the disclosed technology. 開示の技術の実施形態に係る細胞培養装置の構成の一例を示す図である。1 is a diagram illustrating an example of a configuration of a cell culture device according to an embodiment of the disclosed technology. 開示の技術の実施形態に係る制御部において実施される処理の流れの一例を示すフローチャートである。11 is a flowchart illustrating an example of a flow of a process performed by a control unit according to an embodiment of the disclosed technology. 開示の技術の実施形態に係る細胞培養装置の構成の一例を示す図である。1 is a diagram illustrating an example of a configuration of a cell culture device according to an embodiment of the disclosed technology. 開示の技術の実施形態に係る細胞培養装置の構成の一例を示す図である。1 is a diagram illustrating an example of a configuration of a cell culture device according to an embodiment of the disclosed technology.
　以下、開示の技術の実施形態の一例を図面を参照しつつ説明する。なお、各図面において同一または等価な構成要素及び部分には同一の参照符号を付与している。 Hereinafter, an example of an embodiment of the disclosed technology will be described with reference to the drawings. In the drawings, the same or equivalent components and portions are denoted by the same reference numerals.
[第１の実施形態]
　図２は、開示の技術の実施形態に係る細胞培養方法における処理の流れの一例を示す工程フロー図である。本実施形態に係る細胞培養方法においては、ｉＰＳ細胞、間葉系胚細胞、ＥＳ細胞等の増殖性を有する幹細胞を培養の対象とする。また、培養容器内において、細胞が、略球状の凝集体（スフェア）を形成して、培地中に浮遊した状態で培養されるものとする。本実施形態に係る細胞培養方法は、分割工程Ａ１、濃縮分離工程Ａ２、混合工程Ａ３、測定工程Ａ４、補充工程Ａ５、回収工程Ａ６を含む。 [First Embodiment]
FIG. 2 is a process flowchart illustrating an example of a processing flow in the cell culture method according to the embodiment of the disclosed technology. In the cell culture method according to the present embodiment, proliferating stem cells such as iPS cells, mesenchymal germ cells, and ES cells are to be cultured. In the culture vessel, the cells form a substantially spherical aggregate (sphere) and are cultured in a state of being suspended in the medium. The cell culture method according to the present embodiment includes a division step A1, a concentration separation step A2, a mixing step A3, a measurement step A4, a replenishment step A5, and a collection step A6.
　分割工程Ａ１では、培養容器から移送された細胞懸濁液に含まれる細胞凝集体を、サイズがより小さい細胞凝集体に分割する分割処理が行われる。分割処理は、細胞凝集体をメッシュに通すことにより行われる。分割工程Ａ１は、例えば、培養容器に収容された細胞凝集体の平均サイズが所定のサイズよりも大きくなった場合に実施される。ｉＰＳ細胞等の幹細胞の培養においては、細胞を浮遊培養することによって生じる細胞凝集体のサイズが過大となると、細胞凝集体同士が接着融合し、細胞が分化を開始したり、細胞凝集体の中心部の細胞が壊死したりする場合がある。分割工程Ａ１において所定のサイズにまで成長した細胞凝集体を分割することで、上記の発生が抑制される。 In the dividing step A1, a dividing process is performed to divide the cell aggregates contained in the cell suspension transferred from the culture vessel into smaller aggregates. The division treatment is performed by passing the cell aggregate through a mesh. The dividing step A1 is performed, for example, when the average size of the cell aggregate contained in the culture container becomes larger than a predetermined size. In culturing stem cells such as iPS cells, when the size of cell aggregates generated by suspension culture of cells becomes excessive, the cell aggregates adhere and fuse with each other, and the cells start to differentiate, or the center of the cell aggregates Some cells may become necrotic. The above-mentioned occurrence is suppressed by dividing the cell aggregate that has grown to a predetermined size in the dividing step A1.
　濃縮分離工程Ａ２では、分割工程Ａ１において分割された細胞凝集体を含む細胞懸濁液の成分が分離され、細胞懸濁液における細胞の濃度が濃縮される。すなわち、濃縮分離工程Ａ２では、分割工程Ａ１において発生した過小サイズの細胞凝集体、細胞凝集体を形成しない単一細胞、死細胞等のデブリスと、適正サイズに分割された細胞凝集体とが分離される。濃縮分離工程Ａ２は、分割工程Ａ１において分割された細胞凝集体を含む細胞懸濁液を濾過する濾過処理を含んでいてもよく、濾過処理は、例えば、濾過膜を用いた膜分離処理を含んでいてもよい。膜分離の方式として、細胞に与えるダメージがデッドエンド方式よりも小さいタンジェンシャルフロー方式を適用することが好ましい。また、濃縮分離工程Ａ２は、分割工程Ａ１において分割された細胞凝集体を含む細胞懸濁液の成分を、遠心分離により分離する遠心分離処理を含んでいてもよい。遠心分離慮離により、分割工程Ａ１において発生した過小サイズの細胞凝集体、細胞凝集体を形成しない単一細胞、死細胞等のデブリスと、適正サイズに分割された細胞凝集体とが分離される。 In the concentration separation step A2, the components of the cell suspension containing the cell aggregates divided in the division step A1 are separated, and the concentration of cells in the cell suspension is concentrated. That is, in the concentration separation step A2, debris such as undersized cell aggregates, single cells that do not form cell aggregates, and dead cells generated in the division step A1 are separated from cell aggregates divided into appropriate sizes. Is done. The concentration separation step A2 may include a filtration treatment for filtering the cell suspension containing the cell aggregates divided in the division step A1, and the filtration treatment includes, for example, a membrane separation treatment using a filtration membrane. You may go out. As a membrane separation method, it is preferable to apply a tangential flow method in which damage to cells is smaller than a dead end method. Further, the concentration separation step A2 may include a centrifugal separation process of separating the components of the cell suspension containing the cell aggregates divided in the division step A1 by centrifugation. By centrifugation separation, debris such as undersized cell aggregates, single cells that do not form cell aggregates, dead cells, etc. generated in the division step A1, and cell aggregates divided into appropriate sizes are separated. .
　混合工程Ａ３では、濃縮分離工程Ａ２において分離された、適正サイズの細胞凝集体を含む細胞懸濁液と、新鮮な培地とを混合する混合処理が行われる。この混合処理により、分割工程Ａ１及び濃縮分離工程Ａ２を経た細胞懸濁液と、新鮮な培地とを混合した混合液が得られる。濃縮分離工程Ａ２を経ることで、細胞懸濁液の細胞濃度は過度に高まるので、濃縮分離工程Ａ２を経た細胞懸濁液と培地とを混合することで、細胞濃度が過度に高い状態が緩和される。 In the mixing step A3, a mixing process of mixing the cell suspension containing the cell aggregates of an appropriate size separated in the concentration separation step A2 with a fresh medium is performed. By this mixing process, a mixed liquid obtained by mixing the cell suspension that has passed through the dividing step A1 and the concentration separation step A2 with a fresh medium is obtained. Since the cell concentration of the cell suspension is excessively increased by passing through the concentration separation step A2, the state where the cell concentration is excessively high is relaxed by mixing the cell suspension and the medium that have passed through the concentration separation step A2. Is done.
　測定工程Ａ４では、混合工程Ａ３において得られた上記混合液における細胞濃度が測定される。細胞濃度は、例えば、上記混合液の単位体積あたりに占める細胞（細胞凝集体）の体積の割合として導出することができる。細胞濃度の導出は、上記混合液を撮像した画像を解析することにより導出してもよい。この場合、画像に含まれる細胞凝集体の個々の面積の積算値と、撮像視野の面積との比から細胞濃度を導出してもよい。 In the measurement step A4, the cell concentration in the mixed solution obtained in the mixing step A3 is measured. The cell concentration can be derived, for example, as a ratio of the volume of cells (cell aggregates) to the unit volume of the mixture. The derivation of the cell concentration may be derived by analyzing an image obtained by imaging the mixed solution. In this case, the cell concentration may be derived from the ratio between the integrated value of the individual areas of the cell aggregates included in the image and the area of the imaging visual field.
　補充工程Ａ５では、測定工程Ａ４において測定された細胞濃度に応じて上記混合液に培地が補充される。すなわち、混合工程Ａ３において混合される培地の量が不十分であり、測定工程Ａ４において測定される混合液の細胞濃度が所定の濃度よりも高い場合、当該混合液に新鮮な培地が追加される。一方、混合工程Ａ３において混合される培地の量が適正であり、測定工程Ａ４において測定される混合液の細胞濃度が適正範囲内である場合、当該混合液に培地は補充されない。この場合、補充工程Ａ５はスキップされる。 In the replenishment step A5, the medium is replenished to the above-mentioned mixture according to the cell concentration measured in the measurement step A4. That is, when the amount of the medium mixed in the mixing step A3 is insufficient and the cell concentration of the mixture measured in the measurement step A4 is higher than a predetermined concentration, a fresh medium is added to the mixture. . On the other hand, when the amount of the medium mixed in the mixing step A3 is appropriate and the cell concentration of the mixed liquid measured in the measuring step A4 is within an appropriate range, the medium is not supplemented to the mixed liquid. In this case, the replenishment step A5 is skipped.
　回収工程Ａ６では、測定工程Ａ４を経た細胞懸濁液（混合液）または補充工程Ａ５を経た細胞懸濁液（混合液）が、元の培養容器またはこれとは別の回収容器に回収される。 In the recovery step A6, the cell suspension (mixture) that has passed through the measurement step A4 or the cell suspension (mixture) that has passed through the replenishment step A5 is recovered in the original culture vessel or another recovery vessel. .
　図３は、上記した細胞培養方法を実現する開示の技術の実施形態に係る細胞培養装置１の構成の一例を示す図である。 FIG. 3 is a diagram illustrating an example of a configuration of a cell culture device 1 according to an embodiment of the disclosed technology for realizing the above-described cell culture method.
　細胞培養装置１は、培養容器１０、回収容器１１、培地収容容器４０、濃縮分離部の一例である濾過部３０、廃液容器３１、分割部２０、モニタ部６０Ａ、６０Ｂ、制御部５０、ポンプＰ１～Ｐ４及びバルブＶ１～Ｖ６を含んで構成されている。培養容器１０、回収容器１１、培地収容容器４０、濾過部３０、廃液容器３１、分割部２０、モニタ部６０Ａ、６０Ｂは、それぞれ流路７０に接続されている。 The cell culture device 1 includes a culture container 10, a collection container 11, a medium storage container 40, a filtration unit 30, which is an example of a concentration separation unit, a waste liquid container 31, a division unit 20, monitor units 60A and 60B, a control unit 50, and a pump P1. To P4 and valves V1 to V6. The culture container 10, the collection container 11, the medium storage container 40, the filtration unit 30, the waste liquid container 31, the division unit 20, and the monitor units 60A and 60B are connected to the flow path 70, respectively.
　培養容器１０には、培養対象である複数の細胞及び培地を含む細胞懸濁液が収容される。培養容器１０において、複数の細胞が、球状の細胞凝集体（スフェア）を形成して培地中に浮遊した状態で培養される。培養容器１０は、例えば、１×１０９個またはそれよりも多くの細胞を収容可能な容積を有する。培養容器１０の形態は、特に限定されず、例えば、ガラス容器または金属容器を培養容器１０として用いることが可能である。培養容器１０は、例えばガス透過性を有するフィルムを含んで構成されるバッグの形態を有していてもよい。 The culture container 10 contains a plurality of cells to be cultured and a cell suspension containing a medium. In the culture vessel 10, a plurality of cells are cultured in a state where they form spherical cell aggregates (spheres) and are suspended in the medium. The culture vessel 10 has, for example, a volume that can accommodate 1 × 10 9 or more cells. The form of the culture container 10 is not particularly limited, and for example, a glass container or a metal container can be used as the culture container 10. The culture vessel 10 may have, for example, a form of a bag including a film having gas permeability.
　分割部２０は、分割工程Ａ１を実施する。すなわち、分割部２０は、培養容器１０から移送された細胞懸濁液に含まれる細胞凝集体を、サイズがより小さい細胞凝集体に分割する分割処理を行う。 The dividing unit 20 performs the dividing step A1. That is, the dividing unit 20 performs a dividing process of dividing the cell aggregates contained in the cell suspension transferred from the culture container 10 into smaller aggregates.
　図４Ａは、分割部２０の構成の一例を示す断面図である。分割部２０は、流入口２０２及び流出口２０３を有するケース２０１と、ケース２０１の内部の、流入口２０２と流出口２０３との間に設けられたメッシュ２１０とを含んで構成されている。図４Ｂは、メッシュ２１０の平面図、図４Ｃは、図４Ｂにおいて破線で囲んだ部分Ｙの拡大図である。メッシュ２１０は、複数の繊維状部材２１２を例えば平織りすることによって形成された複数の開口部（網目）２１１を有する。なお、繊維状部材２１２の織り方は、平織りに限定されない。繊維状部材２１２の材質は、特に限定されるものではないが、耐食性の高い材料で構成されていることが好ましく、例えばナイロンまたはステンレスを好適に用いることができる。 FIG. 4A is a cross-sectional view illustrating an example of the configuration of the dividing unit 20. The dividing unit 20 includes a case 201 having an inlet 202 and an outlet 203, and a mesh 210 provided inside the case 201 between the inlet 202 and the outlet 203. 4B is a plan view of the mesh 210, and FIG. 4C is an enlarged view of a portion Y surrounded by a broken line in FIG. 4B. The mesh 210 has a plurality of openings (mesh) 211 formed by, for example, plain weaving a plurality of fibrous members 212. The weaving method of the fibrous member 212 is not limited to plain weaving. The material of the fibrous member 212 is not particularly limited, but is preferably made of a material having high corrosion resistance. For example, nylon or stainless steel can be suitably used.
　メッシュ２１０は、複数の開口部２１１を有する主面が、細胞懸濁液の流れ方向ＦＬと交差する方向に延在するようにケース２０１内に設置されている。細胞懸濁液が、メッシュ２１０を通過することで、細胞懸濁液に含まれる細胞凝集体が機械的に分割される。分割部２０が有するメッシュ２１０の孔径Ｌは、例えば、分割処理前の細胞凝集体の平均径よりも小さい大きさとされ、分割処理後の細胞凝集体の目標サイズに応じて定められる。細胞凝集体の平均径として、細胞凝集体の各々を球形近似したときの、当該球形の直径の算術平均を適用することが可能である。 The mesh 210 is installed in the case 201 such that the main surface having the plurality of openings 211 extends in a direction intersecting with the flow direction FL of the cell suspension. When the cell suspension passes through the mesh 210, the cell aggregates contained in the cell suspension are mechanically divided. The pore diameter L of the mesh 210 included in the division unit 20 is, for example, smaller than the average diameter of the cell aggregate before the division processing, and is determined according to the target size of the cell aggregate after the division processing. As the average diameter of the cell aggregate, it is possible to apply the arithmetic average of the diameter of the spherical shape when each of the cell aggregates is approximated to a spherical shape.
　濃縮分離部の一例である濾過部３０は、濃縮分離工程Ａ２を実施する。すなわち、濾過部３０は、細胞懸濁液の成分を分離して細胞懸濁液における細胞の濃度を濃縮する。より具体的には、濾過部３０は、分割部２０における分割処理によって発生した過小サイズの細胞凝集体、細胞凝集体を形成しない単一細胞、死細胞等のデブリスと、適正サイズの細胞凝集体とを分離する濾過処理を行う。濾過部３０における濾過処理は、例えば、濾過膜を用いた膜分離によって行われてもよい。この場合、膜分離の方式は、細胞に与えるダメージがデッドエンド方式よりも小さいタンジェンシャルフロー方式であることが好ましい。濾過部３０において分離された過小サイズの細胞凝集体、細胞凝集体を形成しない単一細胞、死細胞等のデブリスは、廃液容器３１に収容される。 濾過 The filtration unit 30, which is an example of the concentration separation unit, performs the concentration separation step A2. That is, the filtration unit 30 separates the components of the cell suspension and concentrates the concentration of the cells in the cell suspension. More specifically, the filtration unit 30 includes debris such as undersized cell aggregates, single cells that do not form cell aggregates, dead cells, and the like generated by the division processing in the division unit 20, and cell aggregates of appropriate size. And a filtration treatment for separating The filtration process in the filtration unit 30 may be performed by, for example, membrane separation using a filtration membrane. In this case, the membrane separation method is preferably a tangential flow method in which damage to cells is smaller than the dead end method. Debris such as undersized cell aggregates, single cells that do not form cell aggregates, and dead cells separated in the filtration unit 30 are stored in the waste liquid container 31.
　培地収容容器４０には、新鮮な培地が収容される。混合工程Ａ３は、濾過部３０においてデブリスが除去された細胞懸濁液と、培地収容容器４０に収容されている培地とを混合することにより行われる。また、補充工程Ａ５は、濾過部３０においてデブリスが除去された細胞懸濁液と培地収容容器４０に収容されている培地とを混合した混合液に、培地収容容器４０に収容された培地を更に追加することにより行われる。 The culture medium container 40 stores a fresh culture medium. The mixing step A3 is performed by mixing the cell suspension from which debris has been removed in the filtration unit 30 with the medium contained in the medium containing container 40. In addition, the replenishment step A5 further includes adding the medium contained in the medium container 40 to a mixed solution obtained by mixing the cell suspension from which debris has been removed in the filtration unit 30 and the medium contained in the medium container 40. It is done by adding.
　モニタ部６０Ａは、流路７０の、培養容器１０と分割部２０との間の区間Ｘ１に設けられ、モニタ部６０Ｂは、流路７０の、濾過部３０と回収容器１１との間の区間Ｘ２に設けられている。モニタ部６０Ａ及び６０Ｂは、それぞれ、区間Ｘ１及びＸ２を通過する細胞懸濁液をモニタする。モニタ部６０Ａ、６０Ｂは、それぞれ、フローセル６１及び撮像装置６２を含んで構成されている。 The monitor section 60A is provided in a section X1 between the culture vessel 10 and the division section 20 in the flow path 70, and the monitor section 60B is provided in a section X2 between the filtration section 30 and the collection vessel 11 in the flow path 70. It is provided in. The monitoring units 60A and 60B monitor the cell suspension passing through the sections X1 and X2, respectively. Each of the monitor units 60A and 60B includes a flow cell 61 and an imaging device 62.
　フローセル６１は、その全体が、ガラスまたはプラスチック等の光透過性を有する材料で構成されている。フローセル６１は、第１の流通口６１ａと、第１の流通口６１ａに連通する第２の流通口６１ｂとを有している。撮像装置６２は、フローセル６１の第１の流通口６１ａと第２の流通口６１ｂとの間の領域に撮像視野が設定されており、フローセル６１の内部を流れる細胞懸濁液に含まれる細胞（細胞凝集体）を、フローセル６１越しに連続的に撮像する。撮像装置６２によって撮像された複数の画像は、制御部５０に送信される。測定工程Ａ４は、モニタ部６０Ｂを用いて実現される。 The entire flow cell 61 is made of a light-transmitting material such as glass or plastic. The flow cell 61 has a first distribution port 61a and a second distribution port 61b communicating with the first distribution port 61a. The imaging device 62 has an imaging field of view set in a region between the first distribution port 61a and the second distribution port 61b of the flow cell 61, and includes cells included in a cell suspension flowing inside the flow cell 61 ( (Cell aggregate) is continuously imaged through the flow cell 61. The plurality of images captured by the imaging device 62 are transmitted to the control unit 50. The measurement step A4 is realized using the monitor unit 60B.
　回収容器１１には、濾過部３０においてデブリスが除去された細胞懸濁液と、培地収容容器４０に収容されている新鮮な培地とを混合した混合液が収容される。回収容器１１の形態は、特に限定されず、例えば、ガラス容器または金属容器を回収容器１１として用いることが可能である。回収容器１１は、例えばガス透過性を有するフィルムを含んで構成されるバッグの形態を有していてもよい。 {Circle around (4)} The collection container 11 contains a mixed liquid obtained by mixing the cell suspension from which debris has been removed in the filtration unit 30 and the fresh medium stored in the medium storage container 40. The form of the collection container 11 is not particularly limited. For example, a glass container or a metal container can be used as the collection container 11. The collection container 11 may have, for example, a form of a bag including a film having gas permeability.
　流路７０は、培養容器１０から分割部２０への細胞懸濁液の移送、分割部２０から濾過部３０への細胞懸濁液の移送、濾過部３０から回収容器１１及び廃液容器３１への細胞懸濁液の移送を可能とするように構成されている。また、流路７０は、培地収容容器４０から抜き出された培地が、濾過部３０においてデブリスが除去された細胞懸濁液、及びモニタ部６０Ｂを通過した細胞懸濁液にそれぞれ合流できるように構成されている。また、流路７０は、モニタ部６０Ａを通過した細胞懸濁液が、分割部２０を経由して濾過部３０に至る第１の経路と、分割部２０を経由することなく濾過部３０に至る第２の経路を形成するように構成されている。 The flow path 70 transfers the cell suspension from the culture container 10 to the dividing unit 20, transfers the cell suspension from the dividing unit 20 to the filtering unit 30, and transfers the cell suspension from the filtering unit 30 to the collecting container 11 and the waste liquid container 31. It is configured to allow transfer of the cell suspension. The flow path 70 allows the medium extracted from the medium storage container 40 to join the cell suspension from which debris has been removed in the filtration unit 30 and the cell suspension that has passed through the monitor unit 60B. It is configured. In addition, the flow path 70 is configured such that the cell suspension that has passed through the monitor unit 60 </ b> A reaches the filtration unit 30 via the division unit 20 and the filtration unit 30 without passing through the division unit 20. It is configured to form a second path.
　ポンプＰ１～Ｐ４及びバルブＶ１～Ｖ６は、流路７０の各所に適宜配置される。ポンプＰ１～Ｐ４は、制御部５０から供給される制御信号に応じて駆動し、バルブＶ１～Ｖ６は、制御部５０から供給される制御信号に応じて開閉する。なお、流路７０の構成及びポンプＰ１～Ｐ４、バルブＶ１～Ｖ６の配置は、図３に例示されたものに限定されず、本実施形態に係る細胞培養方法を実施できるように構成されていればよい。 (4) The pumps P1 to P4 and the valves V1 to V6 are appropriately disposed at various points in the flow path 70. The pumps P1 to P4 are driven according to a control signal supplied from the control unit 50, and the valves V1 to V6 are opened and closed according to the control signal supplied from the control unit 50. The configuration of the flow path 70 and the arrangement of the pumps P1 to P4 and the valves V1 to V6 are not limited to those illustrated in FIG. 3, but may be configured so that the cell culture method according to the present embodiment can be performed. I just need.
　制御部５０は、制御信号を用いてポンプＰ１～Ｐ４の駆動制御及びバルブＶ１～Ｖ３の開閉制御を行うことにより、流路７０を介した細胞懸濁液の移送を制御する。 The controller 50 controls the transfer of the cell suspension through the flow path 70 by controlling the driving of the pumps P1 to P4 and controlling the opening and closing of the valves V1 to V3 using the control signal.
　図５は、細胞培養装置１において細胞培養を行う場合に、制御部５０において実施される処理の流れの一例を示すフローチャートである。 FIG. 5 is a flowchart illustrating an example of a flow of processing performed in the control unit 50 when performing cell culture in the cell culture device 1.
　ステップＳ１において、制御部５０は、ポンプＰ１を駆動させることで、培養容器１０に収容されている細胞懸濁液をモニタ部６０Ａに移送する。モニタ部６０Ａは、流路７０の区間Ｘ１を通過する細胞懸濁液をモニタする。すなわち、撮像装置６２は、フローセル６１を通過する細胞懸濁液に含まれる細胞（細胞凝集体）を連続的に撮像する。モニタ部６０Ａの撮像装置６２は、例えば、流路７０の区間Ｘ１を通過する細胞懸濁液に含まれる全ての細胞（細胞凝集体）を撮像可能な間隔で撮像を行う。なお、撮像装置６２は、流路７０の区間Ｘ１を通過する細胞懸濁液に含まれる一部の細胞（細胞凝集体）を撮像してもよい。 In step S1, the control unit 50 drives the pump P1 to transfer the cell suspension contained in the culture container 10 to the monitor unit 60A. The monitoring unit 60A monitors the cell suspension passing through the section X1 of the channel 70. That is, the imaging device 62 continuously images cells (cell aggregates) contained in the cell suspension passing through the flow cell 61. The imaging device 62 of the monitor unit 60A captures, for example, all the cells (cell aggregates) included in the cell suspension passing through the section X1 of the flow channel 70 at intervals at which the imaging can be performed. Note that the imaging device 62 may image a part of cells (cell aggregates) included in the cell suspension passing through the section X1 of the flow channel 70.
　ステップＳ２において、制御部５０は、モニタ部６０Ａの撮像装置６２によって撮像された細胞の画像を取得する。 In step S2, the control unit 50 acquires an image of the cell captured by the imaging device 62 of the monitor unit 60A.
　ステップＳ３において、制御部５０は、ステップＳ２において取得した画像に基づいて、分割処理の要否を判断する。制御部５０は、分割処理の要否の判断を例えば、以下のようにして行う。制御部５０は、例えば、ステップＳ２においてモニタ部６０Ａから取得した画像に含まれる細胞凝集体の平均サイズを導出し、導出した平均サイズが所定のサイズよりも大きい場合には、分割処理が必要であると判断し、導出した平均サイズが所定のサイズよりも小さい場合には、分割処理が不要であると判断する。細胞凝集体の平均サイズとして、細胞凝集体の各々を球形近似したときの、当該球形の直径の算術平均を適用することが可能である。制御部５０は、分割処理が必要であると判断した場合、処理をステップＳ４に移行し、分割処理が必要ではないと判断した場合、処理をステップＳ５に移行する。 In step S3, the control unit 50 determines whether or not the division processing is necessary based on the image acquired in step S2. The control unit 50 determines whether or not the division processing is necessary, for example, as follows. The control unit 50 derives, for example, the average size of the cell aggregates included in the image acquired from the monitor unit 60A in step S2, and when the derived average size is larger than a predetermined size, a division process is necessary. It is determined that there is, and if the derived average size is smaller than the predetermined size, it is determined that the dividing process is unnecessary. As the average size of the cell aggregate, it is possible to apply the arithmetic mean of the diameter of the spherical shape when each of the cell aggregates is approximated to a spherical shape. When the control unit 50 determines that the division process is necessary, the process proceeds to step S4, and when it determines that the division process is not required, the process proceeds to step S5.
　制御部５０は、分割処理が必要であると判断した場合、ステップＳ４において、バルブＶ１、Ｖ２を開状態、バルブＶ３、Ｖ４を閉状態に制御することで、細胞懸濁液を分割部２０に移送する。分割部２０に移送された細胞懸濁液に含まれる細胞凝集体は、分割部２０のメッシュ２１０（図４Ａ、図４Ｂ、図４Ｃ参照）を通過することにより、サイズのより小さい細胞凝集体に分割される。分割された細胞凝集体を含む細胞懸濁液は、ステップＳ５において濾過部３０に移送される。 When the control unit 50 determines that the division process is necessary, in step S4, the control unit 50 controls the valves V1 and V2 to be open and the valves V3 and V4 to be closed so that the cell suspension is transmitted to the division unit 20. Transfer. The cell aggregates contained in the cell suspension transferred to the dividing unit 20 pass through the mesh 210 of the dividing unit 20 (see FIGS. 4A, 4B, and 4C) to form smaller cell aggregates. Divided. The cell suspension containing the divided cell aggregates is transferred to the filtration unit 30 in step S5.
　一方、制御部５０は、分割処理が必要ではないと判断した場合、ステップＳ５において、バルブＶ３、Ｖ４を開状態、バルブＶ１、Ｖ２を閉状態に制御することで、細胞懸濁液を、分割部２０を経由させることなく濾過部３０に移送する。 On the other hand, if the control unit 50 determines that the dividing process is not necessary, in step S5, the valves V3 and V4 are opened and the valves V1 and V2 are closed, thereby dividing the cell suspension. It is transferred to the filtration unit 30 without passing through the unit 20.
　濾過部３０は、移送された細胞懸濁液に対して濾過処理を行う。すなわち、濾過部３０は、移送された細胞懸濁液に含まれる、過小サイズの細胞凝集体、細胞凝集体を形成しない単一細胞、死細胞等のデブリスと、適正サイズの細胞凝集体とを分離する。細胞懸濁液が、分割部２０を経由して濾過部３０に移送される場合、分割部２０における分割処理によって発生した、過小サイズの細胞凝集体、単一細胞、死細胞等のデブリスが、濾過部３０において除去される。 The filtration unit 30 performs a filtration process on the transferred cell suspension. That is, the filtration unit 30 separates debris such as undersized cell aggregates, single cells that do not form cell aggregates, dead cells, etc., contained in the transferred cell suspension, and cell aggregates of appropriate size. To separate. When the cell suspension is transferred to the filtration unit 30 via the division unit 20, debris such as undersized cell aggregates, single cells, and dead cells generated by the division processing in the division unit 20, It is removed in the filtration unit 30.
　ステップＳ６において、制御部５０は、ポンプＰ２を駆動させることで、濾過部３０によって分離された、過小サイズの細胞凝集体、単一細胞、死細胞等のデブリスを廃液容器３１に移送する。 In step S <b> 6, the control unit 50 drives the pump P <b> 2 to transfer the debris such as the undersized cell aggregates, single cells, and dead cells separated by the filtration unit 30 to the waste liquid container 31.
　ステップＳ７において、制御部５０は、ポンプＰ３を駆動させることで、デブリスが除去された細胞懸濁液をモニタ部６０Ｂに移送する。 In step S7, the control unit 50 drives the pump P3 to transfer the cell suspension from which debris has been removed to the monitor unit 60B.
　ステップＳ７と並行して実施されるステップＳ８において、制御部５０は、ポンプＰ４を駆動させ、バルブＶ５を開状態、バルブＶ６を閉状態に制御することで、培地収容容器４０に収容されている新鮮な培地を培地収容容器４０から抜き出し、この培地と、濾過部３０からモニタ部６０Ｂに向けて流れるデブリスが除去された細胞懸濁液とを流路７０上で合流させ、これらを混合する。 In step S8, which is performed in parallel with step S7, the control unit 50 drives the pump P4 to control the valve V5 to open and the valve V6 to close, so that the control unit 50 is stored in the culture medium storage container 40. A fresh culture medium is extracted from the culture medium container 40, and the culture medium and the cell suspension from which the debris flows from the filtration unit 30 to the monitor unit 60B are removed on the flow path 70 and are mixed.
　モニタ部６０Ｂは、流路７０の区間Ｘ２を通過する、デブリスが除去された細胞懸濁液と培地収容容器４０から抜き出された新鮮な培地とを混合した混合液をモニタする。すなわち、モニタ部６０Ｂの撮像装置６２は、フローセル６１を通過する混合液に含まれる細胞（細胞凝集体）を連続的に撮像する。撮像装置６２は、例えば、流路７０の区間Ｘ２を通過する細胞懸濁液に含まれる全ての細胞（細胞凝集体）を撮像可能な間隔で撮像を行う。なお、撮像装置６２は、流路７０の区間Ｘ２を通過する細胞懸濁液に含まれる一部の細胞（細胞凝集体）を撮像してもよい。 The monitor unit 60 </ b> B monitors a liquid mixture that passes through the section X <b> 2 of the channel 70 and is a mixture of the cell suspension from which debris has been removed and the fresh medium extracted from the medium container 40. That is, the imaging device 62 of the monitor unit 60B continuously captures images of cells (cell aggregates) contained in the mixed solution passing through the flow cell 61. The imaging device 62 images all cells (cell aggregates) included in the cell suspension passing through the section X2 of the flow path 70 at intervals at which imaging can be performed, for example. Note that the imaging device 62 may image a part of cells (cell aggregates) included in the cell suspension passing through the section X2 of the flow path 70.
　ステップＳ９において、制御部５０は、モニタ部６０Ｂの撮像装置６２によって撮像された細胞の画像を取得する。 In step S9, the control unit 50 acquires an image of the cell captured by the imaging device 62 of the monitor unit 60B.
　ステップＳ１０において、制御部５０は、ステップＳ９において取得した上記混合液に含まれる細胞を撮像した画像に基づいて、当該混合液に対する培地の補充が必要であるか否かを判断する。制御部５０は、培地の補充の要否の判断を例えば、以下のようにして行う。制御部５０は、例えば、ステップＳ９においてモニタ部６０Ｂから取得した画像から、当該混合液における細胞濃度を導出し、導出した細胞濃度が所定値よりも大きい場合には、培地の補充が必要であると判断し、導出した細胞濃度が所定値よりも小さい場合には、培地の補充が必要ではないと判断する。混合液における細胞濃度は、例えば、ステップＳ９において取得した画像に含まれる細胞凝集体の個々のサイズの積算値と、撮像装置６２の撮像視野の面積との比から導出することが可能である。制御部５０は、培地の補充が必要であると判断した場合、処理をステップＳ１１に移行し、培地の補充が必要ではないと判断した場合、処理を終了させる。 In step S10, the control unit 50 determines whether or not it is necessary to replenish the mixture with the medium based on the image obtained by imaging the cells contained in the mixture obtained in step S9. The control unit 50 determines whether or not the medium needs to be replenished, for example, as follows. For example, the control unit 50 derives the cell concentration in the mixed solution from the image acquired from the monitor unit 60B in step S9, and when the derived cell concentration is higher than a predetermined value, the medium needs to be replenished. If the derived cell concentration is smaller than the predetermined value, it is determined that the medium need not be replenished. The cell concentration in the mixed solution can be derived, for example, from the ratio of the integrated value of the individual size of the cell aggregate included in the image acquired in step S9 and the area of the imaging visual field of the imaging device 62. Control unit 50 shifts the processing to step S11 when judging that medium replenishment is necessary, and ends processing when judging that medium replenishment is not necessary.
　制御部５０は、培地の補充が必要であると判断した場合、ステップＳ１１において、ステップＳ１０において導出した混合液の細胞濃度に基づいて、当該混合液の細胞濃度を所定の濃度とするための培地の補充量を導出する。 If the control unit 50 determines that supplementation of the culture medium is necessary, in step S11, based on the cell concentration of the mixture liquid derived in step S10, the control unit 50 adjusts the cell concentration of the mixture liquid to a predetermined concentration. The replenishment amount is derived.
　ステップＳ１２において、制御部５０は、ポンプＰ４を駆動させ、バルブＶ６を開状態に制御することで、培地収容容器４０に収容されている新鮮な培地をステップＳ１１において導出した補充量に相当する分量だけ抜き出し、抜き出した培地と、モニタ部６０Ｂを通過した混合液とを流路７０上で合流させる。これにより、混合液に、新鮮な培地が補充され、混合液における細胞濃度が適正な濃度に調整される。このときバルブＶ５は開状態に維持される。すなわち、濾過部３０においてデブリスが除去された細胞懸濁液と培地収容容器４０に収容されている培地とを混合して混合液を得る混合処理と、培地収容容器４０に収容されている培地を上記混合液に補充して細胞濃度を調整する濃度調整処理が並行して行われる。細胞濃度の調整は、例えば、バルブＶ５及びＶ６の開度を制御することで実現することができる。適正な濃度に調整された混合液は、回収容器１１に移送される。回収容器１１に移送された細胞は、回収容器１１内において継続して培養される。なお、本実施形態においては、バルブＶ５及びＶ６を通過する培地の送液を、共通の培地収容容器４０及び共通のポンプＰ４を用いて行う場合を例示しているが、別々の培地収容容器及びポンプを用いることも可能である。 In step S12, the control unit 50 drives the pump P4 and controls the valve V6 to the open state, so that the fresh medium stored in the medium storage container 40 is dispensed in an amount corresponding to the replenishment amount derived in step S11. Then, the extracted medium and the mixed solution that has passed through the monitor unit 60B are joined on the flow path 70. Thus, the mixture is supplemented with fresh medium, and the cell concentration in the mixture is adjusted to an appropriate concentration. At this time, the valve V5 is kept open. That is, a mixing process of mixing the cell suspension from which debris has been removed in the filtration unit 30 and the culture medium contained in the culture medium container 40 to obtain a mixed solution, and the culture medium contained in the culture medium container 40 Concentration adjustment processing for adjusting the cell concentration by replenishing the mixture is performed in parallel. Adjustment of the cell concentration can be realized, for example, by controlling the degree of opening of the valves V5 and V6. The mixed solution adjusted to an appropriate concentration is transferred to the collection container 11. The cells transferred to the collection container 11 are continuously cultured in the collection container 11. Note that, in the present embodiment, the case where the feeding of the culture medium passing through the valves V5 and V6 is performed using the common culture medium storage container 40 and the common pump P4 is described. It is also possible to use a pump.
　以上のように、開示の技術の第１の実施形態に係る細胞培養方法及び細胞培養装置１によれば、分割工程Ａ１の後に、濃縮分離工程Ａ２が実施される。分割工程Ａ１において発生する過小サイズの細胞凝集体、単一細胞、死細胞等のデブリスは、濃縮分離工程Ａ２において除去される。従って、回収容器１１に回収される細胞懸濁液への不要成分（デブリス）の混入が抑制される。これにより、回収容器１１内で培養される細胞の増殖率及び生存率を高めることができる。 As described above, according to the cell culture method and the cell culture device 1 according to the first embodiment of the disclosed technology, the concentration separation step A2 is performed after the division step A1. Debris such as undersized cell aggregates, single cells, dead cells and the like generated in the dividing step A1 are removed in the concentration separation step A2. Therefore, mixing of unnecessary components (debris) into the cell suspension collected in the collection container 11 is suppressed. Thereby, the growth rate and the survival rate of the cells cultured in the collection container 11 can be increased.
　また、開示の技術の実施形態に係る細胞培養方法及び細胞培養装置によれば、測定工程Ａ４及び補充工程Ａ５によって、適正な細胞濃度の細胞懸濁液が回収容器１１に回収されることが担保される。また、分割処理後の細胞懸濁液における細胞濃度の予測が困難な状況においても、測定工程Ａ４及び補充工程Ａ５が回収工程Ａ６の直前に実施されることで、回収容器１１に回収される細胞懸濁における細胞濃度を適正範囲にコントロールすることが可能となる。 In addition, according to the cell culture method and the cell culture device according to the embodiment of the disclosed technology, it is ensured that the cell suspension having an appropriate cell concentration is collected in the collection container 11 by the measurement step A4 and the replenishment step A5. Is done. Further, even in a situation where it is difficult to predict the cell concentration in the cell suspension after the division process, the measurement step A4 and the replenishment step A5 are performed immediately before the collection step A6, so that the cells collected in the collection container 11 are obtained. It becomes possible to control the cell concentration in the suspension within an appropriate range.
　なお、本実施形態では、補充用の培地と濃度調整前の混合液とを流路７０上で合流させた後に、これらを回収容器１１に移送する場合を例示したが、濃度調整前の混合液を回収容器１１に移送した後に、補充用の培地を回収容器１１に移送してもよい。 In the present embodiment, the case where the replenishing medium and the mixed solution before the concentration adjustment are combined on the flow path 70 and then transferred to the collection container 11 has been described as an example. May be transferred to the collection container 11 after transferring to the collection container 11.
　また、図３には、各工程を経た細胞懸濁液を回収容器１１に回収する場合の構成が、例示されているが、図６に示すように、細胞培養装置１は、各工程を経た細胞懸濁液が培養容器１０に回収されるように構成されていてもよい。 FIG. 3 illustrates an example of a configuration in which the cell suspension that has passed through each step is collected in the collection container 11. As illustrated in FIG. 6, the cell culture device 1 has passed through each step. The cell suspension may be configured to be collected in the culture container 10.
　また、本実施形態では、濃縮分離工程Ａ１を実施する濃縮分離部の一例として濾過部３０を例示したが、この態様に限定されない。濃縮分離工程Ａ１を実施する濃縮分離部は、例えば、細胞懸濁液の成分を、遠心分離によって分離する遠心分離機構を含んでいてもよい。遠心分離機構は、分割部２０における分割処理によって発生した過小サイズの細胞凝集体、細胞凝集体を形成しない単一細胞、死細胞等のデブリスと、適正サイズの細胞凝集体とを、遠心分離により分離する。 Also, in the present embodiment, the filtration unit 30 is illustrated as an example of the concentration separation unit that performs the concentration separation step A1, but the present invention is not limited to this mode. The concentration separation unit that performs the concentration separation step A1 may include, for example, a centrifugal separation mechanism that separates components of the cell suspension by centrifugation. The centrifugation mechanism separates the undersized cell aggregates generated by the division process in the division unit 20, single cells that do not form the cell aggregates, debris such as dead cells, and the appropriate size cell aggregates by centrifugation. To separate.
［第２の実施形態］
　図７は、開示の技術の第２の実施形態に係る細胞培養装置１Ａの構成の一例を示す図である。細胞培養装置１Ａは、希釈液収容容器４１、濃縮分離部の一例である２つの濾過部３０Ａ、３０Ｂ、３つモニタ部６０Ａ、６０Ｂ、６０Ｃ、２つの回収容器１１Ａ、１１Ｂを含む点が、上記した第１の実施形態に係る細胞培養装置１と異なる。 [Second embodiment]
FIG. 7 is a diagram illustrating an example of a configuration of a cell culture device 1A according to the second embodiment of the disclosed technology. The point that the cell culture device 1A includes the diluent storage container 41, two filtration units 30A and 30B, which are examples of a concentration separation unit, three monitoring units 60A, 60B and 60C, and two collection containers 11A and 11B is described above. This is different from the cell culture device 1 according to the first embodiment described above.
　希釈液収容容器４１は、希釈液を収容される容器である。希釈液の基本成分は、培養容器１０内で行われる細胞培養に用いられる培地の基本成分と同じであってもよい。培養容器１０内で行われる細胞培養に用いられる培地には、細胞の栄養補給を行うためのサプリメント、培地の粘度を高めるための増粘剤等の添加剤が添加され得るが、希釈液は、このような添加剤を含んでいないことが好ましい。すなわち、希釈液の粘度は、培養容器１０に収容される培地の粘度よりも低いことが好ましい。 The diluent storage container 41 is a container for storing the diluent. The basic components of the diluent may be the same as the basic components of the medium used for the cell culture performed in the culture vessel 10. The culture medium used for cell culture performed in the culture vessel 10 may be supplemented with a supplement for supplying cells with nutrients, and an additive such as a thickener for increasing the viscosity of the culture medium. It is preferable not to include such additives. That is, the viscosity of the diluent is preferably lower than the viscosity of the culture medium contained in the culture vessel 10.
　濾過部３０Ｂは、濾過部３０Ａに対して、細胞懸濁液の流れ方向の下流側に設けられている。濾過部３０Ａ及び３０Ｂにおける濾過処理は、濾過膜を用いた膜分離処理によって行われる。濾過部３０Ａ及び３０Ｂにおける膜分離の方式は、タンジェンシャルフロー方式であることが好ましい。本実施形態において、濾過部３０Ａの濾過膜の孔径は、濾過部３０Ｂの濾過膜の孔径よりも小さくなっている。 The filtration unit 30B is provided downstream of the filtration unit 30A in the flow direction of the cell suspension. The filtration process in the filtration units 30A and 30B is performed by a membrane separation process using a filtration membrane. The method of membrane separation in the filtration units 30A and 30B is preferably a tangential flow method. In the present embodiment, the pore size of the filtration membrane of the filtration unit 30A is smaller than the pore size of the filtration membrane of the filtration unit 30B.
　濾過部３０Ａは、分割部２０における分割処理によって発生する単一細胞、死細胞等のデブリスと、デブリスよりもサイズの大きい細胞凝集体とを分離する。濾過部３０Ａの濾過膜を透過したデブリスは、廃液容器３１に収容される。一方、濾過部３０Ａの濾過膜によって阻止された細胞凝集体は、濾過部３０Ｂに移送される。 The filtration unit 30A separates debris, such as single cells and dead cells, generated by the division processing in the division unit 20, and cell aggregates larger in size than the debris. The debris that has passed through the filtration membrane of the filtration unit 30A is stored in the waste liquid container 31. On the other hand, the cell aggregate blocked by the filtration membrane of the filtration unit 30A is transferred to the filtration unit 30B.
　濾過部３０Ｂは、濾過部３０Ａから移送された細胞凝集体を、相対的にサイズが大きい細胞凝集体と、相対的にサイズが小さい細胞凝集体とに分離する。濾過部３０Ｂの濾過膜を透過した相対的にサイズが小さい細胞凝集体は、モニタ部６０Ｃを経由して、回収容器１１Ｂに移送される。一方、濾過部３０Ｂの濾過膜によって阻止された相対的にサイズが大きい細胞凝集体は、モニタ部６０Ｂを経由して、回収容器１１Ａに移送される。 The filtration unit 30B separates the cell aggregate transferred from the filtration unit 30A into a cell aggregate having a relatively large size and a cell aggregate having a relatively small size. The cell aggregate having a relatively small size that has passed through the filtration membrane of the filtration unit 30B is transferred to the collection container 11B via the monitor unit 60C. On the other hand, the relatively large cell aggregate blocked by the filtration membrane of the filtration unit 30B is transferred to the collection container 11A via the monitor unit 60B.
　なお、濾過部３０Ａにおいて実施される膜分離処理は、開示の技術における第１の膜分離処理の一例であり、濾過部３０Ｂにおいて実施される膜分離処理は、開示の技術における第２の膜分離処理の一例である。濾過部３０Ａの濾過膜は、開示の技術における第１の濾過膜の一例であり、濾過部３０Ｂの濾過膜は、開示の技術における第２の濾過膜の一例である。 The membrane separation process performed in the filtration unit 30A is an example of the first membrane separation process in the disclosed technology, and the membrane separation process performed in the filtration unit 30B is the second membrane separation process in the disclosed technology. It is an example of processing. The filtration membrane of the filtration unit 30A is an example of a first filtration membrane in the disclosed technology, and the filtration membrane of the filtration unit 30B is an example of a second filtration membrane in the disclosed technology.
　モニタ部６０Ａは、流路７０の、培養容器１０と分割部２０との間の区間Ｘ１に設けられている。モニタ部６０Ｂは、流路７０の、濾過部３０Ｂと回収容器１１Ａとの間の区間Ｘ２に設けられている。モニタ部６０Ｃは、流路７０の、濾過部３０Ｂと回収容器１１Ｂとの間の区間Ｘ３に設けられている。モニタ部６０Ａ、６０Ｂ及び６０Ｃは、それぞれ、区間Ｘ１、Ｘ２及びＸ３を通過する細胞懸濁液をモニタする。モニタ部６０Ａ、６０Ｂ、６０Ｃは、それぞれ、フローセル６１及び撮像装置６２を含んで構成されている。 The monitor unit 60 </ b> A is provided in a section X <b> 1 of the flow channel 70 between the culture vessel 10 and the division unit 20. The monitor unit 60B is provided in a section X2 of the flow path 70 between the filtration unit 30B and the collection container 11A. The monitor unit 60C is provided in a section X3 of the flow path 70 between the filtration unit 30B and the collection container 11B. The monitoring units 60A, 60B and 60C monitor the cell suspension passing through the sections X1, X2 and X3, respectively. Each of the monitor units 60A, 60B, and 60C includes a flow cell 61 and an imaging device 62.
　流路７０は、希釈液収容容器４１に収容されている希釈液が、モニタ部６０Ａを通過した細胞懸濁液に合流できるように構成されている。また、流路７０は、分割部２０から濾過部３０Ａへの細胞懸濁液の移送、濾過部３０Ａから濾過部３０Ｂ及び廃液容器３１への細胞懸濁液の移送、濾過部３０Ｂから回収容器１１Ａ及び１１Ｂへの細胞懸濁液の移送を可能とするように構成されている。また、流路７０は、培地収容容器４０に収容されている培地が、濾過部３０Ｂによって分離された各成分に合流できるように構成されている。 The channel 70 is configured such that the diluent stored in the diluent storage container 41 can join the cell suspension that has passed through the monitor unit 60A. In addition, the flow path 70 transfers the cell suspension from the dividing section 20 to the filtering section 30A, transfers the cell suspension from the filtering section 30A to the filtering section 30B and the waste liquid container 31, and transfers the cell suspension from the filtering section 30B to the collecting vessel 11A. And 11B to allow the transfer of the cell suspension. The flow path 70 is configured so that the medium contained in the medium container 40 can be combined with the components separated by the filtration unit 30B.
　図８は、細胞培養装置１Ａにおいて細胞培養を行う場合に、制御部５０において実施される処理の流れの一例を示すフローチャートである。 FIG. 8 is a flowchart illustrating an example of a flow of processing performed by the control unit 50 when performing cell culture in the cell culture device 1A.
　ステップＳ２１において、制御部５０は、ポンプＰ１を駆動させることで、培養容器１０に収容されている細胞懸濁液をモニタ部６０Ａに移送する。モニタ部６０Ａは、流路７０の区間Ｘ１を通過する細胞懸濁液をモニタする。すなわち、撮像装置６２は、フローセル６１を通過する細胞懸濁液に含まれる細胞（細胞凝集体）を連続的に撮像する。 In step S21, the control unit 50 drives the pump P1 to transfer the cell suspension contained in the culture container 10 to the monitor unit 60A. The monitoring unit 60A monitors the cell suspension passing through the section X1 of the channel 70. That is, the imaging device 62 continuously images cells (cell aggregates) contained in the cell suspension passing through the flow cell 61.
　ステップＳ２２において、制御部５０は、モニタ部６０Ａの撮像装置６２によって撮像された細胞の画像を取得する。 In step S22, the control unit 50 acquires an image of the cell captured by the imaging device 62 of the monitor unit 60A.
　ステップＳ２３において、制御部５０は、ステップＳ２２において取得した画像に基づいて、分割処理の要否を判断する。制御部５０は、分割処理が必要であると判断した場合、処理をステップＳ２４に移行し、分割処理が必要ではないと判断した場合、処理をステップＳ２５に移行する。 In step S23, the control unit 50 determines whether the division processing is necessary based on the image acquired in step S22. When the control unit 50 determines that the division process is necessary, the process proceeds to step S24, and when it determines that the division process is not necessary, the process proceeds to step S25.
　制御部５０は、分割処理が必要であると判断した場合、ステップＳ２４において、バルブＶ１、Ｖ２を開状態、バルブＶ３、Ｖ４を閉状態に制御することで、細胞懸濁液を分割部２０に移送する。ステップＳ２４と並行して実施されるステップＳ２４ａにおいて制御部５０は、ポンプＰ５を駆動することで、希釈液収容容器４１に収容されている希釈液を希釈液収容容器４１から抜き出し、希釈液と、分割部２０に向けて流れる細胞懸濁液とを流路７０上で合流させ、これらを混合する。分割処理前の細胞懸濁液には、比較的サイズが大きい細胞凝集体を多く含むため、分割処理前の細胞懸濁液は、細胞濃度が過度に高まっていることが想定される。分割処理前の細胞懸濁液と希釈液とを混合することで、分割処理前の細胞懸濁液における細胞濃度を適正な濃度することができる。また、分割処理前の細胞懸濁液と希釈液とを混合することで、分割処理前の細胞懸濁液の粘度を低下させることができ、これにより、分割部２０のメッシュの目詰まりの発生を抑制することができる。 When the control unit 50 determines that the dividing process is necessary, in step S24, the valves V1 and V2 are opened and the valves V3 and V4 are closed so that the cell suspension is transferred to the dividing unit 20. Transfer. In step S24a performed in parallel with step S24, the control unit 50 drives the pump P5 to extract the diluent stored in the diluent storage container 41 from the diluent storage container 41, and The cell suspension flowing toward the dividing section 20 and the cell suspension are combined on the flow path 70, and they are mixed. Since the cell suspension before the splitting treatment contains many relatively large cell aggregates, it is assumed that the cell suspension before the splitting treatment has an excessively high cell concentration. By mixing the cell suspension before the division treatment with the diluent, the cell concentration in the cell suspension before the division treatment can be adjusted to an appropriate concentration. In addition, by mixing the cell suspension before the division processing and the diluent, the viscosity of the cell suspension before the division processing can be reduced, thereby causing clogging of the mesh of the division unit 20. Can be suppressed.
　分割部２０に移送された細胞懸濁液に含まれる細胞凝集体は、分割部２０のメッシュ２１０（図４Ａ、図４Ｂ、図４Ｃ参照）を通過することにより、サイズのより小さい細胞凝集体に分割される。分割された細胞凝集体を含む細胞懸濁液は、ステップＳ２５において濾過部３０Ａに移送される。一方、制御部５０は、分割処理が必要ではないと判断した場合、ステップＳ２５において、バルブＶ３、Ｖ４を開状態、バルブＶ１、Ｖ２を閉状態に制御することで、細胞懸濁液を分割部２０を経由させることなく濾過部３０Ａに移送する。 The cell aggregates contained in the cell suspension transferred to the dividing unit 20 pass through the mesh 210 of the dividing unit 20 (see FIGS. 4A, 4B, and 4C) to form smaller cell aggregates. Divided. The cell suspension containing the divided cell aggregates is transferred to the filtration unit 30A in step S25. On the other hand, when the control unit 50 determines that the dividing process is not necessary, in step S25, the valves V3 and V4 are opened and the valves V1 and V2 are closed so that the cell suspension is divided. It is transferred to the filtration unit 30A without passing through the filter unit 20.
　濾過部３０Ａは、移送された細胞懸濁液に含まれる、単一細胞、死細胞等のデブリスと、デブリスよりもサイズが大きい細胞凝集体とを分離する。 The filtration unit 30A separates debris, such as single cells and dead cells, contained in the transferred cell suspension, and cell aggregates larger in size than the debris.
　ステップＳ２６において、制御部５０は、ポンプＰ２を駆動させることで、濾過部３０Ａの濾過膜を透過したデブリスを廃液容器３１に移送する。濾過部３０Ａの濾過膜によって阻止された細胞凝集体を含む細胞懸濁液は、濾過部３０Ｂに移送される。濾過部３０Ｂは、濾過部３０Ａから移送された細胞凝集体を、相対的にサイズが大きい細胞凝集体と、相対的にサイズが小さい細胞凝集体とに分離する。 In step S26, the control unit 50 drives the pump P2 to transfer the debris that has passed through the filtration membrane of the filtration unit 30A to the waste liquid container 31. The cell suspension containing the cell aggregate blocked by the filtration membrane of the filtration unit 30A is transferred to the filtration unit 30B. The filtration unit 30B separates the cell aggregate transferred from the filtration unit 30A into a relatively large cell aggregate and a relatively small cell aggregate.
　ステップＳ２７において、制御部５０は、ポンプＰ３ａ及びＰ３ｂを駆動させることで、濾過部３０Ｂの濾過膜によって阻止された相対的にサイズが大きい細胞凝集体を含む細胞懸濁液をモニタ部６０Ｂに移送し、濾過部３０Ｂの濾過膜を透過した相対的にサイズが小さい細胞凝集体を含む細胞懸濁液をモニタ部６０Ｃに移送する。 In step S27, the control unit 50 drives the pumps P3a and P3b to transfer the cell suspension containing the relatively large cell aggregate blocked by the filtration membrane of the filtration unit 30B to the monitoring unit 60B. Then, the cell suspension containing the cell aggregate having a relatively small size that has passed through the filtration membrane of the filtration unit 30B is transferred to the monitor unit 60C.
　ステップＳ２７と並行して実施されるステップＳ２８において、制御部５０は、ポンプＰ４ａ及びＰ４ｂを駆動させ、バルブＶ５及びＶ７を開状態、バルブＶ６及びＶ８を閉状態に制御することで、培地収容容器４０に収容されている新鮮な培地を培地収容容器４０から抜き出し、この培地と、濾過部３０Ｂからモニタ部６０Ｂ及び６０Ｃの各々に向けて流れる細胞懸濁液とを流路７０上で合流させ、これらを混合する。かかる混合処理により、相対的にサイズが大きい細胞凝集体を含む細胞懸濁液と、培地収容容器４０から移送された培地とを混合した第１の混合液と、相対的にサイズが小さい細胞凝集体を含む細胞懸濁液と、培地収容容器４０から移送された培地とを混合した第２の混合液とが生成される。 In step S28, which is performed in parallel with step S27, the control unit 50 drives the pumps P4a and P4b to control the valves V5 and V7 to open and the valves V6 and V8 to close, so that the culture medium container The fresh medium contained in 40 is extracted from the medium containing container 40, and this medium and the cell suspension flowing from the filtration unit 30B to each of the monitor units 60B and 60C are merged on the channel 70, Mix these. By the mixing process, the first mixed solution obtained by mixing the cell suspension containing the cell aggregate having a relatively large size and the medium transferred from the medium storage container 40 is mixed with the cell mixture having a relatively small size. A second liquid mixture in which the cell suspension containing the aggregate and the medium transferred from the medium storage container 40 are mixed is generated.
　モニタ部６０Ｂは、流路７０の区間Ｘ２を通過する第１の混合液をモニタし、モニタ部６０Ｃは、流路７０の区間Ｘ３を通過する第２の混合液をモニタする。 The monitor unit 60B monitors the first mixed liquid passing through the section X2 of the flow path 70, and the monitor unit 60C monitors the second mixed liquid passing through the section X3 of the flow path 70.
　ステップＳ２９において、制御部５０は、モニタ部６０Ｂ及び６０Ｃの撮像装置６２によって撮像された細胞の画像を取得する。 In step S29, the control unit 50 acquires an image of a cell captured by the imaging device 62 of the monitor units 60B and 60C.
　ステップＳ３０において、制御部５０は、ステップＳ２９において取得した画像に基づいて、第１の混合液及び第２の混合液の各々について、培地の補充が必要であるか否かを判断する。制御部５０は、第１の混合液及び第２の混合液の少なくとも一方について、培地の補充が必要であると判断した場合、処理をステップＳ３１に移行し、培地の補充が必要ではないと判断した場合、処理を終了させる。 In step S30, the control unit 50 determines whether the replenishment of the culture medium is necessary for each of the first mixed liquid and the second mixed liquid based on the image acquired in step S29. When determining that at least one of the first mixture and the second mixture requires replenishment of the medium, the control unit 50 shifts the processing to step S31 and determines that the replenishment of the medium is not necessary. If so, the process ends.
　制御部５０は、第１の混合液及び第２の混合液の少なくとも一方について培地の補充が必要であると判断した場合、ステップＳ３１において、当該混合液の細胞濃度を所定の濃度とするための培地の補充量を導出する。 When the controller 50 determines that at least one of the first mixture and the second mixture requires replenishment of the culture medium, in step S31, the controller 50 sets the cell concentration of the mixture to a predetermined concentration. Deriving the replenishment amount of the medium.
　第１の混合液に培地を補充する場合、ステップＳ３２において、制御部５０は、ポンプＰ４ａを駆動させ、バルブＶ６を開状態に制御することで、培地収容容器４０に収容されている新鮮な培地をステップＳ３１において導出した補充量に相当する分量だけ抜き出し、抜き出した培地と、モニタ部６０Ｂを通過した第１の混合液とを流路７０上で合流させる。第２の混合液に培地を補充する場合、ステップＳ３２において、制御部５０は、ポンプＰ４ｂを駆動させ、バルブＶ８を開状態に制御することで、培地収容容器４０に収容されている新鮮な培地をステップＳ３１において導出した補充量に相当する分量だけ抜き出し、抜き出した培地と、モニタ部６０Ｃを通過した第２の混合液とを流路７０上で合流させる。これにより、当該混合液に、新鮮な培地が補充され、当該混合液における細胞濃度が適正な濃度に調整される。上記のように培地の補充を行う場合、バルブＶ５及びＶ７は開状態に維持される。すなわち、濾過部３０Ａ及び３０Ｂにおいてデブリスが除去された細胞懸濁液と培地収容容器４０に収容されている培地とを混合して混合液を得る混合処理と、培地収容容器４０に収容されている培地を上記混合液に補充して細胞濃度を調整する濃度調整処理が並行して行われる。細胞濃度の調整は、例えば、バルブＶ５～Ｖ８の開度を制御することで実現することができる。適正な濃度に調整された第１の混合液は、回収容器１１Ａに移送され、適正な濃度に調整された第２の混合液は、回収容器１１Ｂに移送される。なお、本実施形態においては、バルブＶ５及びＶ６を通過する培地の送液を、共通の培地収容容器４０及び共通のポンプＰ４ａを用いて行う場合を例示しているが、別々の培地収容容器及びポンプを用いることも可能である。同様に、バルブＶ７及びＶ８を通過する培地の送液を、共通の培地収容容器４０及び共通のポンプＰ４ｂを用いて行う場合を例示しているが、別々の培地収容容器及びポンプを用いることも可能である。 When replenishing the first mixed solution with the culture medium, in step S32, the control unit 50 drives the pump P4a and controls the valve V6 to be in the open state, whereby the fresh culture medium stored in the culture medium storage container 40 is controlled. Is extracted by an amount corresponding to the replenishment amount derived in step S31, and the extracted medium and the first mixed solution that has passed through the monitor unit 60B are combined on the flow path 70. When replenishing the second mixed solution with the culture medium, in step S32, the control unit 50 drives the pump P4b and controls the valve V8 to the open state, so that the fresh culture medium stored in the culture medium storage container 40 is opened. Is extracted by an amount corresponding to the replenishment amount derived in step S31, and the extracted medium and the second mixed solution that has passed through the monitor unit 60C are combined on the flow path 70. As a result, the mixture is supplemented with fresh medium, and the cell concentration in the mixture is adjusted to an appropriate concentration. When the medium is replenished as described above, the valves V5 and V7 are kept open. That is, the cell suspension from which debris has been removed in the filtration units 30A and 30B is mixed with the culture medium stored in the culture medium container 40 to obtain a mixed solution, and the mixture is stored in the culture medium container 40. A concentration adjusting process for adjusting the cell concentration by replenishing the medium with the mixed solution is performed in parallel. The adjustment of the cell concentration can be realized, for example, by controlling the degree of opening of the valves V5 to V8. The first mixed solution adjusted to the appropriate concentration is transferred to the collection container 11A, and the second mixed solution adjusted to the appropriate concentration is transferred to the collection container 11B. In addition, in this embodiment, the case where the supply of the culture medium passing through the valves V5 and V6 is performed using the common culture medium storage container 40 and the common pump P4a is illustrated. It is also possible to use a pump. Similarly, the case where the feeding of the culture medium passing through the valves V7 and V8 is performed using the common culture medium storage container 40 and the common pump P4b is illustrated, but it is also possible to use separate culture medium storage containers and pumps. It is possible.
　開示の技術の第２の実施形態に係る細胞培養装置１Ａによれば、第１の実施形態に係る細胞培養装置１と同様、回収容器１１Ａ及び１１Ｂに回収される細胞懸濁液への不要成分（デブリス）の混入が抑制される。これにより、回収容器１１Ａ及び１１Ｂ内でそれぞれ培養される細胞の増殖率及び生存率を高めることができる。 According to the cell culture device 1A according to the second embodiment of the disclosed technology, similarly to the cell culture device 1 according to the first embodiment, unnecessary components in the cell suspension collected in the collection containers 11A and 11B. (Debris) is suppressed. Thereby, the growth rate and the survival rate of the cells cultured in the collection containers 11A and 11B, respectively, can be increased.
　また、分割処理後の細胞懸濁液においては、当該細胞懸濁液に含まれる成分の相互間のサイズ差が小さくなるため、分割処理後に濾過処理を実施した場合には、濾過処理における分離性が低下する。しかしながら、本実施形態に係る細胞培養装置１Ａによれば、互いに孔径が異なる濾過膜を備えた２つの濾過部３０Ａ及び３０Ｂによって、２段階の濾過処理を行うことで、濾過処理における分離性を高めることができる。また、後段の濾過部３０Ｂの濾過膜を透過した成分には、その後の培養で増殖を見込める細胞が多く含まれている可能性もあるため、濾過部３０Ｂの濾過膜によって阻止された成分のみならず、濾過部３０Ｂの濾過膜を透過した成分をも回収することで、細胞のロスを少なくすることができる。また、濾過部３０Ｂの濾過膜によって阻止されたサイズが相対的に大きい細胞凝集体と、濾過部３０Ｂの濾過膜を透過したサイズが相対的に小さい細胞凝集体とを別々の回収容器１１Ａ、１１Ｂに回収することで、各サイズの細胞凝集体に適した環境で培養を行うことができ、細胞の生存率及び増殖率を向上させることができる。 Further, in the cell suspension after the division treatment, since the size difference between the components contained in the cell suspension becomes small, when the filtration treatment is performed after the division treatment, the separation property in the filtration treatment is reduced. Decrease. However, according to the cell culture device 1A according to the present embodiment, the two-stage filtration is performed by the two filtration units 30A and 30B having the filtration membranes having different pore sizes from each other, thereby improving the separation property in the filtration. be able to. In addition, since the components that have passed through the filtration membrane of the subsequent filtration unit 30B may contain many cells that can be expected to proliferate in the subsequent culture, if only the components blocked by the filtration membrane of the filtration unit 30B are used, In addition, by recovering the components that have passed through the filtration membrane of the filtration unit 30B, cell loss can be reduced. Separate collection vessels 11A and 11B separate the cell aggregate having a relatively large size blocked by the filtration membrane of the filtration unit 30B and the cell aggregate having a relatively small size transmitted through the filtration membrane of the filtration unit 30B. By collecting the cells in a suitable size, the cells can be cultured in an environment suitable for the cell aggregate of each size, and the survival rate and proliferation rate of the cells can be improved.
　なお、本実施形態では、補充用の培地と第１及び第２の混合液とを流路７０上で合流させた後に、これらをそれぞれ回収容器１１Ａ及び１１Ｂに移送する場合を例示したが、濃度調整前の第１及び第２の混合液を、それぞれ回収容器１１Ａ及び１１Ｂに移送した後に、補充用の培地を回収容器１１Ａ及び１１Ｂに移送してもよい。 Note that, in the present embodiment, a case has been exemplified in which, after the replenishment medium and the first and second mixed liquids are merged on the flow path 70, they are transferred to the collection containers 11A and 11B, respectively. After transferring the first and second mixed liquids before the adjustment to the collection containers 11A and 11B, respectively, the supplemental medium may be transferred to the collection containers 11A and 11B.
　また、図７には、濾過部３０Ｂの濾過膜によって阻止された成分を回収容器１１Ａに回収する場合の構成が例示されているが、図９に示すように、細胞培養装置１Ａは、濾過部３０Ｂの濾過膜によって阻止された成分が培養容器１０に回収されるように構成されていてもよい。 FIG. 7 illustrates a configuration in which components blocked by the filtration membrane of the filtration unit 30B are collected in the collection container 11A. As shown in FIG. 9, the cell culture device 1A includes a filtration unit. The components blocked by the 30B filtration membrane may be collected in the culture vessel 10.
　また、細胞培養装置１Ａは、図１０に示すように、濾過部３０Ａと濾過部３０Ｂとの間に配置された流路に接続された希釈液収容器４２を含んでいてもよい。濾過部３０Ａの濾過膜によって阻止された細胞懸濁液は、希釈液収容容器４２に収容されている希釈液と混合され、濾過部３０Ｂに移送される。濾過部３０Ａの濾過膜によって阻止された細胞懸濁液は、細胞濃度が過度に高まっていることが想定される。濾過部３０Ａの濾過膜によって阻止された細胞懸濁液と希釈液収容容器４２に収容されている希釈液とを混合することで、濾過部３０Ｂに移送される細胞懸濁液における細胞濃度を適正な濃度することができる。 Also, as shown in FIG. 10, the cell culture device 1A may include a diluent container 42 connected to a flow path disposed between the filtration unit 30A and the filtration unit 30B. The cell suspension blocked by the filtration membrane of the filtration unit 30A is mixed with the diluent stored in the diluent storage container 42 and transferred to the filtration unit 30B. It is assumed that the cell concentration of the cell suspension blocked by the filtration membrane of the filtration unit 30A is excessively high. By mixing the cell suspension blocked by the filtration membrane of the filtration unit 30A with the diluent stored in the diluent storage container 42, the cell concentration in the cell suspension transferred to the filtration unit 30B can be adjusted appropriately. High concentration.
　濃縮分離部として、２つの濾過部３０Ａ、３０Ｂを例示したが、この態様に限定されない。濃縮分離部は、２つの遠心分離機構を含んで構成されていてもよい。すなわち、濃縮分離部は、細胞懸濁液の成分を、第１の条件による遠心分離により分離する第１の遠心分離機構と、第１の遠心分離機構により処理された細胞懸濁液の成分を、第１の条件とは異なる第２の条件による遠心分離により分離する第２の遠心分離機構と、を含んでいてもよい。遠心分離の条件としては、例えば、遠心分離機構が発生させる遠心力及び遠心分離処理の処理時間などが挙げられる。 (4) Although the two filtration units 30A and 30B are illustrated as the concentration separation unit, the present invention is not limited to this embodiment. The concentration and separation unit may include two centrifugal separation mechanisms. That is, the concentration / separation unit separates the components of the cell suspension by a first centrifugation mechanism that separates the components of the cell suspension by centrifugation under the first condition, and the components of the cell suspension processed by the first centrifugation mechanism. And a second centrifugal separation mechanism for separating by centrifugation under a second condition different from the first condition. The conditions for centrifugation include, for example, the centrifugal force generated by the centrifugal separation mechanism and the processing time of centrifugation.

Claims (20)

  1. 　細胞凝集体を分割する分割工程と、
    　前記分割工程において分割された細胞凝集体を含む細胞懸濁液の成分を分離して細胞懸濁液における細胞の濃度を濃縮する濃縮分離工程と、
    　前記濃縮分離工程を経た細胞懸濁液と培地とを混合した混合液を得る混合工程と、
    　前記混合液における細胞の濃度を測定する測定工程と、
    　前記測定工程の後に、前記混合液を回収する回収工程と、
    　を含む細胞培養方法。     A dividing step of dividing the cell aggregate,
    A concentration separation step of separating the components of the cell suspension containing the cell aggregates divided in the division step and concentrating the concentration of cells in the cell suspension,
    A mixing step of obtaining a mixed solution obtained by mixing the cell suspension and the medium after the concentration separation step,
    A measurement step of measuring the concentration of cells in the mixture,
    After the measuring step, a collecting step of collecting the mixed solution,
    A cell culture method comprising:
  2. 　前記測定工程において測定された濃度に基づいて、前記混合液に培地を補充する補充工程を更に含む
    　請求項１に記載の細胞培養方法。     The cell culture method according to claim 1, further comprising a replenishing step of replenishing the mixture with a medium based on the concentration measured in the measuring step.
  3. 　前記濃縮分離工程は、前記分割工程において分割された細胞凝集体を含む細胞懸濁液を膜分離する膜分離処理を含む
    　請求項１または請求項２に記載の細胞培養方法。     The cell culture method according to claim 1, wherein the concentration separation step includes a membrane separation process of membrane separating a cell suspension containing the cell aggregates divided in the division step.
  4. 　前記膜分離処理は、
    　前記分割工程において分割された細胞凝集体を含む細胞懸濁液を第１の濾過膜を用いて膜分離する第１の膜分離処理と、
    　前記第１の濾過膜によって阻止された成分を、第２の濾過膜を用いて膜分離する第２の膜分離処理と、
    　を含む請求項３に記載の細胞培養方法。     The membrane separation process,
    A first membrane separation process of membrane-separating the cell suspension containing the cell aggregates divided in the division step using a first filtration membrane;
    A second membrane separation process for separating the components blocked by the first filtration membrane using a second filtration membrane;
    The cell culture method according to claim 3, comprising:
  5. 　前記第１の濾過膜の孔径は、前記第２の濾過膜の孔径よりも小さい
    　請求項４に記載の細胞培養方法。     The cell culture method according to claim 4, wherein the pore size of the first filtration membrane is smaller than the pore size of the second filtration membrane.
  6. 　前記回収工程において、前記第２の濾過膜によって阻止された成分と、前記第２の濾過膜を透過した成分とを別々に回収する
    　請求項４または請求項５に記載の細胞培養方法。     The cell culture method according to claim 4, wherein, in the collecting step, a component blocked by the second filtration membrane and a component that has passed through the second filtration membrane are separately collected.
  7. 　前記第１の濾過膜によって阻止された成分と希釈液とを混合したものを前記第２の濾過膜を用いて膜分離する
    　請求項４から請求項６のいずれか１項に記載の細胞培養方法。     The cell culture method according to any one of claims 4 to 6, wherein a mixture of a component blocked by the first filtration membrane and a diluent is subjected to membrane separation using the second filtration membrane. .
  8. 　前記濃縮分離工程は、前記分割工程において分割された細胞凝集体を含む細胞懸濁液の成分を遠心分離により分離する遠心分離処理を含む
    　請求項１または請求項２に記載の細胞培養方法。     The cell culture method according to claim 1, wherein the concentration and separation step includes a centrifugal separation process of separating, by centrifugation, components of the cell suspension containing the cell aggregates divided in the division step.
  9. 　前記遠心分離処理は、
    　前記分割工程において分割された細胞凝集体を含む細胞懸濁液の成分を、第１の条件による遠心分離により分離する第１の遠心分離処理と、
    　前記第１の遠心分離処理が施された細胞懸濁液の成分を、前記第１の条件とは異なる第２の条件による遠心分離により分離する第２の遠心分離処理と、
    　を含む請求項８に記載の細胞培養方法。     The centrifugation process,
    A first centrifugation process of separating the components of the cell suspension containing the cell aggregates divided in the division step by centrifugation under the first condition;
    A second centrifugation process for separating the components of the cell suspension subjected to the first centrifugation process by centrifugation under a second condition different from the first condition;
    The cell culture method according to claim 8, comprising:
  10. 　前記分割工程において分割される前の細胞凝集体を含む細胞懸濁液に希釈液を添加する希釈工程を更に含む
    　請求項１から請求項９のいずれか１項に記載の細胞培養方法。     The cell culture method according to any one of claims 1 to 9, further comprising a dilution step of adding a diluent to a cell suspension containing cell aggregates before the division in the division step.
  11. 　細胞凝集体を分割する分割部と、
    　細胞懸濁液の成分を分離して細胞懸濁液における細胞の濃度を濃縮する濃縮分離部と、
    　培地を収容するための培地収容容器と、
    　細胞懸濁液をモニタするモニタ部と、
    　前記分割部、前記濃縮分離部、前記培地収容容器及び前記モニタ部に接続された流路と、
    　前記流路を介した細胞懸濁液及び培地の移送を制御する制御部と、
    　を含み、
    　前記制御部は、
    　前記分割部において分割された細胞凝集体を含む細胞懸濁液を前記濃縮分離部に移送し、
    　前記濃縮分離部において濃縮された細胞懸濁液と前記培地収容容器に収容されている培地とを混合した混合液を前記モニタ部に移送し、
    　前記モニタ部によりモニタされた前記混合液を前記流路に接続された容器に移送する
    　細胞培養装置。     A dividing unit for dividing the cell aggregate,
    A concentration separation unit that separates components of the cell suspension and concentrates the concentration of cells in the cell suspension,
    A medium storage container for storing a medium,
    A monitor for monitoring the cell suspension;
    A flow path connected to the dividing unit, the concentration / separation unit, the culture medium container and the monitor unit,
    A control unit that controls the transfer of the cell suspension and the culture medium through the flow path,
    Including
    The control unit includes:
    Transfer the cell suspension containing the cell aggregates divided in the dividing section to the concentration separation section,
    Transfer the mixed solution obtained by mixing the cell suspension concentrated in the concentration separation unit and the medium contained in the medium containing container to the monitor unit,
    A cell culture device for transferring the mixed solution monitored by the monitor unit to a container connected to the flow path.
  12. 　前記流路に接続された培養容器を更に含み、
    　前記制御部は、前記培養容器に収容されている細胞凝集体を含む細胞懸濁液を前記分割部に移送し、前記モニタ部によりモニタされた前記混合液を前記培養容器に移送する
    　請求項１１に記載の細胞培養装置。     Further comprising a culture vessel connected to the flow path,
    The said control part transfers the cell suspension containing the cell aggregate accommodated in the said culture container to the said division | segmentation part, and transfers the said mixed liquid monitored by the said monitor part to the said culture container. The cell culture device according to item 1.
  13. 　前記流路に接続された培養容器と、
    　前記流路に接続された回収容器と、
    　を更に含み、
    　前記制御部は、前記培養容器に収容されている細胞凝集体を含む細胞懸濁液を前記分割部に移送し、前記モニタ部によりモニタされた前記混合液を前記回収容器に移送する
    　請求項１１に記載の細胞培養装置。     A culture vessel connected to the flow path,
    A collection container connected to the flow path,
    Further comprising
    The said control part transfers the cell suspension containing the cell aggregate accommodated in the said culture container to the said division | segmentation part, and transfers the said mixed liquid monitored by the said monitor part to the said collection container. The cell culture device according to item 1.
  14. 　前記制御部は、前記モニタ部によるモニタの結果に基づいて導出された前記混合液に含まれる細胞の濃度に基づいて、前記培地収容容器に収容されている培地を前記混合液に補充する
    　請求項１１から請求項１３のいずれか１項に記載の細胞培養装置。     The control unit replenishes the mixed solution with a medium contained in the medium containing container based on a concentration of cells contained in the mixed solution derived based on a result of monitoring by the monitor unit. The cell culture device according to any one of claims 11 to 13.
  15. 　前記濃縮分離部は、細胞懸濁液を膜分離する濾過膜を備えた濾過部を含む
    　請求項１１から請求項１４のいずれか１項に記載の細胞培養装置。     The cell culture device according to any one of claims 11 to 14, wherein the concentration / separation unit includes a filtration unit provided with a filtration membrane for membrane-separating the cell suspension.
  16. 　前記濃縮分離部は、
    　細胞懸濁液を膜分離する第１の濾過膜を備えた第１の濾過部と、
    　前記第１の濾過膜によって阻止された成分を膜分離する第２の濾過膜を備えた第２の濾過部と、
    　を含む請求項１５に記載の細胞培養装置。     The concentration separation section,
    A first filtration unit including a first filtration membrane for membrane-separating the cell suspension;
    A second filtration unit comprising a second filtration membrane for membrane-separating components blocked by the first filtration membrane;
    The cell culture device according to claim 15, comprising:
  17. 　前記第１の濾過膜の孔径は、前記第２の濾過膜の孔径よりも小さい
    　請求項１６に記載の細胞培養装置。     The cell culture device according to claim 16, wherein the pore size of the first filtration membrane is smaller than the pore size of the second filtration membrane.
  18. 　前記第２の濾過膜によって阻止された成分が収容される第１の回収容器と、
    　前記第２の濾過膜を透過した成分が収容される第２の回収容器と、
    　を含む
    　請求項１６または請求項１７に記載の細胞培養装置。     A first collection container containing a component blocked by the second filtration membrane;
    A second collection container in which a component that has passed through the second filtration membrane is stored;
    The cell culture device according to claim 16 or 17, comprising:
  19. 　前記濃縮分離部は、細胞懸濁液の成分を遠心分離により分離する遠心分離機構を有する
    　請求項１１から請求項１４のいずれか１項に記載の細胞培養装置。     The cell culture device according to any one of claims 11 to 14, wherein the concentration and separation unit has a centrifugal separation mechanism that separates components of the cell suspension by centrifugation.
  20. 　前記濃縮分離部は、
    　細胞懸濁液の成分を、第１の条件による遠心分離により分離する第１の遠心分離機構と、
    　前記第１の遠心分離機構により処理された細胞懸濁液の成分を、前記第１の条件とは異なる第２の条件による遠心分離により分離する第２の遠心分離機構と、
    　を含む請求項１９に記載の細胞培養装置。     The concentration separation section,
    A first centrifugation mechanism for separating components of the cell suspension by centrifugation under the first condition;
    A second centrifugal separation mechanism that separates components of the cell suspension processed by the first centrifugal separation mechanism by centrifugation under a second condition different from the first condition;
    The cell culture device according to claim 19, comprising:
PCT/JP2019/030859 2018-08-27 2019-08-06 Cell culture method and cell culture device WO2020044984A1 (en)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
JP2018-158598 2018-08-27
JP2018158598 2018-08-27

Publications (1)

Publication Number Publication Date
WO2020044984A1 true WO2020044984A1 (en) 2020-03-05

Family

ID=69643538

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/JP2019/030859 WO2020044984A1 (en) 2018-08-27 2019-08-06 Cell culture method and cell culture device

Country Status (1)

Country Link
WO (1) WO2020044984A1 (en)

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2010526530A (en) * 2007-05-04 2010-08-05 スンドストローム，エリック Slicing device
JP2016131538A (en) * 2015-01-20 2016-07-25 富士フイルム株式会社 Cell culture apparatus and cell culture method
WO2017038887A1 (en) * 2015-08-31 2017-03-09 アイ・ピース株式会社 Pluripotent stem cell production system
WO2017191775A1 (en) * 2016-05-06 2017-11-09 富士フイルム株式会社 Method for subculturing pluripotent stem cells
WO2018003476A1 (en) * 2016-06-30 2018-01-04 富士フイルム株式会社 Cell-suspension membrane separation method and cell culture device

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2010526530A (en) * 2007-05-04 2010-08-05 スンドストローム，エリック Slicing device
JP2016131538A (en) * 2015-01-20 2016-07-25 富士フイルム株式会社 Cell culture apparatus and cell culture method
WO2017038887A1 (en) * 2015-08-31 2017-03-09 アイ・ピース株式会社 Pluripotent stem cell production system
WO2017191775A1 (en) * 2016-05-06 2017-11-09 富士フイルム株式会社 Method for subculturing pluripotent stem cells
WO2018003476A1 (en) * 2016-06-30 2018-01-04 富士フイルム株式会社 Cell-suspension membrane separation method and cell culture device

Similar Documents

Publication Publication Date Title
US11725174B2 (en) Cell culture apparatus and cell culture method
JP7102391B2 (en) Fast harvest with alternating tangential flow
WO2018003476A1 (en) Cell-suspension membrane separation method and cell culture device
CN106255542B (en) For the continuously ultra filtration unit of buffering or Medium Exchange from protein solution
WO2016117615A1 (en) Cell culture apparatus and cell culture method
JP2023182712A (en) Automated production and collection
JPH02168960A (en) Treating device for cell contained
CN112262209A (en) Cell culture system and cell culture method
WO2020044984A1 (en) Cell culture method and cell culture device
WO2020039911A1 (en) Cell culture method and cell culture device
JP2018070519A (en) Cell formulation production method and apparatus as well as cell formulation produced by production apparatus
JP7030849B2 (en) Cell culture device and cell culture method
JP6815618B1 (en) Culture system and culture method
US11685888B2 (en) Method for producing product
JP2019162035A (en) Cell treatment device
WO2021049117A1 (en) Cell culture device
US10232104B2 (en) Cell filter separation system
EP3893956B1 (en) Cell washing apparatus
US20230407229A1 (en) A method and system for configuring and/or setup of a downstream process for processing a biomass
CN117795053A (en) Method for producing biological products
JP2000032977A (en) Cultivation of cell and device therefor
SE527078C2 (en) Isolating protein fractions from skimmed milk by microfiltration, involves heating milk prior to microfiltration

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 19854210

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 19854210

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: JP