WO2022065400A1 - Cell culture apparatus and method for producing cell groups - Google Patents
Cell culture apparatus and method for producing cell groups Download PDFInfo
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- WO2022065400A1 WO2022065400A1 PCT/JP2021/034943 JP2021034943W WO2022065400A1 WO 2022065400 A1 WO2022065400 A1 WO 2022065400A1 JP 2021034943 W JP2021034943 W JP 2021034943W WO 2022065400 A1 WO2022065400 A1 WO 2022065400A1
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Classifications
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/483—Physical analysis of biological material
- G01N33/487—Physical analysis of biological material of liquid biological material
- G01N33/48707—Physical analysis of biological material of liquid biological material by electrical means
- G01N33/48735—Investigating suspensions of cells, e.g. measuring microbe concentration
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
- C12M1/00—Apparatus for enzymology or microbiology
- C12M1/02—Apparatus for enzymology or microbiology with agitation means; with heat exchange means
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
- C12M1/00—Apparatus for enzymology or microbiology
- C12M1/34—Measuring or testing with condition measuring or sensing means, e.g. colony counters
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
- C12M3/00—Tissue, human, animal or plant cell, or virus culture apparatus
- C12M3/02—Tissue, human, animal or plant cell, or virus culture apparatus with means providing suspensions
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
Definitions
- the present disclosure relates to a cell culture apparatus and a method for producing a cell group.
- the target sample for example, cells, humoral factors, etc.
- the target sample for example, cells, humoral factors, etc.
- the target sample for example, cells, humoral factors, etc.
- cells collected from a subject are grown in a medium, then undesired cells and impurities are separated from the medium, and the target cells and humoral factors are recovered and used. ..
- the cell culture process previously involved a lot of manual work. For example, during the culturing process, the suspension is collected at predetermined time intervals, and the cell concentration or the number of cells contained in the suspension is observed (measured) with a microscope or the like, so that the culturing proceeds smoothly.
- the confirmation work such as whether or not contamination such as germs has occurred, was mainly performed manually.
- the presence of frequent manual work in the culture process causes problems such as a decrease in the yield of cultured cells due to frequent collection of suspensions and an increase in the risk of contamination such as germs.
- various automation technologies have been proposed for the purpose of reducing such problems.
- Japanese Patent Application Laid-Open No. 2020-54234 discloses a technique for acquiring statistical data of cells (cell mass) contained in a suspension using an imaging unit.
- JP-A-2010-99011 and JP-A-2006-320226 when the cells are seeded, the distribution state (dispersion state) of the cells in the culture vessel is measured by using an image pickup device, and then the cells are seeded.
- a technique for controlling the swing of a culture vessel by a driving device according to the distribution state of cells is disclosed.
- suspension culture mass culture
- cell masses which are aggregates of a plurality of cells, are generated as the suspension culture progresses.
- the cells carried on the culture carrier proliferate on the culture carrier as the suspension culture progresses, so that the cells are cultured with the cells.
- the specific gravity of the cell complex with the carrier gradually increases. As a result, the cell mass and the cell complex gradually settle on the bottom surface of the culture vessel.
- a cell culture apparatus capable of suppressing the sedimentation of cell masses, cell complexes and other masses in a culture vessel and culturing cells with high yield and time efficiency. , And a method for producing a cell population.
- the cell culture apparatus includes a culture vessel containing at least a suspension containing cells and an adherend to which the cells adhere, a driving device for swinging the suspension, and the above-mentioned.
- a measuring device for measuring the mass distribution information value for the mass in at least a part of the region of the suspension, the rate of change of the mass distribution information value per predetermined unit time, and the rate of change.
- a control device for controlling the swing speed of the drive device based on the relationship with at least one set threshold value is provided.
- the cell culture apparatus is a culture containing a suspension containing at least one of a cell mass which is an aggregate of a plurality of cells and a cell complex which is a complex of the cells and a culture carrier. At least one of the cell distribution information value for the cell and the carrier distribution information value for the culture carrier in at least a part of the region of the suspension, the driving device for rocking the suspension, and the container is measured. Based on the relationship between the measuring device to be used, the rate of change of at least one of the cell distribution information value and the carrier distribution information value per predetermined unit time, and at least one or more thresholds set for the rate of change. , A control device for controlling the swing speed of the drive device.
- the measuring device is a first measuring device having an electrode for forming an electric field in at least a part of the region and a capacitance measuring unit for measuring the capacitance in the electric field.
- the first measuring device measures the cell distribution information value in at least a part of the region based on the capacitance.
- the first measuring apparatus further includes a conductivity measuring unit for measuring conductivity in at least a part of the region.
- the first measuring device further includes a pH measuring unit for measuring pH in at least a part of the region.
- the measuring device includes a second measuring device including an imaging unit that captures an image of at least a part of the region and acquires one or more images. The measuring device measures at least one of the cell distribution information value and the carrier distribution information value in the at least a part region based on the image.
- the cell distribution information value is the number of living cells or the concentration of living cells among the cells in the suspension in the at least a part of the region.
- the carrier distribution information value is the number of the culture carriers in the suspension or the concentration of the culture carriers in the at least a part of the region.
- the threshold value includes a first threshold value set to 0% with respect to the rate of change. Further, in the cell culture apparatus according to one aspect, the threshold value further has a second threshold value set to a value larger than 0% with respect to the rate of change.
- the control device when the control device determines that the rate of change is equal to or higher than the first threshold value, the control device increases the swing speed of the drive device, and the rate of change is the first. If it is determined to be less than one threshold value, the rocking speed of the driving device is reduced. Further, in the cell culture device according to one embodiment, when the control device continuously determines that the rate of change is less than the first threshold value over a plurality of the predetermined unit times, the control device shakes the drive device. The moving speed is set to 0. Further, in the cell culture device according to one aspect, when the rate of change is determined to be equal to or higher than the second threshold value, the control device maintains the swing speed at the time of the determination.
- the driving device is a stirrer or a shaker that stirs the suspension.
- the capacity of the culture container is 2.0 L or more.
- the at least a part of the region is the central portion in the suspension in the depth direction of the culture vessel, and the culture in the suspension. Includes at least one of the bottom portions of the container.
- the cell culture apparatus includes a culture vessel containing at least a suspension containing cells and a mass of an adherend to which the cells adhere, a driving device for rocking the suspension, and the like.
- a measuring device for measuring the mass distribution information value for the mass in the plurality of regions in the suspension, the first mass distribution information value in the first region of the plurality of regions, and the above.
- the mass distribution information difference value calculated by comparing the second mass distribution information value in the second region of the plurality of regions and at least set for the mass distribution information difference value.
- a control device for controlling the swing speed of the drive device based on the relationship with one or more thresholds is provided.
- the cell culture apparatus contains a suspension containing at least one of a cell mass which is an aggregate of a plurality of cells and a cell complex which is a complex of the cells and a culture carrier.
- the driving device for rocking the suspension the measuring device for measuring the carrier distribution information value for the culture carrier in the plurality of regions in the suspension, and the plurality of regions.
- the carrier distribution information difference value calculated by comparing the first carrier distribution information value in the first region with the second carrier distribution information value in the second region of the plurality of regions, and the carrier.
- a control device for controlling the swing speed of the drive device based on the relationship with at least one or more threshold values set for the distribution information difference value is provided.
- the measuring device includes a third measuring device having an imaging device for capturing an image of the plurality of regions and acquiring one or more images, and the third measurement.
- the apparatus measures the first carrier distribution information value based on the image in the first region, and measures the second carrier distribution information value based on the image in the second region.
- the carrier distribution information value is the number of the culture carriers in the suspension or the concentration of the culture carriers in each of the plurality of regions, and is the first.
- the 1 carrier distribution information value is the number of the culture carriers in the suspension or the concentration of the culture carrier in the first region
- the second carrier distribution information value is the number of the culture carriers in the second region. The number of the culture carriers or the concentration of the culture carriers in the suspension.
- the driving device is a stirrer or a shaker that stirs the suspension.
- the capacity of the culture container is 2.0 L or more.
- the first region is a central portion in the suspension in the depth direction of the culture vessel, and the second region is the suspension. It is in the turbid liquid and is the bottom portion of the culture vessel.
- a method for producing a cell group is to house a suspension containing at least a mass of cells and an adherend to which the cells adhere in a culture vessel, and to prepare the suspension by a driving device. Shaking, measuring the mass distribution information value for the mass in at least a part of the region of the suspension, the rate of change of the mass distribution information value per predetermined unit time, and the change. The relationship with at least one or more thresholds set for the rate is determined, and the rocking speed of the driving device is determined based on the result of the determination, and the determined rocking speed is used as described above. Includes outputting to the drive.
- the method for producing the cell group includes a suspension containing at least one of a cell mass which is an aggregate of a plurality of cells and a cell complex which is a complex of the cells and a culture carrier. Containment in a culture vessel, rocking the suspension by a driving device, cell distribution information values for the cells in at least a portion of the suspension, and carrier distribution information for the culture carrier. Measuring at least one of the values, the rate of change of at least one of the cell distribution information value and the carrier distribution information value per predetermined unit time, and at least one or more thresholds set for the rate of change. It is carried out by a culture method including determining a relationship, determining the rocking speed of the driving device based on the result of the determination, and outputting the determined rocking speed to the driving device. ..
- a method for producing a cell group is to house a suspension containing at least a mass of cells and an adherend to which the cells adhere in a culture vessel, and the suspension by a driving device.
- To measure the mass distribution information value for the mass in the plurality of regions in the suspension and to measure the mass distribution information in the first region of the plurality of regions.
- With respect to the mass distribution information difference value calculated by comparing the value with the second mass distribution information value in the second region of the plurality of regions and the mass distribution information difference value. The relationship with at least one set threshold value is determined, the rocking speed of the drive device is determined based on the result of the determination, and the determined rocking speed is output to the drive device. Including to do.
- a method for producing a cell group comprises a suspension containing at least one of a cell mass which is an aggregate of a plurality of cells and a cell complex which is a complex of the cells and a culture carrier. Containment in a culture vessel, rocking the suspension by a driving device, measuring carrier distribution information values for the culture carrier in a plurality of regions in the suspension, the plurality of regions. The carrier distribution information difference value calculated by comparing the first carrier distribution information value in the first region of the above with the second carrier distribution information value in the second region of the plurality of regions. , The relationship with at least one threshold set for the carrier distribution information difference value is determined, and the rocking speed of the driving device is determined based on the result of the determination, and determined. It is carried out by a culture method including outputting the rocking speed to the driving device.
- a cell culture apparatus capable of culturing cells with high yield and time efficiency by suppressing sedimentation of cell masses, cell complexes, etc. in a culture vessel, and A method of producing a cell culture can be provided.
- FIG. 2 is an enlarged view of a region surrounded by a dotted line, and is a schematic diagram schematically showing an enlarged part of a first measuring device. It is a schematic diagram schematically showing an example of the embodiment of another driving device different from the driving device shown in FIGS. 1 and 2 in the cell culture device according to one embodiment. It is a block diagram schematically showing an example of the function of the control device which concerns on one Embodiment.
- FIG. 1 It is a figure which shows the change of the capacitance and the number of viable cells with the passage of time with respect to the cell proliferation state by the cell culture apparatus which concerns on one Embodiment. It is a schematic diagram schematically showing the structure in the case of the cell culture apparatus which concerns on modification 1 and the 2nd measuring apparatus is used as a measuring apparatus. It is a figure which shows an example of the image imaged by the image pickup apparatus (imaging unit) of the 3rd measuring apparatus in the cell culture apparatus which concerns on modification 2. FIG. It is a schematic diagram which shows typically the structure of the cell culture apparatus which concerns on modification 3.
- FIG. 1 is a schematic diagram schematically showing the configuration of the cell culture apparatus 1 according to the embodiment.
- FIG. 2 is a schematic diagram schematically showing a configuration when the first measuring device 300 is used as the measuring device 30 in the cell culture device 1 according to the embodiment.
- FIG. 3 is an enlarged view of the region P surrounded by the dotted line in FIG. 2, and is a schematic diagram schematically showing an enlarged part of the first measuring device 300.
- FIG. 4 is a schematic diagram schematically showing an example of another mode of the driving device 20 different from the driving device 20 shown in FIGS. 1 and 2 in the cell culture device 1 according to the embodiment.
- FIG. 1 is a schematic diagram schematically showing the configuration of the cell culture apparatus 1 according to the embodiment.
- FIG. 2 is a schematic diagram schematically showing a configuration when the first measuring device 300 is used as the measuring device 30 in the cell culture device 1 according to the embodiment.
- FIG. 3 is an enlarged view of the region P surrounded by the dotted line in FIG. 2, and is a schematic diagram
- 5 is a block diagram schematically showing an example of the function of the control device 40 according to the embodiment.
- 6 and 7 are diagrams schematically showing an example of the relationship between the determination result by the determination unit 43 of the control device 40 according to the embodiment and the swing speed of the drive device 20 determined by the output unit 44.
- the cell to be cultured in the cell culture apparatus 1 is not particularly limited as long as it is a cell capable of suspension culture, and may be a floating cell or an adhesive cell. Further, the cells are cultured if a plurality of cells can contact each other to form an aggregate during suspension culture or adhere to a culture carrier to form a complex of one or a plurality of cells and the culture carrier. It may float in the suspension as a single cell inside.
- single cell means one independent cell.
- cell mass which is an aggregate of a plurality of cells means a mass formed by contacting cells with each other, and is not particularly limited, and is a spheroid formed sterically during the cell proliferation phase and other factors.
- the cells are preferably animal-derived cells, more preferably mammalian-derived cells. Examples of mammals include humans, monkeys, chimpanzees, cows, pigs, horses, sheep, goats, rabbits, rats, mice, guinea pigs, dogs, cats and the like.
- the cell may be, for example, a cell derived from a tissue such as skin, liver, kidney, muscle, bone, blood vessel, blood, or nervous tissue. Usually, one type of cell is subjected to culture alone, but two or more types may be used in combination for culture.
- the cell may be a primary cell from tissue or a cell line established by immortalization.
- adhesive cells examples include somatic cells, stem cells and the like.
- somatic cells for example, endothelial cells, epidermal cells, epithelial cells, myocardial cells, myoblasts, nerve cells, bone cells, osteoblasts, fibroblasts, fat cells, hepatocytes, renal cells, pancreatic cells, adrenal cells.
- Root membrane cells for example, gingival cells, bone membrane cells, skin cells, dendritic cells, macrophages and the like.
- the adherent cells may be stem cells.
- stem cells include somatic stem cells such as mesenchymal stem cells, hematopoietic stem cells, neural stem cells, bone marrow stem cells, and germ stem cells, and can be mesenchymal stem cells or bone marrow mesenchymal stem cells. ..
- Mesenchymal stem cells broadly mean somatic stem cells that are present in various tissues of the human body and can be differentiated into all or some of mesenchymal cells such as osteoblasts, chondrocytes and adipocytes. do.
- the stem cells may further include induced pluripotent stem cells (iPS cells) and embryonic stem cells (ES cells).
- iPS cells induced pluripotent stem cells
- ES cells embryonic stem cells
- the floating cell may be any cell. Examples thereof include, but are not limited to, Chinese hamster ovary cells (CHO cells), Vero cells, Jurkat, HL60, FM3A and the like.
- the suspension can be obtained by suspending these cells in a medium according to the cell type.
- the cell concentration of the suspension at the start of culture is, for example, 1 ⁇ 10 3 to 2 ⁇ 10 5 cells / mL, 5 ⁇ 10 3 to 1 ⁇ 10 5 cells / when the culture carrier is mixed in the suspension. It can be mL or 1 ⁇ 10 4-5 ⁇ 10 4 cells / mL, 5 ⁇ 10 3-2 ⁇ 10 8 cells / mL, 1 ⁇ 10 4 if the culture carrier is not mixed into the suspension. It can be ⁇ 1 ⁇ 10 7 cells / mL or 5 ⁇ 10 4 ⁇ 1 ⁇ 10 6 cells / mL.
- the cell culture device 1 is mainly composed of a culture container 10, a driving device 20, a measuring device 30, and a control device 40, but may include other than these.
- a heater or the like for heating the suspension contained in the culture vessel to a predetermined temperature may be separately provided.
- the measuring device 30 and the control device 40 are provided separately in FIG. 1, but the present invention is not limited to this, and at least a part of the measuring device 30 and the control device 40 are integrally formed. May be used.
- the details of the main components of the cell culture apparatus 1 will be described.
- the culture vessel 10 and the suspension in the culture vessel 10 As the culture vessel 10 according to the embodiment, a generally known bioreactor can be used.
- the capacity of the culture vessel 10 can be, for example, 0.3 L or more, 0.5 L or more, 1 L or more, 2 L or more, 5 L or more, 8 L or more, or 10 L or more in order to enable suspension culture and mass culture. ..
- the upper limit of the capacity of the culture vessel 10 is not particularly limited, but may be, for example, 50 L or less, 40 L or less, or 30 L or less from the viewpoint of efficiency and economy, but 80 L or more, 90 L or more, and 100 L. It may be the above or more.
- the shape of the culture vessel 10 is not particularly limited, and for example, a columnar, prismatic, or bag-shaped one can be used. Further, the side surface and the bottom surface of the culture vessel 10 are made of a material, color, and shape that allow irradiation light emitted from the image pickup device 360 to pass through for image pickup of the suspension by the image pickup device 360 (imaging unit 360) described later. Is preferable. Further, for the same reason, other components (for example, the above-mentioned heater and the like) arranged around the culture vessel 10 are located at a position different from the path of the irradiation light emitted from the image pickup apparatus 360 (out of the path). It is preferable that the culture vessel 10 is arranged on at least one of the side surface and the bottom surface of the culture vessel 10.
- a suspension containing cells is housed in the culture vessel 10 and subjected to suspension culture.
- the culture carrier is also appropriately mixed in the suspension depending on the type of cells to be cultured.
- the culture carrier for example, microbeads having a diameter of several hundred ⁇ m are known as microcarriers used for suspension culture of cells.
- the drive of the drive device 20 may be stopped for a certain period of time depending on the type, state, etc. of the cells immediately after or after the culture carrier is put into the culture container 10, and the drive may be intermittently driven. It may be made to drive continuously, or it may be driven continuously.
- the intermittent drive means that the swing of the drive device 20 is executed for a predetermined time and the swing of the drive device 20 is stopped for a predetermined time alternately. Alternately performing the swing may be a combination of the swing execution and the swing stop repeated two or more times (“execute swing” followed by “stop swing”.
- the time corresponding to the execution of the swing and the time corresponding to the stop of the swing may be the same or different. Further, in the two or more repetitions, the time corresponding to the execution of the swing may always be the same or different between the times. Similarly, between each round, the time corresponding to the stoppage of the swing may always be the same or different.
- the time for stopping the shaking may be the time for allowing the suspension to stand in the culture vessel 10.
- the swing speed of the drive device 20 when "swinging is executed" in the intermittent drive may be the same as or different from the specific swing speed of the drive device 20 described later.
- the time corresponding to the execution of the rocking can be, for example, 0.5 to 60 minutes, preferably 1 to 20 minutes, and more preferably 3 to 10 minutes.
- the time corresponding to the stop of the rocking may be, for example, 0.1 to 10 hours, preferably 0.5 to 6 hours, and more preferably 1 to 3 hours.
- “execute shaking” and subsequent “stop shaking” "swing for 0.5 to 60 minutes” followed by "swing for 0.1 to 10 hours”.
- “Stop shaking” is mentioned, and “” Shake for 1 to 20 minutes “and subsequent” Stop swinging for 0.5 to 6 hours "” is preferable, and “Swing for 3 to 10 minutes” is preferable. It is more preferable to "make it stop” and then "stop shaking for 1 to 3 hours”.
- the time for performing intermittent drive (total time for performing "exercise of rocking" and subsequent "stopping rocking" two or more times) is, for example, 1 to 80 hours, preferably 10 to. It is 40 hours, more preferably 20 to 30 hours.
- Continuous drive means to continuously swing for a predetermined time.
- the shaking time (excluding the case where the culture is stopped due to the above-mentioned determination of "abnormality") is, for example, 1 to 14 days, preferably 2 to 10 days, and more preferably 3 to 7 days. It's a day.
- shaking may be temporarily stopped due to addition of medium to the suspension, medium exchange, or the like.
- temporary suspension means that the rocking is temporarily suspended due to processing such as addition of medium to the suspension or medium replacement, rather than simply allowing the suspension to stand still.
- the swinging time does not have to be the sum of the swinging time before the temporary stop and the swinging time after the temporary stop. That is, the measurement may be performed by dividing the time during which the swing is performed by stopping the swing.
- the above-mentioned intermittent drive and continuous drive may be performed continuously, or may include not swinging for a predetermined time between the intermittent drive and the continuous drive.
- the predetermined time is, for example, 0.1 to 24 hours, preferably 0.5 to 5 hours, and more preferably 1 to 2 hours. Not shaking may be to allow the suspension to stand still.
- the intermittent drive may be performed, or the combination of the intermittent drive and the continuous drive may be repeatedly performed.
- the total culture time including intermittent drive and continuous drive (excluding the case where the culture is stopped due to the above-mentioned determination of "abnormality") is appropriately set according to the type, purpose, culture conditions, etc. of the cells to be cultured. be able to.
- the material of the culture carrier may be, for example, an organic substance, an inorganic substance, or a composite material thereof, and may be either soluble or insoluble.
- an organic substance for example, as an organic substance, for example, synthesis of polystyrene, polyester, polyurethane, polyethylene, polypropylene, polyvinyl alcohol, (meth) acrylic polymer, (meth) acrylamide polymer, silicone polymer, epoxy resin, urethane resin and the like.
- examples thereof include, but are not limited to, natural polymers such as polymers, cellulose, dextran, collagen, polygalacturonic acid, polyarginic acid, gelatin and proteins.
- the inorganic substance include, but are not limited to, glass, ceramics, metals, alloys, metal oxides and the like.
- one containing polystyrene may be used as the culture carrier of one embodiment.
- a cationic functional group may be introduced on the surface of the culture carrier.
- the cationic functional group include a group containing a substituted or unsubstituted amino group such as a dimethylamino group, a diethylamino group and an amino group.
- a cell adhesion polymer may be arranged on the surface of the culture carrier.
- collagen, gelatin, alginic acid, Matrigel TM (BD Biosciences), hyaluronic acid, laminin, fibronectin, vitronectin, elastin, heparan sulfate, dextran, dextran sulfate, chondroitin sulfate and the like are arranged. good.
- the culture carrier preferably contains a spherical culture carrier.
- the culture carrier may be a porous culture carrier having pores inside or a culture carrier having no cells inside.
- the average particle size (D50) of the culture carrier is, for example, 50 to 1,000 ⁇ m, preferably 100 to 500 ⁇ m, and more preferably 150 to 250 ⁇ m from the viewpoint of promoting cell proliferation.
- the average particle size of the culture carrier is a value measured as the median diameter (D50) in physiological saline or a medium.
- the average particle size of the culture carrier can be measured by a laser diffraction / scattering type particle size distribution measuring device.
- the concentration of the culture carrier in the suspension can be appropriately adjusted based on the shape, size, surface area, etc. of the culture carrier, and is, for example, 0.01 to 100 g / L, 0.5 to 50 g / L, or It can be 1 to 20 g / L.
- the suspension contains at least one of a cell mass and a cell complex.
- the cell suspension may contain one or both of the cells in a single state and the culture carrier to which the cells are not adhered.
- the adherend may contain, for example, cells, a culture carrier, or the like, and the mass may include, for example, the above-mentioned cell mass, cell complex, or the like.
- the mass may be a floating single cell.
- the wording of the cell distribution information value and the carrier distribution information value described later can be read as the mass distribution information value, and the wording of the carrier distribution information difference value described later is a mass. It can be read as a difference in body distribution information.
- the drive device 20 can be used without particular limitation as long as it swings the suspension contained in the culture vessel 10.
- the drive device 20 includes, for example, as shown in FIGS. 1 and 2, a motor 21, a shaft 22 connected to a rotating shaft (not shown) of the motor 21, and a stirring blade 23 connected to the tip of the shaft 22.
- a stirrer can be used.
- the driving device 20 for example, a generally known shaking machine as shown in FIG. 4 may be used, and the suspension may be shaken by shaking the culture vessel 10 from the outside by the shaking machine. .. Therefore, in the present disclosure, “swinging” can include any movement mode in which the suspension is shaken, such as stirring and shaking. Similarly, in the present disclosure, the “swing speed” can include speeds relating to any motion mode in which the suspension is shaken, such as stirring speed, shaking speed, and the like.
- the drive device 20 receives a command from a control device 40 described later that controls the swing speed (stirring speed) of the drive source (in the case where the stirrer is used as the drive device 20, the motor 21 shown in FIG. 1).
- a receiving unit (not shown) is provided separately.
- the drive device 20 can transmit the swing speed corresponding to the command of the control device 40 to the drive source.
- the measuring device 30 is in the process of culturing at least one of the cell distribution information value for cells and the carrier distribution information value for a culture carrier in at least a part of the region in the suspension in the culture vessel 10. It is measured (calculated).
- the cell distribution information value can be rephrased as, for example, the number of living cells or the concentration of living cells in the suspension in at least a part of the region, but is not limited thereto. That is, the cell distribution information value may be a value indicating how much live cells are distributed in the suspension of at least a part of the region, and in some cases, it is formed by agglomeration of a plurality of cells. It may be the number of cell clusters to be formed.
- the carrier distribution information value can be translated into, for example, the number of cultured carriers in the suspension or the concentration of the cultured carriers in at least a part of the region, but is not limited thereto. That is, the carrier distribution information value may be a value indicating how much the culture carrier is distributed in the suspension of at least a part of the region.
- the first measuring device 300 as shown in FIG. 2 or the like can be used as the measuring device 30 of the cell culture device 1 according to the embodiment.
- the first measuring device 300 described in detail below can measure only the cell distribution information value, but the second measuring device 350 according to the modification 1 described later can measure both the cell distribution information value and the carrier distribution information value. It is possible to measure.
- Whether to use the first measuring device 300 or the second measuring device 350 described later as the measuring device 30 may be appropriately determined based on the cell type to be cultured, the size of the culture container 10, and the like.
- the first measuring device 300 is suitable for directly observing (managing) the number of living cells or the concentration of living cells, and the second measuring device 350 directly observes (manages) a cell mass or a cell complex. ) Is suitable.
- the first measuring device 300 includes a substantially rod-shaped main portion 301 inserted into the suspension in the culture vessel 10 in the region P surrounded by the dotted line in FIG. 2, and the same. Based on an electrode 310 provided on the main portion 301 that oscillates an electric signal RF of a predetermined frequency to form an electric field in the suspension of the region P, and the amount of live cells Ce polarized in the formed electric field. Based on the capacitance measurement unit 320 that measures the capacitance (capacitance) in the electric field and the capacitance measured by the capacitance measurement unit 320, the cell distribution information value in the region P (specifically, the living cell in the region P).
- the main unit 301 includes at least one of a conductivity measuring unit 340 for measuring the conductivity of the suspension in the region P and a pH measuring unit 341 for measuring the pH of the suspension in the region P, or these. Both may be arranged.
- a conductivity measuring unit 340 and the pH measuring unit 341 generally known known ones can be used.
- a plurality of main portions 301 may be provided, and correspondingly, the electrode 310, the capacitance measuring unit 320, and the conductivity
- a plurality of rate measuring units 340, pH measuring unit 341, and the like may also be provided.
- the frequency of the electric signal RF oscillated from the electrode 310 is not particularly limited, but for example, one of 50 kHz to 20 MHz, preferably 100 kHz to 1 MHz, and more preferably 400 kHz to 600 kHz can be used. It is preferable that electric signals having a plurality of types of frequencies are oscillated from the electrode 310. By oscillating electric signal RF of various frequencies from the electrode 310, it is possible to measure the number of viable cells or the concentration of viable cells for each viable cell having various particle sizes, and thus it is included in the suspension of the region P. Accurate viable cell number or viable cell concentration can be measured.
- the location of the region P in the suspension where the electric field is formed by the electrode 310 and the capacitance is measured by the capacitance measuring unit 320 is higher than the liquid level of the suspension when the suspension is housed in the culture vessel 10. As long as it is in the lower position, it may be in any position in the depth direction inside the culture vessel 10.
- the location of the region P may be the central portion in the culture vessel 10 in the depth direction or the bottom portion 10x in the culture vessel 10, as shown in FIGS. 2 and 3.
- the number of regions P in the suspension may be provided at only one location in the culture vessel 10 or at a plurality of locations. When a plurality of regions P are provided in the culture vessel 10, they may be arranged at different positions or at the same position in the depth direction in the culture vessel 10.
- the portion for measuring the capacitance is measured at a plurality of points in the culture vessel 10.
- the first measuring device 300 is used at the plurality of locations. It may be designed to have a plurality of electrodes 310 corresponding to the above and a plurality of capacitance measuring units 320 (and a main unit 301 corresponding to these).
- the first measuring device 300 can accurately calculate the number of living cells or the concentration of living cells in the suspension.
- the device for measuring the number of living cells or the concentration of living cells using capacitance is already known (for example, Harriet E. Cole et al., “The Application”. Of Dielectric Spectroscopy and Biocalorimetry for the Monitoring of Biomass in Immobilized Mammalian Cell Cultures ”, Processes 2015, 3, 384-405, etc.), so further detailed description of the first measuring device 300 will be omitted.
- the cell distribution information value (number of living cells or concentration of living cells) calculated by the main body 330 of the first measuring device 300 is obtained by the control device 40 described later at a predetermined time interval (for example, an interval of a predetermined unit time). Will be sent.
- Control device 40 The control device 40 according to the embodiment may use, for example, general hardware to which a central processing unit (CPU), a main storage device, an input / output interface, an input device, an output device, and the like are connected by a data bus or the like. can. As shown in FIG. 1, the control device 40 may be provided separately from the measuring device 30 as a terminal device, or at least a part of the measuring device 30 (for example, the main body 330 in the first measuring device 300 described above). ) And the control device 40 may be integrally configured.
- CPU central processing unit
- main storage device for example, a central processing unit (CPU), a main storage device, an input / output interface, an input device, an output device, and the like are connected by a data bus or the like. can.
- the control device 40 may be provided separately from the measuring device 30 as a terminal device, or at least a part of the measuring device 30 (for example, the main body 330 in the first measuring device 300 described above). ) And the control device 40 may be integral
- the function of the control device 40 is mainly composed of a communication unit 41, a storage unit 42, a determination unit 43, and an output unit 44.
- the communication unit 41 receives the cell distribution information value from the measuring device 30 (the first measuring device 300 in one embodiment), and transmits the received cell distribution information value to the storage unit 42. Further, as will be described later, the communication unit 41 gives the drive device 20 a control command for the drive device 20 to determine (calculate) the swing speed of the drive device 20 determined (calculated) by the output unit 44 based on the determination result of the determination unit 43. It can be transmitted (the drive device 20 is equipped with a function of receiving a control command from the communication unit 41).
- the storage unit 42 can store the cell distribution information value received from the communication unit 41, the control command for the driving device 20, the threshold value described later, and the like.
- the determination unit 43 is based on the cell distribution information value received by the communication unit 41 from the measuring device 30 (stored by the storage unit 42), and the change rate of the cell distribution information value per predetermined unit time (hereinafter, simply “change rate”). Also called.) To monitor. Further, the determination unit 43 compares the rate of change with a threshold value preset for the rate of change, and determines the relationship between the rate of change and the threshold value every predetermined unit time. For example, the determination unit 43 determines whether the rate of change is equal to or greater than the threshold value or is less than the threshold value.
- the rate of change for example, when the predetermined unit time is set to 1 hour (60 minutes), the cell distribution information value at the start of the 1 hour and the end time (that is, 60 minutes after the start). By comparing with the cell distribution information value of the above, the rate of change of the cell distribution information value in the one hour can be calculated. Further, for example, when the predetermined unit time is set to 1 hour (60 minutes), a total of 7 times (that is, at the start time, 10 minutes later, 20 minutes later, 30 minutes later) every 10 minutes from the start of the 1 hour.
- the rate of change in capacitance per predetermined unit time may be used instead of the rate of change in the cell distribution information value per predetermined unit time. ..
- the measuring device 30 uses the measured capacitance information (capacitance value) together with the cell distribution information value calculated by the main body 330 or in place of the cell distribution information value calculated by the main body 330. It may be transmitted to the control device 40 described later at a predetermined time interval (for example, an interval of a predetermined unit time) (hence, in the present disclosure, the “change rate of cell distribution information value” also includes a “capacitance change rate”. It shall be included).
- the predetermined unit time regarding the rate of change is not particularly limited, but may be appropriately set, for example, between 10 minutes and 24 hours.
- the rocking speed of the drive device 20 is finely controlled to maximize the yield and efficiency of the cells to be cultured (the aggregate of cells to be cultured is also referred to as "cell group" in the present disclosure). From this point of view, it is preferable that the predetermined unit time is, for example, 30 minutes to 1 hour.
- the threshold value for example, one is set to "0%” with respect to the above-mentioned change rate (the threshold value in this case is also referred to as "first threshold value").
- the rate of change of 0% means that the above-mentioned cell distribution information value to be measured is constant at a predetermined unit time (at the start and end of the predetermined unit time), and the rate of change is If it is larger than 0%, it means that the cell distribution information value is increasing, and if the rate of change is less than 0%, it means that the cell distribution information value is decreasing.
- the determination unit 43 described above determines, for example, that cell proliferation is "normal” when the rate of change is 0% or more (first threshold value or higher), and when the rate of change is less than 0% (less than the first threshold value). Cell proliferation can be determined to be "abnormal". The specific contents of the determination of "normal” and "abnormal” may be changed as appropriate.
- another threshold value in addition to the above-mentioned first threshold value, another threshold value (another threshold value in this case is also referred to as a "second threshold value”) may be further set to a value larger than 0%.
- the second threshold value can be set, for example, for any value in the range of 10% to 90% of the rate of change.
- the determination unit 43 determines that the cell proliferation is "normal” when the rate of change is equal to or higher than the second threshold value, and the cell proliferation is “slightly” when the rate of change is equal to or greater than the first threshold value and less than the second threshold value.
- cell proliferation can be determined to be "abnormal".
- the specific determination contents of "normal”, “slightly low", and "abnormal” may be changed as appropriate.
- the output unit 44 determines the value of the swing speed of the drive device 20 (in the case where the stirrer is used as the drive device 20, the stirring speed) based on the determination result of the determination unit 43 described above.
- the relationship between the determination result of the determination unit 43 and the swing speed of the drive device 20 determined by the output unit 44 will be described with reference to FIGS. 6 and 7.
- the length of the predetermined unit time (predetermined unit time A to predetermined unit time J in FIG. 6 and predetermined unit time A to predetermined unit time I in FIG. 7) is all constant (for example,). It should be understood that the end time and the start time of the adjacent predetermined unit time are substantially the same (that is, the end time of the predetermined unit time A and the start time of the predetermined unit time B). Substantially the same).
- the output unit 44 is "normal” (the rate of change is the first threshold value "0" or more” as described above. )
- a new rocking speed is output so as to increase the rocking speed of the drive device 20 at the time of the judgment (that is, when the predetermined unit time A) (note that the new rocking speed is output).
- the time point is the start time of the next predetermined unit time B).
- the determination unit 43 is "abnormal" as described above (the rate of change is less than the first threshold value "0"). If it is determined, a new swing speed is output so as to reduce the swing speed of the drive device 20 at the time of the determination (that is, at the time of the predetermined unit time E) (note that a new swing speed is output). The time point is the start time of the next predetermined unit time F).
- the rate of change becomes equal to or higher than the first threshold value (the rate of change is the first threshold value "0") as a result of executing the reduction of the swing speed
- the determination unit 43 determines that it is "normal". As in the case, a new swing speed that increases the swing speed of the drive device 20 is output (see the predetermined unit time F to the predetermined unit time G in FIG. 6).
- the output unit 44 stops driving the drive device 20 when the determination unit 43 determines that the determination for each predetermined unit time is "abnormal" (that is, the rate of change is less than the first threshold value) a plurality of times in a row. Therefore, the swing speed 0 is output (see the predetermined unit time G to the predetermined unit time J in FIG. 6).
- the determination of the predetermined unit time is determined to be "abnormal” a plurality of times in a row, the living cells are caused by the occurrence of contamination such as germs in the culture vessel 10 and the shaking speed of the driving device 20 being too fast. It is necessary to stop the cell culture immediately because there is a possibility that a problem may occur due to damage to the cells.
- the above-mentioned "multiple times in a row” means that the determination for each predetermined unit time may be two times in a row, three times in a row, or four or more times in a row. It may be appropriately set according to the cell type and the culture conditions. In one embodiment, the above-mentioned is based on the measured value of at least one of the conductivity in the suspension measured by the conductivity measuring unit 340 and the pH of the suspension measured by the pH measuring unit 341. You may try to grasp the problem such as.
- a second threshold value Y is set in addition to the above-mentioned first threshold value as a threshold value for the rate of change (Xt) will be described with reference to FIG. 7.
- the determination unit 43 is "slightly low” as described above (change rate Xt is "Y> Xt ⁇ "). If it is determined to be "0"), a new rocking speed is output so as to increase the rocking speed of the drive device 20 at the time of the judgment (at the time of the predetermined unit time A) (note that the new rocking speed is calculated).
- the output time is the start time of the next predetermined unit time B).
- the determination unit 43 is “normal” as described above (change rate Xt is “Y ⁇ Xt”). If it is determined, the rocking speed at the time of the determination (at the time of the predetermined unit time D) is maintained.
- the output unit 44 determines that the determination unit 43 is “abnormal” (change rate Xt is “Xt ⁇ 0”) as described above.
- a new rocking speed is output so as to reduce the rocking speed at the time of the judgment (at the time of the predetermined unit time F) (note that the time when the new rocking speed is output is the next time. It is the start of the predetermined unit time G).
- the output unit 44 has a swing speed of 0 in order to stop the drive device 20. Is output.
- the specific rocking speed of the driving device 20 and the degree of increase / decrease in the rocking speed are appropriately set according to the cell type, the capacity of the culture vessel 10, and the like. do it.
- the specific rocking speed is 10 mm / s to 100 mm / s, preferably 20 mm / s to 80 mm / s. , More preferably in the range of 30 mm / s to 60 mm / s.
- the swing speed at this time means the rotation speed of the stirring blade 23 when the stirrer is used as the drive device 20, and the above swing speed is used as the moving distance of the outer peripheral end of the stirring blade 23. It may be converted into a rotation speed.
- FIG. 8 is a flow chart showing an example of a part of the operation performed in the cell culture apparatus 1 according to the embodiment.
- 9 and 10 are diagrams showing changes in capacitance and the number of living cells over time with respect to the proliferation status of cells (for example, mesenchymal stem cells) by the cell culture apparatus 1 according to the embodiment.
- the first threshold value and the second threshold value described above are preset as the threshold value.
- the dotted line indicates the capacitance value (pF / cm)
- the black circle indicates the number of living cells (cells / mL).
- the cells to be cultured are housed in the culture vessel 10 containing the medium, and the suspension culture is started. Then, after a lapse of a predetermined time, in step (hereinafter referred to as “ST”) 500, a suspension containing a cell mass which is an aggregate of a plurality of cells is formed (contained) in the culture vessel 10 (containment step). ).
- ST a predetermined time
- a cell complex which is a complex of the cells and the culture carrier, is contained in the culture vessel 10 after a predetermined time has passed after the start of suspension culture. Is formed (contained) in.
- the drive device 20 swings the suspension in the culture vessel 10 by a predetermined drive method (stop for a certain period of time, intermittent drive, continuous drive, etc.) (rocking step).
- the swing of the suspension by the drive device 20 may be started before the ST500.
- the measuring device 30 measures the cell distribution information value (number of living cells or living cell concentration) in at least a part of the region (region P) in the suspension (the number of living cells or the concentration of living cells). Measurement process).
- the carrier distribution information value described in detail in the modification described later may be measured by the measuring device 30 (second measuring device 350).
- the measuring device 30 transmits the measured cell distribution information value (live cell number or live cell concentration) to the control device 40.
- the measuring device 30 controls the measured carrier distribution information value (number of cultured carriers or culture carrier concentration) 40. May be sent to.
- the determination unit 43 of the control device 40 relates the rate of change of the cell distribution information value per predetermined unit time to a preset threshold value (for example, the above-mentioned first threshold value and second threshold value). Is determined every predetermined unit time, and the determination result is transmitted to the output unit 44.
- a preset threshold value for example, the above-mentioned first threshold value and second threshold value.
- the output unit 44 of the control device 40 determines the swing speed of the drive device 20 based on the judgment result of the determination unit 43 (swing speed determination step).
- the communication unit 41 of the control device 40 outputs the swing speed of the drive device 20 determined by the output unit 44 to the drive device 20 as a control command to the drive device 20 (output step). ..
- the drive device 20 outputs the swing speed according to the control command.
- the cell culture apparatus 1 repeats each operation related to ST502 to ST506 as long as the cell culture process is continued (ST507).
- the rate of change of the cell distribution information value becomes constant at "0" for a predetermined period regardless of the shaking speed of the driving device 20, or
- the determination by the determination unit 43 for each predetermined unit time becomes "abnormal" a plurality of times in succession, a series of operations is terminated (stopped).
- target value of the number of living cells varies depending on the type of cells to be cultured, the culture scale, the intended use, etc., but is, for example, 5 to 100 times the cell concentration at the start of culture.
- the number of viable cells (live cell concentration) can be doubled, 5 times to 50 times, or 10 to 30 times.
- the predetermined unit time is set to 1 hour (60 minutes).
- the “capacitance change rate” is used instead of the "cell distribution information value change rate”.
- the rate of change (rate of change in capacitance) in a predetermined unit time is equal to or higher than the preset second threshold value (Y described above) over the time t1 to t2, it is driven during this period.
- the rocking speed (rotational speed of the stirring blade) of the device 20 increases every predetermined unit time (every hour).
- the rate of change is also increased to the second threshold value or higher. From this, it is understood that the sedimentation of cell masses, cell complexes and the like is suppressed in the culture vessel 10, and cell proliferation (production of cell groups) is efficiently executed.
- the rate of change of the capacitance is "0" over a certain period regardless of the swing speed of the drive device 20, so that the cell proliferation (production of the cell group) is substantially completed (the production of the cell group). It is understood that the number of living cells has reached the target value).
- FIG. 9 shows a place where the capacitance value temporarily drops, but this is a temporary fluctuation in the capacitance value due to the culture medium exchange, and the swing speed of the drive device 20 is increased. It should be added that it does not affect the control.
- the case shown in FIG. 10 is basically the same as the case shown in FIG. 9, but the rate of change of the capacitance sharply decreases near the time t3, and after the time t3.
- the rate of change is less than the first threshold value (0) (abnormal) when the determination for each predetermined unit time is performed a plurality of times in succession. Therefore, in the case shown in FIG. 10, the control device 40 is finally set to 0 in order to stop the swing speed of the drive device 20.
- a separate threshold value may be further set for a value (for example, -10%) in which the rate of change is less than 0. good.
- the cell culture apparatus 1 can appropriately stop the culture when it is determined to be abnormal, so that the cause of the determination of abnormality can be investigated, unnecessary progress of culture can be prevented, and the like. It is useful in terms of time efficiency from the viewpoint of.
- FIG. 11 is a schematic diagram schematically showing the configuration of the cell culture device 1 according to the modified example 1 in the case where the second measuring device 350 is used as the measuring device 30.
- the cell culture device 1 according to the modified example 1 is substantially the same as the cell culture device 1 according to the embodiment described above, but the second measurement device 350 is applied instead of the first measurement device 300. be. Therefore, in the cell culture device 1 according to the modified example 1, the details of the second measuring device 350 will be described as follows, and the detailed description of other components will be omitted.
- the second measuring device 350 applied to the cell culture device 1 according to the first modification captures an image of the culture carrier (reference numeral MC in FIG. 11) contained in the suspension of at least a part of the region in the culture vessel 10. It may have an image pickup unit 360 for acquiring an image (which may be a moving image), and a main body unit 370 for measuring a carrier distribution information value in the region based on the image captured by the image pickup unit 360. can.
- the "measurement" of the carrier distribution information value by the second measuring device 350 includes “calculating" the carrier distribution information value based on the image.
- the image pickup unit 360 a generally known image sensor or the like that can continuously image an object at predetermined intervals and acquire a two-dimensional or three-dimensional image can be used.
- the imaging unit 360 may be a large one capable of uniformly imaging the inside of the culture vessel 10 in the depth direction, or as shown in FIG. 11, a plurality of (for example, in FIG. 11) may be used. 4) small imaging units 361 to 364 may be arranged side by side with respect to the culture vessel 10 in the depth direction, or these four imaging units 361 to 364 may be arranged on the outer peripheral surface of the culture vessel 10.
- the imaging unit 361 is the “12 o'clock position”
- the imaging unit 362 is the “3 o'clock position”
- the imaging unit 363 is the “6 o'clock position”
- the imaging unit 364 is the "9 o'clock position”
- the main body unit 370 continuously obtains the images acquired by the image pickup unit 360, recognizes the culture carrier MC contained in the image, and then determines the number of the culture carrier MC included in the image. , It has a function of being able to be counted as a carrier distribution information value. Specifically, the counting of the carrier distribution information value by the main body unit 370 is based on, for example, continuously obtaining an image acquired by any one of the image pickup units 361 to 364 and taking the image. It may be executed. Alternatively, the counting of the carrier distribution information values by the main body unit 370 is based on, for example, the images acquired by at least two of the image pickup units 361 to 364 continuously obtaining the images acquired from each image pickup unit.
- the average value thereof may be calculated as the final carrier distribution information value. Further, in the main body portion 370, the number of cell clusters in the suspension and the number of cells on the cell complex (number of viable cells) are used as cell distribution information values by the imaging unit 360 (imaging units 361 to 364). It may have a function that can be counted.
- the main body portion 370 has a function of calculating the concentration of the culture carrier contained in the image as a carrier distribution information value based on the color and shading of the image. It may have.
- the main body 370 is trained in advance to learn a large amount of colors and shades corresponding to various culture carrier concentrations, and stores data relating the colors or shades to the culture carrier concentration. Based on the data, the concentration of the culture carrier contained in the image acquired by the imaging unit 360 may be calculated.
- the main body unit 370 transmits the number of culture carrier MCs or the culture carrier concentration calculated as described above to the control device 40, and the control device 40 executes the same processing and control as in one embodiment. As described above, when counting the number of cell clusters (and the number of cells on the cell complex), the main body portion 370 counts the number of cell clusters (and the number of cells on the cell complex). , With the number of culture carrier MCs, or in place of the number of culture carrier MCs, can also be transmitted to the control device 40.
- the second measuring device 350 when the second measuring device 350 calculates the number of culture carrier MCs (or the culture carrier concentration) as the carrier distribution information value, the change in the carrier distribution information value per predetermined unit time.
- a third threshold value for the rate (corresponding to the first threshold value in one embodiment) will be set.
- a fourth threshold value (corresponding to the second threshold value in one embodiment) for the rate of change of the carrier distribution information value per predetermined unit time may be further set in advance. Therefore, the determination by the determination unit 43 in the modification 1 is executed based on the rate of change of the carrier distribution information value per predetermined unit time.
- the third threshold value and the fourth threshold value may be the same as or different from the first threshold value and the second threshold value in one embodiment.
- the second measuring device 350 determines the number of culture carrier MCs (or the culture carrier concentration) as the carrier distribution information value and the number of cell clusters (and the number of cells) as the cell distribution information value.
- the second measuring device 350 may transmit both the cell distribution information value and the carrier distribution information value to the control device 40.
- the determination variation by the determination unit 43 of the control device 40 in the modification 1 increases, and for example, it is possible to set as shown in Table 1 below.
- the cell distribution information value and the carrier distribution information value when set as shown in Table 1 below are, for example, captured by the second image pickup unit 363 from the top or the third image pickup unit 362 from the top in FIG. It shall be acquired based on the image to be used.
- the rocking speed of the drive device 20 corresponding to each determination regarding the situations 1 to 5. Can be set in advance, so that the swing speed of the drive device 20 can be finely controlled. Therefore, it is possible to efficiently suppress the sedimentation of cell masses, cell complexes and the like in the culture vessel 10, and further improve the yield and time efficiency of the production of cell groups.
- FIG. 12 is a diagram showing an example of an image captured by the imaging device 360 (imaging unit 360) of the third measuring device 351 in the cell culture device 1 according to the modified example 2.
- the cell culture device 1 according to the modified example 2 is substantially the same as the cell culture device 1 according to the modified example 1 shown in FIG. 11 described above, but instead of the second measuring device 350, the third measuring device 351 Is applied. Therefore, in the cell culture device 1 according to the modified example 2, the details of the third measuring device 351 will be described as follows, and the detailed description of other components will be omitted.
- the physical configuration of the third measuring device 351 is the same as that of the second measuring device 350 shown in FIG.
- the third measuring device 351 As the third measuring device 351 according to the modified example 2, the same as the image pickup unit 360 according to the modified example 1 can be used as the image pickup device 360. Then, the third measuring device 351 measures the carrier distribution information value in each of the plurality of regions in the suspension in the culture vessel 10, and the first carrier distribution information value in the first region of the plurality of regions. And the second carrier distribution information value in the second region of the plurality of regions are compared and calculated to calculate the carrier distribution information difference value. Then, the rocking speed of the drive device 20 is controlled based on the relationship between the carrier distribution information difference value and at least one or more threshold values set in advance for the difference value.
- the first carrier distribution information value is a carrier distribution information value in the first region (specifically, the number of culture carriers or the concentration of the culture carrier), and the second carrier distribution information value is a second carrier distribution information value. It means the carrier distribution information value in the region 2.
- the image pickup apparatus 360 As shown in FIGS. 11 and 12, a plurality of small ones (for example, four in FIG. 11) may be used, or one image pickup apparatus 360 may be used. A large image pickup device 360 may be used. Then, for example, when the image pickup device 360 is four small image pickup devices 361 to 364 (or at least two of these image pickup devices) as shown in FIGS. 11 and 12, each of these image pickup devices 361 To 364 image the regions Q1 to Q4 (or at least two regions of the regions Q1 to Q4) of the corresponding suspension.
- each of the image pickup devices 361 to 364 transmits the image captured and acquired in each area to the main body unit 371.
- the main body 371 measures each carrier distribution information value in the regions Q1 to Q4 (or at least two regions of these regions), and transfers each measured carrier distribution information value to the control device 40. Send.
- the control device 40 for acquiring each carrier distribution information value in the regions Q1 to Q4 (or at least two regions of these regions) from the main body portion 371 is, for example, a carrier in the region Q3 (corresponding to the first region).
- the distribution information value (first carrier distribution information value) and the carrier distribution information value (second carrier distribution information value) in the region Q1 (corresponding to the second region) are compared and calculated to calculate the carrier distribution information difference value. do.
- the carrier distribution information difference value is obtained, for example, by subtracting the second carrier distribution information value from the first carrier distribution information value.
- the determination unit 43 of the control device 40 monitors the carrier distribution information difference value calculated in this way, and based on the relationship with the threshold value set in advance for the carrier distribution information difference value, the present embodiment Similarly, the determination of "normal”, "abnormal” and the like is executed.
- the cultured carrier is suspended. Since it is considered to be uniformly dispersed in the liquid, it can be judged as "normal”, and if the subtracted value is significantly lower than 0, it is considered that the culture carrier has settled, etc., so it is "abnormal". Can be determined. Therefore, it is possible to efficiently suppress the sedimentation of cell masses, cell complexes and the like in the culture vessel 10, and further improve the yield and time efficiency of the production of cell groups.
- FIG. 13 is a schematic view schematically showing the configuration of the cell culture apparatus 1 according to the modified example 3.
- the cell culture device 1 according to the modified example 3 is applied to the first measuring device 300 applied to the cell culture device 1 according to the embodiment and the cell culture device 1 according to the modified example 1 (and the modified example 2). Both the second measuring device 350 (and the third measuring device 351) are applied. Therefore, the control device 40 according to the modified example 3 has a cell distribution information value and a carrier distribution information value (in some cases, cell distribution information) from the first measuring device 300 and the second measuring device 350 (and the third measuring device 351). You can receive a large number of values for). Therefore, the determination variation of the determination unit 43 of the control device 40 can be maximized. As a result, the swing speed of the drive device 20 can be controlled more finely. This makes it possible to more efficiently suppress the sedimentation of cell masses, cell complexes and the like in the culture vessel 10, and further improve the yield and time efficiency of the production of cell groups.
- the numerical range indicated by using "-" indicates a range including the numerical values before and after "-" as the minimum value and the maximum value, respectively.
- the upper or lower limit of the numerical range at one stage may be optionally combined with the upper or lower limit of the numerical range at another stage.
- the term "process” is included in this term not only as an independent process but also as long as the intended action of the process is achieved even if it cannot be clearly distinguished from other processes. ..
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Abstract
[Problem] To provide a cell culture apparatus that allows cell culture with a high yield and a good time efficiency, while suppressing precipitation of masses such as cell masses, cell complexes etc. in a culture container; and a method for producing cell groups. [Solution] A cell culture apparatus is equipped with: a culture container that accommodates a suspension including at least cell groups that are aggregates of a plurality of cells or cell complexes that are complexes of cells and culture carriers; a driving device that agitates the suspension; a measuring device that measures, in a region of at least one part of the suspension, at least cellular distribution information values related to the cells or carrier distribution information values related to the culture carriers; and a control device that controls the agitation speed of the driving device, based on the relation between the change ratio, per predetermined unit of time, of at least the cellular distribution information values or the carrier distribution information values and at least one threshold value set with reference to the aforementioned change ratio.
Description
本開示は、細胞培養装置、及び、細胞群を生産する方法に関する。
The present disclosure relates to a cell culture apparatus and a method for producing a cell group.
生物医学的研究および臨床の分野においては、被検体から採取した試料を用いて生体外で培養した後、目的の試料(例えば、細胞や液性因子等)の選別および回収を経て、その後の研究および治療に用いることが行われる。例えば再生医療の分野においては、被検体から採取した細胞を培地中で増殖させた後、培地から、目的としない細胞や夾雑物を分離し、目的の細胞や液性因子を回収して使用する。
In the fields of biomedical research and clinical practice, after culturing in vitro using a sample collected from a subject, the target sample (for example, cells, humoral factors, etc.) is selected and recovered, and then further research is performed. And used for treatment. For example, in the field of regenerative medicine, cells collected from a subject are grown in a medium, then undesired cells and impurities are separated from the medium, and the target cells and humoral factors are recovered and used. ..
ここで、細胞の培養プロセスにおいては、従前、多くの手作業が含まれていた。例えば、培養プロセス中、所定の時間間隔で懸濁液を採取して、該懸濁液中に含まれる細胞濃度又は細胞数を顕微鏡等で観察(測定)することで、培養が順調に進んでいるか、雑菌等のコンタミネーションが発生していないか、といった確認作業は、主に手作業で行われていた。しかしながら、培養プロセスにおいて頻繁な手作業が存在すると、懸濁液を頻繁に採取することに起因する培養細胞の収率低下や、雑菌等のコンタミネーションリスクの増加といった問題が生じる。昨今では、このような問題を低減する目的で、様々な自動化技術が提案されている。
Here, the cell culture process previously involved a lot of manual work. For example, during the culturing process, the suspension is collected at predetermined time intervals, and the cell concentration or the number of cells contained in the suspension is observed (measured) with a microscope or the like, so that the culturing proceeds smoothly. The confirmation work, such as whether or not contamination such as germs has occurred, was mainly performed manually. However, the presence of frequent manual work in the culture process causes problems such as a decrease in the yield of cultured cells due to frequent collection of suspensions and an increase in the risk of contamination such as germs. Recently, various automation technologies have been proposed for the purpose of reducing such problems.
例えば、特開2020-54234号公報には、撮像ユニットを用いて、懸濁液中に含まれる細胞(細胞塊)の統計データを取得する技術が開示されている。
For example, Japanese Patent Application Laid-Open No. 2020-54234 discloses a technique for acquiring statistical data of cells (cell mass) contained in a suspension using an imaging unit.
また、特開2010-99011号公報及び特開2006-320226号公報には、細胞の播種作業に際して、培養容器内の細胞の分布状態(分散状態)を、撮像装置を用いて測定したうえで、細胞の分布状態に応じて駆動装置による培養容器の揺動を制御する技術が開示されている。
Further, in JP-A-2010-99011 and JP-A-2006-320226, when the cells are seeded, the distribution state (dispersion state) of the cells in the culture vessel is measured by using an image pickup device, and then the cells are seeded. A technique for controlling the swing of a culture vessel by a driving device according to the distribution state of cells is disclosed.
しかしながら、細胞を浮遊培養(大量培養)する場合、浮遊培養の進行に伴って、複数の細胞の凝集体である細胞塊が生成される。また、懸濁液中において細胞を培養担体に担持させて浮遊培養する場合においては、浮遊培養の進行に伴って、培養担体に担持された細胞が該培養担体上で増殖するため、細胞と培養担体との細胞複合体の比重が次第に大きくなる。これらの結果、細胞塊及び細胞複合体は、培養容器の底面に次第に沈降してしまう。
However, in the case of suspension culture (mass culture), cell masses, which are aggregates of a plurality of cells, are generated as the suspension culture progresses. In addition, in the case where cells are carried on a culture carrier in a suspension for suspension culture, the cells carried on the culture carrier proliferate on the culture carrier as the suspension culture progresses, so that the cells are cultured with the cells. The specific gravity of the cell complex with the carrier gradually increases. As a result, the cell mass and the cell complex gradually settle on the bottom surface of the culture vessel.
培養容器の底面に多くの細胞塊、細胞複合体等が沈降してしまうと、細胞と培地との接触面積の減少、複数の培養担体間の凝集、浮遊状態にある細胞と培養担体との担持効率の低下等が誘引され、結果として、培養効率が低下するという問題が生じる。
When many cell masses, cell complexes, etc. settle on the bottom surface of the culture vessel, the contact area between the cells and the medium decreases, aggregation between multiple culture carriers, and support of suspended cells and culture carriers. A decrease in efficiency or the like is induced, and as a result, there arises a problem that the culture efficiency is decreased.
一方で、浮遊培養(大量培養)の場合においては、培養細胞(細胞群)の収率性と時間効率性の観点から、前述の問題が生じないように、培養容器内を管理することが強く求められる。
On the other hand, in the case of suspension culture (mass culture), it is strongly important to manage the inside of the culture vessel so that the above-mentioned problems do not occur from the viewpoint of yield and time efficiency of cultured cells (cell group). Desired.
そこで、様々な実施形態により、培養容器内での細胞塊、細胞複合体等の塊体の沈降を抑制して、高い収率性と時間効率性で細胞を培養することが可能な細胞培養装置、及び、細胞群を生産する方法を提供する。
Therefore, according to various embodiments, a cell culture apparatus capable of suppressing the sedimentation of cell masses, cell complexes and other masses in a culture vessel and culturing cells with high yield and time efficiency. , And a method for producing a cell population.
一態様に係る細胞培養装置は、細胞と、前記細胞が接着する被接着体との塊体を少なくとも含む懸濁液を収容する培養容器と、前記懸濁液を揺動させる駆動装置と、前記懸濁液中の少なくとも一部の領域における、前記塊体に関する塊体分布情報値を測定する測定装置と、前記塊体分布情報値の所定単位時間あたりの変化率と、前記変化率に対して設定される少なくとも1つ以上の閾値との関係に基づいて、前記駆動装置の揺動速度を制御する制御装置と、を具備する。
The cell culture apparatus according to one embodiment includes a culture vessel containing at least a suspension containing cells and an adherend to which the cells adhere, a driving device for swinging the suspension, and the above-mentioned. A measuring device for measuring the mass distribution information value for the mass in at least a part of the region of the suspension, the rate of change of the mass distribution information value per predetermined unit time, and the rate of change. A control device for controlling the swing speed of the drive device based on the relationship with at least one set threshold value is provided.
また、一態様に係る前記細胞培養装置は、複数の細胞の凝集体である細胞塊、及び前記細胞と培養担体との複合体である細胞複合体の少なくとも一方を含む懸濁液を収容する培養容器と、前記懸濁液を揺動させる駆動装置と、前記懸濁液中の少なくとも一部の領域における、前記細胞に関する細胞分布情報値、及び前記培養担体に関する担体分布情報値の少なくとも一方を測定する測定装置と、前記細胞分布情報値及び前記担体分布情報値の少なくとも一方の所定単位時間あたりの変化率と、前記変化率に対して設定される少なくとも1つ以上の閾値との関係に基づいて、前記駆動装置の揺動速度を制御する制御装置と、を具備する。
また、一態様に係る前記細胞培養装置において、前記測定装置は、前記少なくとも一部の領域において電場を形成させる電極と、前記電場内のキャパシタンスを測定するキャパシタンス測定部と、を有する第1測定装置を含み、前記第1測定装置は、前記キャパシタンスに基づいて、前記少なくとも一部の領域における前記細胞分布情報値を測定する。
また、一態様に係る前記細胞培養装置において、前記第1測定装置は、前記少なくとも一部の領域における導電率を測定する導電率測定部、をさらに有する。 Further, the cell culture apparatus according to one aspect is a culture containing a suspension containing at least one of a cell mass which is an aggregate of a plurality of cells and a cell complex which is a complex of the cells and a culture carrier. At least one of the cell distribution information value for the cell and the carrier distribution information value for the culture carrier in at least a part of the region of the suspension, the driving device for rocking the suspension, and the container is measured. Based on the relationship between the measuring device to be used, the rate of change of at least one of the cell distribution information value and the carrier distribution information value per predetermined unit time, and at least one or more thresholds set for the rate of change. , A control device for controlling the swing speed of the drive device.
Further, in the cell culture device according to one aspect, the measuring device is a first measuring device having an electrode for forming an electric field in at least a part of the region and a capacitance measuring unit for measuring the capacitance in the electric field. The first measuring device measures the cell distribution information value in at least a part of the region based on the capacitance.
Further, in the cell culture apparatus according to one aspect, the first measuring apparatus further includes a conductivity measuring unit for measuring conductivity in at least a part of the region.
また、一態様に係る前記細胞培養装置において、前記測定装置は、前記少なくとも一部の領域において電場を形成させる電極と、前記電場内のキャパシタンスを測定するキャパシタンス測定部と、を有する第1測定装置を含み、前記第1測定装置は、前記キャパシタンスに基づいて、前記少なくとも一部の領域における前記細胞分布情報値を測定する。
また、一態様に係る前記細胞培養装置において、前記第1測定装置は、前記少なくとも一部の領域における導電率を測定する導電率測定部、をさらに有する。 Further, the cell culture apparatus according to one aspect is a culture containing a suspension containing at least one of a cell mass which is an aggregate of a plurality of cells and a cell complex which is a complex of the cells and a culture carrier. At least one of the cell distribution information value for the cell and the carrier distribution information value for the culture carrier in at least a part of the region of the suspension, the driving device for rocking the suspension, and the container is measured. Based on the relationship between the measuring device to be used, the rate of change of at least one of the cell distribution information value and the carrier distribution information value per predetermined unit time, and at least one or more thresholds set for the rate of change. , A control device for controlling the swing speed of the drive device.
Further, in the cell culture device according to one aspect, the measuring device is a first measuring device having an electrode for forming an electric field in at least a part of the region and a capacitance measuring unit for measuring the capacitance in the electric field. The first measuring device measures the cell distribution information value in at least a part of the region based on the capacitance.
Further, in the cell culture apparatus according to one aspect, the first measuring apparatus further includes a conductivity measuring unit for measuring conductivity in at least a part of the region.
また、一態様に係る前記細胞培養装置において、前記第1測定装置は、前記少なくとも一部の領域におけるpHを測定するpH測定部、をさらに有する。
また、一態様に係る前記細胞培養装置において、前記測定装置は、前記少なくとも一部の領域を撮像して1つ以上の画像を取得する撮像部、を有する第2測定装置を含み、前記第2測定装置は、前記画像に基づいて、前記少なくとも一部の領域における前記細胞分布情報値及び前記担体分布情報値の少なくとも一方を測定する。
また、一態様に係る前記細胞培養装置において、前記細胞分布情報値は、前記少なくとも一部の領域における、前記懸濁液中の前記細胞のうちの生細胞数又は生細胞濃度である。 Further, in the cell culture device according to one aspect, the first measuring device further includes a pH measuring unit for measuring pH in at least a part of the region.
Further, in the cell culture device according to one aspect, the measuring device includes a second measuring device including an imaging unit that captures an image of at least a part of the region and acquires one or more images. The measuring device measures at least one of the cell distribution information value and the carrier distribution information value in the at least a part region based on the image.
Further, in the cell culture apparatus according to one embodiment, the cell distribution information value is the number of living cells or the concentration of living cells among the cells in the suspension in the at least a part of the region.
また、一態様に係る前記細胞培養装置において、前記測定装置は、前記少なくとも一部の領域を撮像して1つ以上の画像を取得する撮像部、を有する第2測定装置を含み、前記第2測定装置は、前記画像に基づいて、前記少なくとも一部の領域における前記細胞分布情報値及び前記担体分布情報値の少なくとも一方を測定する。
また、一態様に係る前記細胞培養装置において、前記細胞分布情報値は、前記少なくとも一部の領域における、前記懸濁液中の前記細胞のうちの生細胞数又は生細胞濃度である。 Further, in the cell culture device according to one aspect, the first measuring device further includes a pH measuring unit for measuring pH in at least a part of the region.
Further, in the cell culture device according to one aspect, the measuring device includes a second measuring device including an imaging unit that captures an image of at least a part of the region and acquires one or more images. The measuring device measures at least one of the cell distribution information value and the carrier distribution information value in the at least a part region based on the image.
Further, in the cell culture apparatus according to one embodiment, the cell distribution information value is the number of living cells or the concentration of living cells among the cells in the suspension in the at least a part of the region.
また、一態様に係る前記細胞培養装置において、前記担体分布情報値は、前記少なくとも一部の領域における、前記懸濁液中の前記培養担体の数又は前記培養担体の濃度である。
また、一態様に係る前記細胞培養装置において、前記閾値は、前記変化率に対して、0%に設定される第1閾値を含む。
また、一態様に係る前記細胞培養装置において、前記閾値は、前記変化率に対して、0%よりも大きい値に設定される第2閾値をさらに有する。 Further, in the cell culture apparatus according to one embodiment, the carrier distribution information value is the number of the culture carriers in the suspension or the concentration of the culture carriers in the at least a part of the region.
Further, in the cell culture apparatus according to one aspect, the threshold value includes a first threshold value set to 0% with respect to the rate of change.
Further, in the cell culture apparatus according to one aspect, the threshold value further has a second threshold value set to a value larger than 0% with respect to the rate of change.
また、一態様に係る前記細胞培養装置において、前記閾値は、前記変化率に対して、0%に設定される第1閾値を含む。
また、一態様に係る前記細胞培養装置において、前記閾値は、前記変化率に対して、0%よりも大きい値に設定される第2閾値をさらに有する。 Further, in the cell culture apparatus according to one embodiment, the carrier distribution information value is the number of the culture carriers in the suspension or the concentration of the culture carriers in the at least a part of the region.
Further, in the cell culture apparatus according to one aspect, the threshold value includes a first threshold value set to 0% with respect to the rate of change.
Further, in the cell culture apparatus according to one aspect, the threshold value further has a second threshold value set to a value larger than 0% with respect to the rate of change.
また、一態様に係る前記細胞培養装置において、前記制御装置は、前記変化率が前記第1閾値以上と判定された場合、前記駆動装置の前記揺動速度を増加させ、前記変化率が前記第1閾値未満と判定された場合、前記駆動装置の前記揺動速度を減少させる。
また、一態様に係る前記細胞培養装置において、前記制御装置は、前記変化率が、複数の前記所定単位時間に渡って連続で前記第1閾値未満と判定された場合、前記駆動装置の前記揺動速度を0とする。
また、一態様に係る前記細胞培養装置において、前記制御装置は、前記変化率が前記第2閾値以上と判定された場合、該判定時の前記揺動速度を維持させる。 Further, in the cell culture device according to one aspect, when the control device determines that the rate of change is equal to or higher than the first threshold value, the control device increases the swing speed of the drive device, and the rate of change is the first. If it is determined to be less than one threshold value, the rocking speed of the driving device is reduced.
Further, in the cell culture device according to one embodiment, when the control device continuously determines that the rate of change is less than the first threshold value over a plurality of the predetermined unit times, the control device shakes the drive device. The moving speed is set to 0.
Further, in the cell culture device according to one aspect, when the rate of change is determined to be equal to or higher than the second threshold value, the control device maintains the swing speed at the time of the determination.
また、一態様に係る前記細胞培養装置において、前記制御装置は、前記変化率が、複数の前記所定単位時間に渡って連続で前記第1閾値未満と判定された場合、前記駆動装置の前記揺動速度を0とする。
また、一態様に係る前記細胞培養装置において、前記制御装置は、前記変化率が前記第2閾値以上と判定された場合、該判定時の前記揺動速度を維持させる。 Further, in the cell culture device according to one aspect, when the control device determines that the rate of change is equal to or higher than the first threshold value, the control device increases the swing speed of the drive device, and the rate of change is the first. If it is determined to be less than one threshold value, the rocking speed of the driving device is reduced.
Further, in the cell culture device according to one embodiment, when the control device continuously determines that the rate of change is less than the first threshold value over a plurality of the predetermined unit times, the control device shakes the drive device. The moving speed is set to 0.
Further, in the cell culture device according to one aspect, when the rate of change is determined to be equal to or higher than the second threshold value, the control device maintains the swing speed at the time of the determination.
また、一態様に係る前記細胞培養装置において、前記駆動装置は、前記懸濁液を撹拌する撹拌機又は振盪機である。
また、一態様に係る前記細胞培養装置において、前記培養容器の容量は、2.0L以上である。
また、一態様に係る前記細胞培養装置において、前記少なくとも一部の領域は、前記懸濁液中であって前記培養容器の深さ方向における中央部分、及び前記懸濁液中であって前記培養容器の底面部分の少なくとも一方を含む。 Further, in the cell culture device according to one embodiment, the driving device is a stirrer or a shaker that stirs the suspension.
Further, in the cell culture apparatus according to one aspect, the capacity of the culture container is 2.0 L or more.
Further, in the cell culture apparatus according to one embodiment, the at least a part of the region is the central portion in the suspension in the depth direction of the culture vessel, and the culture in the suspension. Includes at least one of the bottom portions of the container.
また、一態様に係る前記細胞培養装置において、前記培養容器の容量は、2.0L以上である。
また、一態様に係る前記細胞培養装置において、前記少なくとも一部の領域は、前記懸濁液中であって前記培養容器の深さ方向における中央部分、及び前記懸濁液中であって前記培養容器の底面部分の少なくとも一方を含む。 Further, in the cell culture device according to one embodiment, the driving device is a stirrer or a shaker that stirs the suspension.
Further, in the cell culture apparatus according to one aspect, the capacity of the culture container is 2.0 L or more.
Further, in the cell culture apparatus according to one embodiment, the at least a part of the region is the central portion in the suspension in the depth direction of the culture vessel, and the culture in the suspension. Includes at least one of the bottom portions of the container.
別の態様に係る細胞培養装置は、細胞と、前記細胞が接着する被接着体との塊体を少なくとも含む懸濁液を収容する培養容器と、前記懸濁液を揺動させる駆動装置と、前記懸濁液中の複数の領域における、前記塊体に関する塊体分布情報値を各々測定する測定装置と、前記複数の領域のうちの第1の領域における第1塊体分布情報値と、前記複数の領域のうちの第2の領域における第2塊体分布情報値とを比較演算することにより算出される塊体分布情報相違値と、該塊体分布情報相違値に対して設定される少なくとも1つ以上の閾値との関係に基づいて、前記駆動装置の揺動速度を制御する制御装置と、を具備する。
The cell culture apparatus according to another embodiment includes a culture vessel containing at least a suspension containing cells and a mass of an adherend to which the cells adhere, a driving device for rocking the suspension, and the like. A measuring device for measuring the mass distribution information value for the mass in the plurality of regions in the suspension, the first mass distribution information value in the first region of the plurality of regions, and the above. The mass distribution information difference value calculated by comparing the second mass distribution information value in the second region of the plurality of regions and at least set for the mass distribution information difference value. A control device for controlling the swing speed of the drive device based on the relationship with one or more thresholds is provided.
また、別の態様に係る前記細胞培養装置は、複数の細胞の凝集体である細胞塊、及び前記細胞と培養担体との複合体である細胞複合体の少なくとも一方を含む懸濁液を収容する培養容器と、前記懸濁液を揺動させる駆動装置と、前記懸濁液中の複数の領域における、前記培養担体に関する担体分布情報値を各々測定する測定装置と、前記複数の領域のうちの第1の領域における第1担体分布情報値と、前記複数の領域のうちの第2の領域における第2担体分布情報値とを比較演算することにより算出される担体分布情報相違値と、該担体分布情報相違値に対して設定される少なくとも1つ以上の閾値との関係に基づいて、前記駆動装置の揺動速度を制御する制御装置と、を具備する。
また、別の態様に係る前記細胞培養装置において、前記測定装置は、前記複数の領域を撮像して1つ以上の画像を取得する撮像装置、を有する第3測定装置を含み、前記第3測定装置は、前記第1の領域における前記画像に基づいて前記第1担体分布情報値を測定し、且つ前記第2の領域における前記画像に基づいて前記第2担体分布情報値を測定する。
また、別の態様に係る前記細胞培養装置において、前記担体分布情報値は、前記複数の領域の各々における、前記懸濁液中の前記培養担体の数又は前記培養担体の濃度であり、前記第1担体分布情報値は、前記第1の領域における、前記懸濁液中の前記培養担体の数又は前記培養担体の濃度であり、前記第2担体分布情報値は、前記第2の領域における、前記懸濁液中の前記培養担体の数又は前記培養担体の濃度である。 Further, the cell culture apparatus according to another aspect contains a suspension containing at least one of a cell mass which is an aggregate of a plurality of cells and a cell complex which is a complex of the cells and a culture carrier. Of the culture vessel, the driving device for rocking the suspension, the measuring device for measuring the carrier distribution information value for the culture carrier in the plurality of regions in the suspension, and the plurality of regions. The carrier distribution information difference value calculated by comparing the first carrier distribution information value in the first region with the second carrier distribution information value in the second region of the plurality of regions, and the carrier. A control device for controlling the swing speed of the drive device based on the relationship with at least one or more threshold values set for the distribution information difference value is provided.
Further, in the cell culture device according to another aspect, the measuring device includes a third measuring device having an imaging device for capturing an image of the plurality of regions and acquiring one or more images, and the third measurement. The apparatus measures the first carrier distribution information value based on the image in the first region, and measures the second carrier distribution information value based on the image in the second region.
Further, in the cell culture apparatus according to another aspect, the carrier distribution information value is the number of the culture carriers in the suspension or the concentration of the culture carriers in each of the plurality of regions, and is the first. The 1 carrier distribution information value is the number of the culture carriers in the suspension or the concentration of the culture carrier in the first region, and the second carrier distribution information value is the number of the culture carriers in the second region. The number of the culture carriers or the concentration of the culture carriers in the suspension.
また、別の態様に係る前記細胞培養装置において、前記測定装置は、前記複数の領域を撮像して1つ以上の画像を取得する撮像装置、を有する第3測定装置を含み、前記第3測定装置は、前記第1の領域における前記画像に基づいて前記第1担体分布情報値を測定し、且つ前記第2の領域における前記画像に基づいて前記第2担体分布情報値を測定する。
また、別の態様に係る前記細胞培養装置において、前記担体分布情報値は、前記複数の領域の各々における、前記懸濁液中の前記培養担体の数又は前記培養担体の濃度であり、前記第1担体分布情報値は、前記第1の領域における、前記懸濁液中の前記培養担体の数又は前記培養担体の濃度であり、前記第2担体分布情報値は、前記第2の領域における、前記懸濁液中の前記培養担体の数又は前記培養担体の濃度である。 Further, the cell culture apparatus according to another aspect contains a suspension containing at least one of a cell mass which is an aggregate of a plurality of cells and a cell complex which is a complex of the cells and a culture carrier. Of the culture vessel, the driving device for rocking the suspension, the measuring device for measuring the carrier distribution information value for the culture carrier in the plurality of regions in the suspension, and the plurality of regions. The carrier distribution information difference value calculated by comparing the first carrier distribution information value in the first region with the second carrier distribution information value in the second region of the plurality of regions, and the carrier. A control device for controlling the swing speed of the drive device based on the relationship with at least one or more threshold values set for the distribution information difference value is provided.
Further, in the cell culture device according to another aspect, the measuring device includes a third measuring device having an imaging device for capturing an image of the plurality of regions and acquiring one or more images, and the third measurement. The apparatus measures the first carrier distribution information value based on the image in the first region, and measures the second carrier distribution information value based on the image in the second region.
Further, in the cell culture apparatus according to another aspect, the carrier distribution information value is the number of the culture carriers in the suspension or the concentration of the culture carriers in each of the plurality of regions, and is the first. The 1 carrier distribution information value is the number of the culture carriers in the suspension or the concentration of the culture carrier in the first region, and the second carrier distribution information value is the number of the culture carriers in the second region. The number of the culture carriers or the concentration of the culture carriers in the suspension.
また、別の態様に係る前記細胞培養装置において、前記駆動装置は、前記懸濁液を撹拌する撹拌機又は振盪機である。
また、別の態様に係る前記細胞培養装置において、前記培養容器の容量は、2.0L以上である。
また、別の態様に係る前記細胞培養装置において、前記第1の領域は、前記懸濁液中であって前記培養容器の深さ方向における中央部分であり、前記第2の領域は、前記懸濁液中であって前記培養容器の底面部分である。 Further, in the cell culture device according to another aspect, the driving device is a stirrer or a shaker that stirs the suspension.
Further, in the cell culture apparatus according to another aspect, the capacity of the culture container is 2.0 L or more.
Further, in the cell culture apparatus according to another aspect, the first region is a central portion in the suspension in the depth direction of the culture vessel, and the second region is the suspension. It is in the turbid liquid and is the bottom portion of the culture vessel.
また、別の態様に係る前記細胞培養装置において、前記培養容器の容量は、2.0L以上である。
また、別の態様に係る前記細胞培養装置において、前記第1の領域は、前記懸濁液中であって前記培養容器の深さ方向における中央部分であり、前記第2の領域は、前記懸濁液中であって前記培養容器の底面部分である。 Further, in the cell culture device according to another aspect, the driving device is a stirrer or a shaker that stirs the suspension.
Further, in the cell culture apparatus according to another aspect, the capacity of the culture container is 2.0 L or more.
Further, in the cell culture apparatus according to another aspect, the first region is a central portion in the suspension in the depth direction of the culture vessel, and the second region is the suspension. It is in the turbid liquid and is the bottom portion of the culture vessel.
一態様に係る細胞群を生産する方法は、細胞と、前記細胞が接着する被接着体との塊体を少なくとも含む懸濁液を培養容器内に収容すること、駆動装置によって前記懸濁液を揺動させること、前記懸濁液中の少なくとも一部の領域における、前記塊体に関する塊体分布情報値を測定すること、前記塊体分布情報値の所定単位時間あたりの変化率と、前記変化率に対して設定される少なくとも1つ以上の閾値との関係を判定し、該判定の結果に基づいて前記駆動装置の揺動速度を決定すること、及び、決定された前記揺動速度を前記駆動装置に出力すること、を含む。
A method for producing a cell group according to one embodiment is to house a suspension containing at least a mass of cells and an adherend to which the cells adhere in a culture vessel, and to prepare the suspension by a driving device. Shaking, measuring the mass distribution information value for the mass in at least a part of the region of the suspension, the rate of change of the mass distribution information value per predetermined unit time, and the change. The relationship with at least one or more thresholds set for the rate is determined, and the rocking speed of the driving device is determined based on the result of the determination, and the determined rocking speed is used as described above. Includes outputting to the drive.
また、一態様に係る前記細胞群を生産する方法は、複数の細胞の凝集体である細胞塊、及び前記細胞と培養担体との複合体である細胞複合体の少なくとも一方を含む懸濁液を培養容器内に収容すること、駆動装置によって前記懸濁液を揺動させること、前記懸濁液中の少なくとも一部の領域における、前記細胞に関する細胞分布情報値、及び前記培養担体に関する担体分布情報値の少なくとも一方を測定すること、前記細胞分布情報値及び前記担体分布情報値の少なくとも一方の所定単位時間あたりの変化率と、前記変化率に対して設定される少なくとも1つ以上の閾値との関係を判定し、該判定の結果に基づいて前記駆動装置の揺動速度を決定すること、及び、決定された前記揺動速度を前記駆動装置に出力すること、を含む培養方法により実行される。
Further, the method for producing the cell group according to one embodiment includes a suspension containing at least one of a cell mass which is an aggregate of a plurality of cells and a cell complex which is a complex of the cells and a culture carrier. Containment in a culture vessel, rocking the suspension by a driving device, cell distribution information values for the cells in at least a portion of the suspension, and carrier distribution information for the culture carrier. Measuring at least one of the values, the rate of change of at least one of the cell distribution information value and the carrier distribution information value per predetermined unit time, and at least one or more thresholds set for the rate of change. It is carried out by a culture method including determining a relationship, determining the rocking speed of the driving device based on the result of the determination, and outputting the determined rocking speed to the driving device. ..
別の態様に係る細胞群を生産する方法は、細胞と、前記細胞が接着する被接着体との塊体を少なくとも含む懸濁液を培養容器内に収容すること、駆動装置によって前記懸濁液を揺動させること、前記懸濁液中の複数の領域における、前記塊体に関する塊体分布情報値を各々測定すること、前記複数の領域のうちの第1の領域における第1塊体分布情報値と、前記複数の領域のうちの第2の領域における第2塊体分布情報値とを比較演算することにより算出される塊体分布情報相違値と、該塊体分布情報相違値に対して設定される少なくとも1つ以上の閾値との関係を判定し、該判定の結果に基づいて前記駆動装置の揺動速度を決定すること、及び、決定された前記揺動速度を前記駆動装置に出力すること、を含む。
A method for producing a cell group according to another embodiment is to house a suspension containing at least a mass of cells and an adherend to which the cells adhere in a culture vessel, and the suspension by a driving device. To measure the mass distribution information value for the mass in the plurality of regions in the suspension, and to measure the mass distribution information in the first region of the plurality of regions. With respect to the mass distribution information difference value calculated by comparing the value with the second mass distribution information value in the second region of the plurality of regions and the mass distribution information difference value. The relationship with at least one set threshold value is determined, the rocking speed of the drive device is determined based on the result of the determination, and the determined rocking speed is output to the drive device. Including to do.
また、別の態様に係る細胞群を生産する方法は、複数の細胞の凝集体である細胞塊、及び前記細胞と培養担体との複合体である細胞複合体の少なくとも一方を含む懸濁液を培養容器内に収容すること、駆動装置によって前記懸濁液を揺動させること、前記懸濁液中の複数の領域における、前記培養担体に関する担体分布情報値を各々測定すること、前記複数の領域のうちの第1の領域における第1担体分布情報値と、前記複数の領域のうちの第2の領域における第2担体分布情報値とを比較演算することにより算出される担体分布情報相違値と、該担体分布情報相違値に対して設定される少なくとも1つ以上の閾値との関係を判定し、該判定の結果に基づいて前記駆動装置の揺動速度を決定すること、及び、決定された前記揺動速度を前記駆動装置に出力すること、を含む培養方法により実行される。
Further, a method for producing a cell group according to another embodiment comprises a suspension containing at least one of a cell mass which is an aggregate of a plurality of cells and a cell complex which is a complex of the cells and a culture carrier. Containment in a culture vessel, rocking the suspension by a driving device, measuring carrier distribution information values for the culture carrier in a plurality of regions in the suspension, the plurality of regions. The carrier distribution information difference value calculated by comparing the first carrier distribution information value in the first region of the above with the second carrier distribution information value in the second region of the plurality of regions. , The relationship with at least one threshold set for the carrier distribution information difference value is determined, and the rocking speed of the driving device is determined based on the result of the determination, and determined. It is carried out by a culture method including outputting the rocking speed to the driving device.
様々な実施形態によれば、培養容器内での細胞塊、細胞複合体等の沈降を抑制して、高い収率性と時間効率性で細胞を培養することが可能な細胞培養装置、及び、細胞群を生産する方法を提供することができる。
According to various embodiments, a cell culture apparatus capable of culturing cells with high yield and time efficiency by suppressing sedimentation of cell masses, cell complexes, etc. in a culture vessel, and A method of producing a cell culture can be provided.
以下、添付図面を参照して様々な実施形態を説明する。なお、図面において共通した構成要件には同一の参照符号が付されている。また、或る図面に表現された構成要素が、説明の便宜上、別の図面においては省略されていることがある点に留意されたい。さらにまた、添付した図面が必ずしも正確な縮尺で記載されている訳ではないということに注意されたい。
Hereinafter, various embodiments will be described with reference to the attached drawings. The same reference numerals are given to the common constituent requirements in the drawings. It should also be noted that the components represented in one drawing may be omitted in another drawing for convenience of explanation. Furthermore, it should be noted that the attached drawings are not always drawn to the correct scale.
1.細胞培養装置の構成
一実施形態に係る細胞培養装置の全体構成の概要について、図1乃至図7を参照しつつ説明する。図1は、一実施形態に係る細胞培養装置1の構成を模式的に示す概略図である。図2は、一実施形態に係る細胞培養装置1において、測定装置30として第1測定装置300が用いられる場合の構成を模式的に示す概略図である。図3は、図2において点線で囲まれた領域Pを拡大して示すものであって、第1測定装置300の一部を拡大して模式的に示す概略図である。図4は、一実施形態に係る細胞培養装置1において、図1及び図2に示される駆動装置20とは異なる、別の駆動装置20の態様の一例を模式的に示す概略図である。図5は、一実施形態に係る制御装置40の機能の一例を模式的に示すブロック図である。図6及び図7は、一実施形態に係る制御装置40の判定部43による判定結果と出力部44が決定する駆動装置20の揺動速度との関係の一例を模式的に示す図である。 1. 1. Configuration of Cell Culture Device An outline of the overall configuration of the cell culture device according to the embodiment will be described with reference to FIGS. 1 to 7. FIG. 1 is a schematic diagram schematically showing the configuration of thecell culture apparatus 1 according to the embodiment. FIG. 2 is a schematic diagram schematically showing a configuration when the first measuring device 300 is used as the measuring device 30 in the cell culture device 1 according to the embodiment. FIG. 3 is an enlarged view of the region P surrounded by the dotted line in FIG. 2, and is a schematic diagram schematically showing an enlarged part of the first measuring device 300. FIG. 4 is a schematic diagram schematically showing an example of another mode of the driving device 20 different from the driving device 20 shown in FIGS. 1 and 2 in the cell culture device 1 according to the embodiment. FIG. 5 is a block diagram schematically showing an example of the function of the control device 40 according to the embodiment. 6 and 7 are diagrams schematically showing an example of the relationship between the determination result by the determination unit 43 of the control device 40 according to the embodiment and the swing speed of the drive device 20 determined by the output unit 44.
一実施形態に係る細胞培養装置の全体構成の概要について、図1乃至図7を参照しつつ説明する。図1は、一実施形態に係る細胞培養装置1の構成を模式的に示す概略図である。図2は、一実施形態に係る細胞培養装置1において、測定装置30として第1測定装置300が用いられる場合の構成を模式的に示す概略図である。図3は、図2において点線で囲まれた領域Pを拡大して示すものであって、第1測定装置300の一部を拡大して模式的に示す概略図である。図4は、一実施形態に係る細胞培養装置1において、図1及び図2に示される駆動装置20とは異なる、別の駆動装置20の態様の一例を模式的に示す概略図である。図5は、一実施形態に係る制御装置40の機能の一例を模式的に示すブロック図である。図6及び図7は、一実施形態に係る制御装置40の判定部43による判定結果と出力部44が決定する駆動装置20の揺動速度との関係の一例を模式的に示す図である。 1. 1. Configuration of Cell Culture Device An outline of the overall configuration of the cell culture device according to the embodiment will be described with reference to FIGS. 1 to 7. FIG. 1 is a schematic diagram schematically showing the configuration of the
一実施形態に係る細胞培養装置1において培養に供される細胞としては、浮遊培養が可能な細胞であれば特に制限はなく、浮遊性細胞であっても、接着性細胞であってもよい。また、細胞は、浮遊培養中に複数の細胞が接触して凝集体を形成するか、又は培養担体に接着して1若しくは複数の細胞と培養担体との複合体を形成可能であれば、培養中に単一の細胞として懸濁液中に浮遊してもよい。本開示において、「単一の細胞」とは、独立した1個の細胞を意味する。本開示において「複数の細胞の凝集体である細胞塊」は、細胞が互いに接して形成された塊を意味し、特に制限はなく、細胞増殖期に立体的に形成されるスフェロイド、その他の要因により2以上の細胞が集まって形成された塊等を含む。
細胞は、動物由来の細胞であることが好ましく、哺乳動物由来の細胞であることがより好ましい。哺乳動物として、例えば、ヒト、サル、チンパンジー、ウシ、ブタ、ウマ、ヒツジ、ヤギ、ウサギ、ラット、マウス、モルモット、イヌ、ネコ等が挙げられる。細胞は、例えば、皮膚、肝臓、腎臓、筋肉、骨、血管、血液、神経組織等の組織に由来する細胞であってよい。細胞は、通常は1種を単独で培養に供するが、2種以上を組み合わせて培養に供してもよい。細胞は、組織からの初代細胞であってもよく、不死化することによって確立された細胞株であってもよい。 The cell to be cultured in thecell culture apparatus 1 according to the embodiment is not particularly limited as long as it is a cell capable of suspension culture, and may be a floating cell or an adhesive cell. Further, the cells are cultured if a plurality of cells can contact each other to form an aggregate during suspension culture or adhere to a culture carrier to form a complex of one or a plurality of cells and the culture carrier. It may float in the suspension as a single cell inside. In the present disclosure, "single cell" means one independent cell. In the present disclosure, "cell mass which is an aggregate of a plurality of cells" means a mass formed by contacting cells with each other, and is not particularly limited, and is a spheroid formed sterically during the cell proliferation phase and other factors. Includes a mass formed by aggregating two or more cells.
The cells are preferably animal-derived cells, more preferably mammalian-derived cells. Examples of mammals include humans, monkeys, chimpanzees, cows, pigs, horses, sheep, goats, rabbits, rats, mice, guinea pigs, dogs, cats and the like. The cell may be, for example, a cell derived from a tissue such as skin, liver, kidney, muscle, bone, blood vessel, blood, or nervous tissue. Usually, one type of cell is subjected to culture alone, but two or more types may be used in combination for culture. The cell may be a primary cell from tissue or a cell line established by immortalization.
細胞は、動物由来の細胞であることが好ましく、哺乳動物由来の細胞であることがより好ましい。哺乳動物として、例えば、ヒト、サル、チンパンジー、ウシ、ブタ、ウマ、ヒツジ、ヤギ、ウサギ、ラット、マウス、モルモット、イヌ、ネコ等が挙げられる。細胞は、例えば、皮膚、肝臓、腎臓、筋肉、骨、血管、血液、神経組織等の組織に由来する細胞であってよい。細胞は、通常は1種を単独で培養に供するが、2種以上を組み合わせて培養に供してもよい。細胞は、組織からの初代細胞であってもよく、不死化することによって確立された細胞株であってもよい。 The cell to be cultured in the
The cells are preferably animal-derived cells, more preferably mammalian-derived cells. Examples of mammals include humans, monkeys, chimpanzees, cows, pigs, horses, sheep, goats, rabbits, rats, mice, guinea pigs, dogs, cats and the like. The cell may be, for example, a cell derived from a tissue such as skin, liver, kidney, muscle, bone, blood vessel, blood, or nervous tissue. Usually, one type of cell is subjected to culture alone, but two or more types may be used in combination for culture. The cell may be a primary cell from tissue or a cell line established by immortalization.
接着性細胞としては、例えば、体細胞、幹細胞等が挙げられる。体細胞として、例えば、内皮細胞、表皮細胞、上皮細胞、心筋細胞、筋芽細胞、神経細胞、骨細胞、骨芽細胞、線維芽細胞、脂肪細胞、肝細胞、腎細胞、膵細胞、副腎細胞、歯根膜細胞、歯肉細胞、骨膜細胞、皮膚細胞、樹状細胞、マクロファージ等が挙げられる。
Examples of adhesive cells include somatic cells, stem cells and the like. As somatic cells, for example, endothelial cells, epidermal cells, epithelial cells, myocardial cells, myoblasts, nerve cells, bone cells, osteoblasts, fibroblasts, fat cells, hepatocytes, renal cells, pancreatic cells, adrenal cells. , Root membrane cells, gingival cells, bone membrane cells, skin cells, dendritic cells, macrophages and the like.
一実施形態において、接着細胞は、幹細胞であってよい。幹細胞としては、間葉系幹細胞、造血系幹細胞、神経系幹細胞、骨髄幹細胞、生殖幹細胞等の体性幹細胞などを挙げることができ、間葉系幹細胞、又は骨髄間葉系幹細胞とすることができる。間葉系幹細胞とは、人体の様々な組織に存在し、骨芽細胞、軟骨細胞及び脂肪細胞等の間葉系の細胞の全て又はいくつかへの分化が可能な体性幹細胞を広義に意味する。幹細胞には、更に、人工多能性幹細胞(Induced pluripotent stem cells;iPS細胞)、胚性幹細胞(Embryonic stem cell;ES細胞)が含まれていてもよい。
In one embodiment, the adherent cells may be stem cells. Examples of stem cells include somatic stem cells such as mesenchymal stem cells, hematopoietic stem cells, neural stem cells, bone marrow stem cells, and germ stem cells, and can be mesenchymal stem cells or bone marrow mesenchymal stem cells. .. Mesenchymal stem cells broadly mean somatic stem cells that are present in various tissues of the human body and can be differentiated into all or some of mesenchymal cells such as osteoblasts, chondrocytes and adipocytes. do. The stem cells may further include induced pluripotent stem cells (iPS cells) and embryonic stem cells (ES cells).
浮遊性細胞としては、いずれの細胞であってもよい。例えば、チャイニーズハムスター卵巣細胞(Chinese hamster ovary cell;CHO細胞)、ベロ細胞(Vero cell)、Jurkat、HL60、FM3A等が挙げられるが、これらに限定されない。
The floating cell may be any cell. Examples thereof include, but are not limited to, Chinese hamster ovary cells (CHO cells), Vero cells, Jurkat, HL60, FM3A and the like.
懸濁液は、これらの細胞を、細胞の種類に応じた培地に懸濁して得ることができる。培養開始時の懸濁液の細胞の濃度は、例えば培養担体を懸濁液中に混入させる場合、1×103~2×105細胞/mL、5×103~1×105細胞/mL、又は1×104~5×104細胞/mLとすることができ、培養担体を懸濁液中に混入させない場合、5×103~2×108細胞/mL、1×104~1×107細胞/mL、又は5×104~1×106細胞/mLとすることができる。
The suspension can be obtained by suspending these cells in a medium according to the cell type. The cell concentration of the suspension at the start of culture is, for example, 1 × 10 3 to 2 × 10 5 cells / mL, 5 × 10 3 to 1 × 10 5 cells / when the culture carrier is mixed in the suspension. It can be mL or 1 × 10 4-5 × 10 4 cells / mL, 5 × 10 3-2 × 10 8 cells / mL, 1 × 10 4 if the culture carrier is not mixed into the suspension. It can be ~ 1 × 10 7 cells / mL or 5 × 10 4 ~ 1 × 10 6 cells / mL.
一実施形態に係る細胞培養装置1は、培養容器10と、駆動装置20と、測定装置30と、制御装置40と、から主に構成されるが、これら以外のものが含まれてもよい。例えば、培養容器内に収容される懸濁液を所定の温度に加温するためのヒーター等を別途設けてもよい。また、測定装置30と制御装置40とは、図1においては、別々に設けられているが、これに限定されず、測定装置30の少なくとも一部と制御装置40とが一体に形成されたものを用いてもよい。以下、細胞培養装置1の主要な構成要素の詳細を説明する。
The cell culture device 1 according to one embodiment is mainly composed of a culture container 10, a driving device 20, a measuring device 30, and a control device 40, but may include other than these. For example, a heater or the like for heating the suspension contained in the culture vessel to a predetermined temperature may be separately provided. Further, the measuring device 30 and the control device 40 are provided separately in FIG. 1, but the present invention is not limited to this, and at least a part of the measuring device 30 and the control device 40 are integrally formed. May be used. Hereinafter, the details of the main components of the cell culture apparatus 1 will be described.
1-1.培養容器10及び培養容器10内の懸濁液
一実施形態に係る培養容器10は、一般的に知られたバイオリアクタを用いることができる。培養容器10の容量は、浮遊培養且つ大量培養を可能とするために、例えば0.3L以上、0.5L以上、1L以上、2L以上、5L以上、8L以上、又は10L以上とすることができる。培養容器10の容量の上限値としては、特に制限はないが、効率性及び経済性の観点から、例えば50L以下、40L以下、又は30L以下とすることができるが、80L以上、90L以上、100L以上、又は、それ以上であってもよい。 1-1. Theculture vessel 10 and the suspension in the culture vessel 10 As the culture vessel 10 according to the embodiment, a generally known bioreactor can be used. The capacity of the culture vessel 10 can be, for example, 0.3 L or more, 0.5 L or more, 1 L or more, 2 L or more, 5 L or more, 8 L or more, or 10 L or more in order to enable suspension culture and mass culture. .. The upper limit of the capacity of the culture vessel 10 is not particularly limited, but may be, for example, 50 L or less, 40 L or less, or 30 L or less from the viewpoint of efficiency and economy, but 80 L or more, 90 L or more, and 100 L. It may be the above or more.
一実施形態に係る培養容器10は、一般的に知られたバイオリアクタを用いることができる。培養容器10の容量は、浮遊培養且つ大量培養を可能とするために、例えば0.3L以上、0.5L以上、1L以上、2L以上、5L以上、8L以上、又は10L以上とすることができる。培養容器10の容量の上限値としては、特に制限はないが、効率性及び経済性の観点から、例えば50L以下、40L以下、又は30L以下とすることができるが、80L以上、90L以上、100L以上、又は、それ以上であってもよい。 1-1. The
培養容器10の形状については特に制限はなく、例えば、円柱状、角柱状、袋状のものを用いることができる。また、培養容器10の側面及び底面は、後述する撮像装置360(撮像部360)による懸濁液の撮像のために、撮像装置360から発射される照射光を通す材質、色、形状であることが好ましい。また、同様の理由から、培養容器10の周辺に配される他の構成要素(例えば、前述のヒーター等)は、撮像装置360から発射される照射光の進路とは異なる位置(該進路から外れた位置)において、培養容器10の側面及び底面の少なくともいずれかに配置されることが好ましい。
The shape of the culture vessel 10 is not particularly limited, and for example, a columnar, prismatic, or bag-shaped one can be used. Further, the side surface and the bottom surface of the culture vessel 10 are made of a material, color, and shape that allow irradiation light emitted from the image pickup device 360 to pass through for image pickup of the suspension by the image pickup device 360 (imaging unit 360) described later. Is preferable. Further, for the same reason, other components (for example, the above-mentioned heater and the like) arranged around the culture vessel 10 are located at a position different from the path of the irradiation light emitted from the image pickup apparatus 360 (out of the path). It is preferable that the culture vessel 10 is arranged on at least one of the side surface and the bottom surface of the culture vessel 10.
培養容器10内には、細胞を含む懸濁液が収容されて浮遊培養に供される。ここで、培養に供される細胞の種類に応じて、適宜、培養担体も懸濁液中に混入される。培養担体としては、例えば、細胞の浮遊培養に用いられるマイクロキャリアとして公知であり、直径数百μmのマイクロビーズを用いることができる。これにより、目的の細胞が培養される際、培地中を浮遊するマイクロビーズ上に細胞を担持(接触)させて増殖させることで、当該細胞を、高い培養面積、且つ高密度で得ることができる。
A suspension containing cells is housed in the culture vessel 10 and subjected to suspension culture. Here, the culture carrier is also appropriately mixed in the suspension depending on the type of cells to be cultured. As the culture carrier, for example, microbeads having a diameter of several hundred μm are known as microcarriers used for suspension culture of cells. Thereby, when the target cells are cultured, the cells can be obtained in a high culture area and high density by supporting (contacting) the cells on the microbeads floating in the medium and proliferating them. ..
なお、培養担体を用いて培養する場合、培養担体の培養容器10内への投入際又は直後、細胞の種類、状態等に応じて駆動装置20の駆動を一定時間停止させてもよく、間欠駆動させてもよく、連続駆動させてもよい。間欠駆動とは、所定の時間にわたり駆動装置20の揺動を実行させることと、所定の時間にわたり駆動装置20の揺動を停止させることとを交互に実施することをいう。交互に実施することとは、揺動の実行と、揺動の停止との組み合わせを、2回以上繰り返すことであってよい(「揺動を実行させる」とそれに続く「揺動を停止させる」を組み合わせの1回と数える。)。揺動の実行に対応する時間と、揺動の停止に対応する時間は、同じであってもよいし異なっていてもよい。また、2回以上の繰り返しにおいて、各回の間で、揺動の実行に対応する時間が常に同じであってもよいし異なっていてもよい。同様に、各回の間で、揺動の停止に対応する時間が常に同じであってもよいし異なっていてもよい。揺動を停止させる時間は、懸濁液を培養容器10内で静置させる時間であってよい。間欠駆動において「揺動を実行させる」場合の駆動装置20の揺動速度としては、後述する駆動装置20の具体的な揺動速度と同じであってもよいし、異なっていてもよい。
In the case of culturing using the culture carrier, the drive of the drive device 20 may be stopped for a certain period of time depending on the type, state, etc. of the cells immediately after or after the culture carrier is put into the culture container 10, and the drive may be intermittently driven. It may be made to drive continuously, or it may be driven continuously. The intermittent drive means that the swing of the drive device 20 is executed for a predetermined time and the swing of the drive device 20 is stopped for a predetermined time alternately. Alternately performing the swing may be a combination of the swing execution and the swing stop repeated two or more times (“execute swing” followed by “stop swing”. Is counted as one combination.) The time corresponding to the execution of the swing and the time corresponding to the stop of the swing may be the same or different. Further, in the two or more repetitions, the time corresponding to the execution of the swing may always be the same or different between the times. Similarly, between each round, the time corresponding to the stoppage of the swing may always be the same or different. The time for stopping the shaking may be the time for allowing the suspension to stand in the culture vessel 10. The swing speed of the drive device 20 when "swinging is executed" in the intermittent drive may be the same as or different from the specific swing speed of the drive device 20 described later.
揺動の実行に対応する時間としては、例えば、0.5~60分間であり、好ましくは1~20分間、より好ましくは3~10分間とすることができる。揺動の停止に対応する時間としては、例えば、0.1~10時間であり、好ましくは0.5~6時間、より好ましくは1~3時間とすることができる。「揺動を実行させる」とそれに続く「揺動を停止させる」の組み合わせの例としては、「『0.5~60分間、揺動させる』とそれに続く『0.1~10時間、揺動を停止させる』」が挙げられ、「『1~20分間、揺動させる』とそれに続く『0.5~6時間、揺動を停止させる』」が好ましく、「『3~10分間、揺動させる』とそれに続く『1~3時間、揺動を停止させる』」がより好ましい。
The time corresponding to the execution of the rocking can be, for example, 0.5 to 60 minutes, preferably 1 to 20 minutes, and more preferably 3 to 10 minutes. The time corresponding to the stop of the rocking may be, for example, 0.1 to 10 hours, preferably 0.5 to 6 hours, and more preferably 1 to 3 hours. As an example of the combination of "execute shaking" and subsequent "stop shaking", "swing for 0.5 to 60 minutes" followed by "swing for 0.1 to 10 hours". "Stop shaking" is mentioned, and "" Shake for 1 to 20 minutes "and subsequent" Stop swinging for 0.5 to 6 hours "" is preferable, and "Swing for 3 to 10 minutes" is preferable. It is more preferable to "make it stop" and then "stop shaking for 1 to 3 hours".
間欠駆動を行う時間(2回以上の「『揺動を実行させる』とそれに続く『揺動を停止させる』」を行う合計の時間)は、例えば、1~80時間であり、好ましくは10~40時間であり、より好ましくは20~30時間である。
The time for performing intermittent drive (total time for performing "exercise of rocking" and subsequent "stopping rocking" two or more times) is, for example, 1 to 80 hours, preferably 10 to. It is 40 hours, more preferably 20 to 30 hours.
間欠駆動を経た後は、通常の駆動(連続駆動)が実行される。後述する細胞培養装置1の様々な動作等は、基本的に連続駆動時に対応するものと理解されたい。連続駆動とは、所定の時間にわたり続けて揺動を行うことをいう。揺動を行う時間(前述の「異常」との判定により培養を停止する場合を除く。)は、例えば、1~14日であり、好ましくは2~10日であり、より好ましくは3~7日である。細胞の培養においては、例えば、懸濁液への培地の追加、培地交換等のために揺動が一時的に停止されることがある。本開示において一時的な停止とは、単に静置させるものではなく、懸濁液への培地の追加、培地交換等の処理のために揺動が一時的に停止されることを意味する。前記の揺動を行う時間は、一時的な停止前の揺動を行う時間と、一時的な停止後の揺動を行う時間との合計でなくてよい。すなわち、揺動の停止によって、揺動を行う時間を区切って測定してもよい。
After the intermittent drive, normal drive (continuous drive) is executed. It should be understood that various operations of the cell culture apparatus 1, which will be described later, basically correspond to continuous driving. Continuous drive means to continuously swing for a predetermined time. The shaking time (excluding the case where the culture is stopped due to the above-mentioned determination of "abnormality") is, for example, 1 to 14 days, preferably 2 to 10 days, and more preferably 3 to 7 days. It's a day. In cell culture, for example, shaking may be temporarily stopped due to addition of medium to the suspension, medium exchange, or the like. In the present disclosure, the term "temporary suspension" means that the rocking is temporarily suspended due to processing such as addition of medium to the suspension or medium replacement, rather than simply allowing the suspension to stand still. The swinging time does not have to be the sum of the swinging time before the temporary stop and the swinging time after the temporary stop. That is, the measurement may be performed by dividing the time during which the swing is performed by stopping the swing.
前述の間欠駆動と連続駆動とは、連続して行ってもよいし、又は、間欠駆動と連続駆動との間に、所定の時間にわたり揺動しないことを含めてもよい。所定の時間は、例えば、0.1~24時間であり、好ましくは0.5~5時間であり、より好ましくは1~2時間である。揺動しないことは、懸濁液を静置させることであってよい。連続駆動の後に、間欠駆動を行ってもよく、間欠駆動及び連続駆動の組み合わせを繰り返し行ってもよい。
The above-mentioned intermittent drive and continuous drive may be performed continuously, or may include not swinging for a predetermined time between the intermittent drive and the continuous drive. The predetermined time is, for example, 0.1 to 24 hours, preferably 0.5 to 5 hours, and more preferably 1 to 2 hours. Not shaking may be to allow the suspension to stand still. After the continuous drive, the intermittent drive may be performed, or the combination of the intermittent drive and the continuous drive may be repeatedly performed.
間欠駆動及び連続駆動を含む培養時間の合計(前述の「異常」との判定により培養を停止する場合を除く。)は、培養する細胞の種類、目的、培養条件等に応じて、適宜設定することができる。
The total culture time including intermittent drive and continuous drive (excluding the case where the culture is stopped due to the above-mentioned determination of "abnormality") is appropriately set according to the type, purpose, culture conditions, etc. of the cells to be cultured. be able to.
培養担体の材質は、例えば、有機物、無機物又はこれらの複合材料であればよく、溶解性又は不溶解性のいずれであってもよい。有機物として、例えば、有機物としては、例えば、ポリスチレン、ポリエステル、ポリウレタン、ポリエチレン、ポリプロピレン、ポリビニルアルコール、(メタ)アクリル系ポリマー、(メタ)アクリルアミド系ポリマー、シリコーン系ポリマー、エポキシ樹脂、ウレタン樹脂等の合成高分子、セルロース、デキストラン、コラーゲン、ポリガラクツロン酸、ポリアルギン酸、ゼラチン、タンパク質等の天然高分子などが挙げられるがこれらに限定されない。無機物としては、例えば、ガラス、セラミック、金属、合金、金属酸化物等が挙げられるが、これらに限定されない。一態様の培養担体として、ポリスチレンを含むものが用いられてもよい。
The material of the culture carrier may be, for example, an organic substance, an inorganic substance, or a composite material thereof, and may be either soluble or insoluble. As an organic substance, for example, as an organic substance, for example, synthesis of polystyrene, polyester, polyurethane, polyethylene, polypropylene, polyvinyl alcohol, (meth) acrylic polymer, (meth) acrylamide polymer, silicone polymer, epoxy resin, urethane resin and the like. Examples thereof include, but are not limited to, natural polymers such as polymers, cellulose, dextran, collagen, polygalacturonic acid, polyarginic acid, gelatin and proteins. Examples of the inorganic substance include, but are not limited to, glass, ceramics, metals, alloys, metal oxides and the like. As the culture carrier of one embodiment, one containing polystyrene may be used.
また、培養担体には、細胞との担持性(接着性)を向上させるために、培養担体の表面には、カチオン性官能基が導入されていてもよい。カチオン性官能基として、ジメチルアミノ基、ジエチルアミノ基、アミノ基等の置換又は非置換のアミノ基を含む基が挙げられる。また、細胞の付着を促進する観点から、培養担体の表面には、細胞接着性ポリマーが配置されていてもよい。細胞接着性ポリマーとしては、コラーゲン、ゼラチン、アルギン酸、Matrigel(商標)(BD Biosciences)、ヒアルロン酸、ラミニン、フィブロネクチン、ビトロネクチン、エラスチン、ヘパラン硫酸、デキストラン、デキストラン硫酸、コンドロイチン硫酸等が配置されていてもよい。
Further, in order to improve the supportability (adhesiveness) with cells in the culture carrier, a cationic functional group may be introduced on the surface of the culture carrier. Examples of the cationic functional group include a group containing a substituted or unsubstituted amino group such as a dimethylamino group, a diethylamino group and an amino group. Further, from the viewpoint of promoting cell adhesion, a cell adhesion polymer may be arranged on the surface of the culture carrier. As the cell adhesion polymer, collagen, gelatin, alginic acid, Matrigel ™ (BD Biosciences), hyaluronic acid, laminin, fibronectin, vitronectin, elastin, heparan sulfate, dextran, dextran sulfate, chondroitin sulfate and the like are arranged. good.
培養担体の形状としては、例えば、球状、扁平状、円柱状、板条、角柱状等が挙げられる。培養担体は、球状培養担体を含むことが好ましい。培養担体は、内部に細孔を有する多孔質培養担体であっても、内部に細胞を有しない培養担体であってもよい。
Examples of the shape of the culture carrier include spherical, flat, columnar, strip, and prismatic. The culture carrier preferably contains a spherical culture carrier. The culture carrier may be a porous culture carrier having pores inside or a culture carrier having no cells inside.
培養担体の平均粒子径(D50)は、細胞の増殖促進の観点から、例えば50~1,000μmであり、100~500μmであることが好ましく、150~250μmであることがより好ましい。培養担体の平均粒子径は、生理食塩水又は培地中のメジアン径(D50)として測定した値とする。培養担体の平均粒子径は、レーザー回折散乱式の粒子径分布測定装置により測定することができる。
The average particle size (D50) of the culture carrier is, for example, 50 to 1,000 μm, preferably 100 to 500 μm, and more preferably 150 to 250 μm from the viewpoint of promoting cell proliferation. The average particle size of the culture carrier is a value measured as the median diameter (D50) in physiological saline or a medium. The average particle size of the culture carrier can be measured by a laser diffraction / scattering type particle size distribution measuring device.
懸濁液中の培養担体の濃度は、培養担体の形状、大きさ、表面積等に基づいて適宜調製可能であるが、例えば、0.01~100g/L、0.5~50g/L、又は1~20g/Lとすることができる。
The concentration of the culture carrier in the suspension can be appropriately adjusted based on the shape, size, surface area, etc. of the culture carrier, and is, for example, 0.01 to 100 g / L, 0.5 to 50 g / L, or It can be 1 to 20 g / L.
ここで、培養容器10内において、時間経過に伴って細胞の増殖(浮遊培養)が進むと、複数の細胞が凝集することで細胞塊が次第に形成される。また、懸濁液に培養担体が含められる場合においては、細胞と培養担体との複合体である細胞複合体が形成され、細胞は該細胞複合体上で増殖する。したがって、懸濁液には、細胞塊と細胞複合体の少なくとも一方が含まれる。なお、細胞懸濁液には、単一状態の細胞と、細胞が接着していない培養担体とのいずれか一方又は双方が含まれていてもよい。
Here, as cell proliferation (suspension culture) progresses with the passage of time in the culture vessel 10, a plurality of cells aggregate to gradually form a cell mass. When the culture carrier is included in the suspension, a cell complex which is a complex of cells and the culture carrier is formed, and the cells proliferate on the cell complex. Therefore, the suspension contains at least one of a cell mass and a cell complex. The cell suspension may contain one or both of the cells in a single state and the culture carrier to which the cells are not adhered.
なお、本開示において、細胞と当該細胞が接着する被接着体とから形成されるものを塊体と称するものとする。ここで、被接着体には、例えば細胞、培養担体等が含まれ、塊体には、例えば前述の細胞塊、細胞複合体等が含まれうる。塊体としては、浮遊する単一細胞であってもよい。これに関連して、本開示において、後述する細胞分布情報値及び担体分布情報値なる文言は、塊体分布情報値に読み替えることが可能であり、後述する担体分布情報相違値なる文言は、塊体分布情報相違値に読み替えることが可能である。
In the present disclosure, what is formed from a cell and an adherend to which the cell adheres is referred to as a mass. Here, the adherend may contain, for example, cells, a culture carrier, or the like, and the mass may include, for example, the above-mentioned cell mass, cell complex, or the like. The mass may be a floating single cell. In this regard, in the present disclosure, the wording of the cell distribution information value and the carrier distribution information value described later can be read as the mass distribution information value, and the wording of the carrier distribution information difference value described later is a mass. It can be read as a difference in body distribution information.
1-2.駆動装置20
一実施形態に係る駆動装置20は、培養容器10内に収容される懸濁液を揺動させるものであれば、特に制限なく用いることができる。駆動装置20としては、例えば図1及び図2に示すように、モータ21、モータ21の回転軸(図示せず)に連結するシャフト22、及びシャフト22の先端に連結する撹拌翼23、を有する撹拌機を用いることができる。 1-2.Drive device 20
Thedrive device 20 according to the embodiment can be used without particular limitation as long as it swings the suspension contained in the culture vessel 10. The drive device 20 includes, for example, as shown in FIGS. 1 and 2, a motor 21, a shaft 22 connected to a rotating shaft (not shown) of the motor 21, and a stirring blade 23 connected to the tip of the shaft 22. A stirrer can be used.
一実施形態に係る駆動装置20は、培養容器10内に収容される懸濁液を揺動させるものであれば、特に制限なく用いることができる。駆動装置20としては、例えば図1及び図2に示すように、モータ21、モータ21の回転軸(図示せず)に連結するシャフト22、及びシャフト22の先端に連結する撹拌翼23、を有する撹拌機を用いることができる。 1-2.
The
また、駆動装置20として、例えば図4に示すような、一般的に知られる振盪機を用いて、該振盪機によって培養容器10を外側から振盪させることで懸濁液を揺動させてもよい。したがって、本開示において「揺動」とは、撹拌、振盪等、懸濁液を揺動させるあらゆる運動態様を含むことができる。同様に、本開示において、「揺動速度」とは、撹拌速度、振盪速度等、懸濁液を揺動させるあらゆる運動態様に係る速度を含むことができる。
Further, as the driving device 20, for example, a generally known shaking machine as shown in FIG. 4 may be used, and the suspension may be shaken by shaking the culture vessel 10 from the outside by the shaking machine. .. Therefore, in the present disclosure, "swinging" can include any movement mode in which the suspension is shaken, such as stirring and shaking. Similarly, in the present disclosure, the "swing speed" can include speeds relating to any motion mode in which the suspension is shaken, such as stirring speed, shaking speed, and the like.
駆動装置20には、駆動源(駆動装置20として撹拌機が用いられる場合においては、図1に示すモータ21)の揺動速度(撹拌速度)を制御する後述する制御装置40からの指令を受信する受信部(図示せず)が別途設けられている。これにより、駆動装置20は、制御装置40の指令に対応する揺動速度を駆動源に伝達することができる。
The drive device 20 receives a command from a control device 40 described later that controls the swing speed (stirring speed) of the drive source (in the case where the stirrer is used as the drive device 20, the motor 21 shown in FIG. 1). A receiving unit (not shown) is provided separately. As a result, the drive device 20 can transmit the swing speed corresponding to the command of the control device 40 to the drive source.
1-3.測定装置30
一実施形態に係る測定装置30は、培養容器10内の懸濁液中の少なくとも一部の領域における、細胞に関する細胞分布情報値、及び培養担体に関する担体分布情報値の少なくとも一方を、培養進行中に測定(算出)するものである。 1-3. Measuringdevice 30
The measuringdevice 30 according to the embodiment is in the process of culturing at least one of the cell distribution information value for cells and the carrier distribution information value for a culture carrier in at least a part of the region in the suspension in the culture vessel 10. It is measured (calculated).
一実施形態に係る測定装置30は、培養容器10内の懸濁液中の少なくとも一部の領域における、細胞に関する細胞分布情報値、及び培養担体に関する担体分布情報値の少なくとも一方を、培養進行中に測定(算出)するものである。 1-3. Measuring
The measuring
ここで、細胞分布情報値とは、例えば、少なくとも一部の領域における、懸濁液中の生細胞数又は生細胞濃度に換言することができるが、これに限定されない。つまり、細胞分布情報値は、少なくとも一部の領域の懸濁液中に、生細胞がどの程度分布しているかを表す値であればよく、場合によっては、複数の細胞が凝集することで形成される細胞塊の数としてもよい。
Here, the cell distribution information value can be rephrased as, for example, the number of living cells or the concentration of living cells in the suspension in at least a part of the region, but is not limited thereto. That is, the cell distribution information value may be a value indicating how much live cells are distributed in the suspension of at least a part of the region, and in some cases, it is formed by agglomeration of a plurality of cells. It may be the number of cell clusters to be formed.
他方、担体分布情報値とは、例えば、少なくとも一部の領域における、懸濁液中の培養担体の数又は培養担体の濃度に換言することができるが、これに限定されない。つまり、担体分布情報値とは、少なくとも一部の領域の懸濁液中に、培養担体がどの程度分布しているかを表す値であればよい。
On the other hand, the carrier distribution information value can be translated into, for example, the number of cultured carriers in the suspension or the concentration of the cultured carriers in at least a part of the region, but is not limited thereto. That is, the carrier distribution information value may be a value indicating how much the culture carrier is distributed in the suspension of at least a part of the region.
一実施形態に係る細胞培養装置1の測定装置30としては、例えば、図2等に示すような、第1測定装置300を用いることができる。なお、以下詳述する第1測定装置300は、細胞分布情報値のみ測定可能であるが、後述する変形例1に係る第2測定装置350は、細胞分布情報値及び担体分布情報値の両方を測定することが可能である。測定装置30として、第1測定装置300及び後述する第2測定装置350のどちらを利用するかについては、培養される細胞種、培養容器10の大きさ等に基づいて適宜に決定すればよいが、第1測定装置300は、生細胞数又は生細胞濃度を直接的に観察(管理)する場合に好適であり、第2測定装置350は、細胞塊や細胞複合体を直接的に観察(管理)する場合に好適である。
As the measuring device 30 of the cell culture device 1 according to the embodiment, for example, the first measuring device 300 as shown in FIG. 2 or the like can be used. The first measuring device 300 described in detail below can measure only the cell distribution information value, but the second measuring device 350 according to the modification 1 described later can measure both the cell distribution information value and the carrier distribution information value. It is possible to measure. Whether to use the first measuring device 300 or the second measuring device 350 described later as the measuring device 30 may be appropriately determined based on the cell type to be cultured, the size of the culture container 10, and the like. The first measuring device 300 is suitable for directly observing (managing) the number of living cells or the concentration of living cells, and the second measuring device 350 directly observes (manages) a cell mass or a cell complex. ) Is suitable.
第1測定装置300は、図2及び図3に示すように、図2において点線で囲まれた領域Pにおける培養容器10内の懸濁液中に挿入される略棒状の主部301と、該主部301上に設けられ、所定周波数の電気信号RFを発振して領域Pの懸濁液中に電場を形成させる電極310と、形成された電場内で分極した生細胞Ceの量に基づいて電場内のキャパシタンス(静電容量)を測定するキャパシタンス測定部320と、キャパシタンス測定部320によって測定されたキャパシタンスに基づいて、領域P内の細胞分布情報値(具体的には領域P内の生細胞数又は生細胞濃度)を演算する本体部330と、を有することができる。さらに、主部301には、領域Pの懸濁液の導電率を測定する導電率測定部340と、領域Pにおける懸濁液のpHを測定するpH測定部341と、の少なくとも一方、又はこれら双方が配置されていてもよい。導電率測定部340及びpH測定部341は、一般的に知られる公知のものを用いることができる。なお、図2に示すように、第1測定装置300において、主部301は複数(図2においては2つ)設けられてもよく、これに対応して、電極310、キャパシタンス測定部320、導電率測定部340、及びpH測定部341等も複数設けられてよい。
As shown in FIGS. 2 and 3, the first measuring device 300 includes a substantially rod-shaped main portion 301 inserted into the suspension in the culture vessel 10 in the region P surrounded by the dotted line in FIG. 2, and the same. Based on an electrode 310 provided on the main portion 301 that oscillates an electric signal RF of a predetermined frequency to form an electric field in the suspension of the region P, and the amount of live cells Ce polarized in the formed electric field. Based on the capacitance measurement unit 320 that measures the capacitance (capacitance) in the electric field and the capacitance measured by the capacitance measurement unit 320, the cell distribution information value in the region P (specifically, the living cell in the region P). It can have a main body unit 330, which calculates the number or the concentration of living cells). Further, the main unit 301 includes at least one of a conductivity measuring unit 340 for measuring the conductivity of the suspension in the region P and a pH measuring unit 341 for measuring the pH of the suspension in the region P, or these. Both may be arranged. As the conductivity measuring unit 340 and the pH measuring unit 341, generally known known ones can be used. As shown in FIG. 2, in the first measuring device 300, a plurality of main portions 301 (two in FIG. 2) may be provided, and correspondingly, the electrode 310, the capacitance measuring unit 320, and the conductivity A plurality of rate measuring units 340, pH measuring unit 341, and the like may also be provided.
電極310から発振される電気信号RFの周波数は、特に制限はないが、例えば、50kHz~20MHz、好ましくは100kHz~1MHz、より好ましくは400kHz~600kHzのものを用いることができる。なお、電極310からは複数種類の周波数の電気信号が発振されることが好ましい。電極310から多種の周波数の電気信号RFが発振されることにより、様々な粒径の生細胞ごとの生細胞数又は生細胞濃度を測定することができるため、領域Pの懸濁液に含まれる正確な生細胞数又は生細胞濃度を測定することができる。
The frequency of the electric signal RF oscillated from the electrode 310 is not particularly limited, but for example, one of 50 kHz to 20 MHz, preferably 100 kHz to 1 MHz, and more preferably 400 kHz to 600 kHz can be used. It is preferable that electric signals having a plurality of types of frequencies are oscillated from the electrode 310. By oscillating electric signal RF of various frequencies from the electrode 310, it is possible to measure the number of viable cells or the concentration of viable cells for each viable cell having various particle sizes, and thus it is included in the suspension of the region P. Accurate viable cell number or viable cell concentration can be measured.
電極310によって電場が形成され、キャパシタンス測定部320によってキャパシタンスが測定される懸濁液中の領域Pの場所は、培養容器10内に懸濁液を収容したときに懸濁液の液面よりも下となる位置であれば、培養容器10内部における深さ方向のいずれの位置にあってもよい。例えば、領域Pの場所は、図2及び図3に示すように、培養容器10内の深さ方向における中央部分であってもよいし、培養容器10内の底面部分10xであってもよい。懸濁液中の領域Pの数は、培養容器内10に1箇所だけ設けてもよいし、複数箇所設けてもよい。領域Pを培養容器内10に複数箇所設ける場合には、培養容器10内の深さ方向において、互いに異なる位置に配置してもよいし、同じ位置に配置してもよい。後述する制御装置40による判定の正確性の観点からすれば、キャパシタンスを測定する部分(キャパシタンス測定部320)は、培養容器10内の複数の箇所で測定されることが好ましい。培養容器10内の懸濁液中、複数の箇所(複数の領域であって、例えば、後述する領域Q1~Q4)においてキャパシタンスを測定する場合には、第1測定装置300は、当該複数の箇所に対応する複数の電極310と複数のキャパシタンス測定部320(及びこれらに対応する主部301)を有するように設計すればよい。
The location of the region P in the suspension where the electric field is formed by the electrode 310 and the capacitance is measured by the capacitance measuring unit 320 is higher than the liquid level of the suspension when the suspension is housed in the culture vessel 10. As long as it is in the lower position, it may be in any position in the depth direction inside the culture vessel 10. For example, the location of the region P may be the central portion in the culture vessel 10 in the depth direction or the bottom portion 10x in the culture vessel 10, as shown in FIGS. 2 and 3. The number of regions P in the suspension may be provided at only one location in the culture vessel 10 or at a plurality of locations. When a plurality of regions P are provided in the culture vessel 10, they may be arranged at different positions or at the same position in the depth direction in the culture vessel 10. From the viewpoint of the accuracy of the determination by the control device 40 described later, it is preferable that the portion for measuring the capacitance (capacitance measuring unit 320) is measured at a plurality of points in the culture vessel 10. When measuring the capacitance at a plurality of locations (a plurality of regions, for example, regions Q1 to Q4 described later) in the suspension in the culture vessel 10, the first measuring device 300 is used at the plurality of locations. It may be designed to have a plurality of electrodes 310 corresponding to the above and a plurality of capacitance measuring units 320 (and a main unit 301 corresponding to these).
ところで、死滅細胞(細胞膜が破れた細胞)は、電場内で分極しないことが知られている。したがって、第1測定装置300は、懸濁液中の生細胞数又は生細胞濃度を正確に算出することができる。なお、キャパシタンスを用いて生細胞数又は生細胞濃度を測定する装置(キャパシタンスと生細胞数には比例関係があること)自体は、既に公知(例えば、Harriet E. Cole et al.、 “The Application of Dielectric Spectroscopy and Biocalorimetry for the Monitoring of Biomass in Immobilized Mammalian Cell Cultures”、Processes 2015, 3, 384-405、等参照)であるので、第1測定装置300の更なる詳細な説明は省略する。
By the way, it is known that dead cells (cells whose cell membrane is broken) do not polarize in an electric field. Therefore, the first measuring device 300 can accurately calculate the number of living cells or the concentration of living cells in the suspension. The device for measuring the number of living cells or the concentration of living cells using capacitance (there is a proportional relationship between capacitance and the number of living cells) is already known (for example, Harriet E. Cole et al., “The Application”. Of Dielectric Spectroscopy and Biocalorimetry for the Monitoring of Biomass in Immobilized Mammalian Cell Cultures ”, Processes 2015, 3, 384-405, etc.), so further detailed description of the first measuring device 300 will be omitted.
なお、第1測定装置300の本体部330によって演算された細胞分布情報値(生細胞数又は生細胞濃度)は、後述する制御装置40に所定の時間間隔(例えば、所定単位時間の間隔)で送信される。
The cell distribution information value (number of living cells or concentration of living cells) calculated by the main body 330 of the first measuring device 300 is obtained by the control device 40 described later at a predetermined time interval (for example, an interval of a predetermined unit time). Will be sent.
1-4.制御装置40
一実施形態に係る制御装置40は、例えば、中央処理装置(CPU)、主記憶装置、入出力インターフェース、入力装置、出力装置等がデータバス等により接続される一般的なハードウェアを用いることができる。制御装置40は、図1に示すように、端末装置として測定装置30と別体に設けられてもよいし、測定装置30の少なくとも一部(例えば、前述の第1測定装置300における本体部330)と制御装置40とが、一体的に構成されるようにしてもよい。 1-4.Control device 40
Thecontrol device 40 according to the embodiment may use, for example, general hardware to which a central processing unit (CPU), a main storage device, an input / output interface, an input device, an output device, and the like are connected by a data bus or the like. can. As shown in FIG. 1, the control device 40 may be provided separately from the measuring device 30 as a terminal device, or at least a part of the measuring device 30 (for example, the main body 330 in the first measuring device 300 described above). ) And the control device 40 may be integrally configured.
一実施形態に係る制御装置40は、例えば、中央処理装置(CPU)、主記憶装置、入出力インターフェース、入力装置、出力装置等がデータバス等により接続される一般的なハードウェアを用いることができる。制御装置40は、図1に示すように、端末装置として測定装置30と別体に設けられてもよいし、測定装置30の少なくとも一部(例えば、前述の第1測定装置300における本体部330)と制御装置40とが、一体的に構成されるようにしてもよい。 1-4.
The
一実施形態に係る制御装置40の機能としては、図5に示すように、主に、通信部41、記憶部42、判定部43、及び出力部44から構成される。
As shown in FIG. 5, the function of the control device 40 according to the embodiment is mainly composed of a communication unit 41, a storage unit 42, a determination unit 43, and an output unit 44.
通信部41は、測定装置30(一実施形態においては第1測定装置300)から、細胞分布情報値を受信し、受信した細胞分布情報値を記憶部42へ伝達する。また、通信部41は、後述するとおり、判定部43の判定結果に基づいて出力部44が決定(算出)する駆動装置20の揺動速度を、駆動装置20に対する制御指令として、駆動装置20に送信することができる(駆動装置20には、通信部41からの制御指令を受信する機能が搭載されている)。
The communication unit 41 receives the cell distribution information value from the measuring device 30 (the first measuring device 300 in one embodiment), and transmits the received cell distribution information value to the storage unit 42. Further, as will be described later, the communication unit 41 gives the drive device 20 a control command for the drive device 20 to determine (calculate) the swing speed of the drive device 20 determined (calculated) by the output unit 44 based on the determination result of the determination unit 43. It can be transmitted (the drive device 20 is equipped with a function of receiving a control command from the communication unit 41).
記憶部42は、通信部41から受信する細胞分布情報値、駆動装置20に対する制御指令、後述する閾値等を記憶することができる。
The storage unit 42 can store the cell distribution information value received from the communication unit 41, the control command for the driving device 20, the threshold value described later, and the like.
判定部43は、測定装置30から通信部41が受信する(記憶部42が記憶する)細胞分布情報値に基づいて、所定単位時間あたりの細胞分布情報値の変化率(以下、単に「変化率」ともいう。)をモニタリングする。さらに、判定部43は、当該変化率と、当該変化率に対して予め設定される閾値とを比較し、当該変化率と閾値との関係を所定単位時間毎に判定する。例えば、判定部43は、当該変化率が閾値以上であるか、又は閾値未満であるか、を判定する。ここで、変化率の算出にあたっては、例えば所定単位時間を1時間(60分)に設定する場合、当該1時間の開始時の細胞分布情報値と終了時(つまり、開始時から60分後)の細胞分布情報値とを比較演算することで、当該1時間における細胞分布情報値の変化率を算出することができる。また、例えば所定単位時間を1時間(60分)に設定する場合において、当該1時間の開始時から10分おきに計7回(つまり、開始時、10分後、20分後、30分後、40分後、50分後、及び終了時)細胞分布情報値を取得して、開始時から10分後までの細胞分布情報値の変化率、10分後から20分後までの細胞分布情報値の変化率、20分後から30分後までの細胞分布情報値の変化率、30分後から40分後までの細胞分布情報値の変化率、40分後から50分後までの細胞分布情報値の変化率、50分後から終了時までの細胞分布情報値の変化率、を算出し、これら計7回の変化率の平均値を所定単位時間あたりの細胞分布情報値の変化率としてもよい。なお、前述したとおり、キャパシタンスと生細胞数には比例関係があることから、所定単位時間あたりの細胞分布情報値の変化率に代えて、所定単位時間あたりのキャパシタンスの変化率を用いてもよい。この場合、測定装置30は、測定したキャパシタンスに関する情報(キャパシタンス値)を、本体部330にて演算された細胞分布情報値とともに、又は本体部330にて演算された細胞分布情報値に代えて、後述する制御装置40に所定の時間間隔(例えば、所定単位時間の間隔)で送信すればよい(したがって、本開示において、「細胞分布情報値の変化率」には、「キャパシタンスの変化率」も包含されるものとする)。
The determination unit 43 is based on the cell distribution information value received by the communication unit 41 from the measuring device 30 (stored by the storage unit 42), and the change rate of the cell distribution information value per predetermined unit time (hereinafter, simply “change rate”). Also called.) To monitor. Further, the determination unit 43 compares the rate of change with a threshold value preset for the rate of change, and determines the relationship between the rate of change and the threshold value every predetermined unit time. For example, the determination unit 43 determines whether the rate of change is equal to or greater than the threshold value or is less than the threshold value. Here, in calculating the rate of change, for example, when the predetermined unit time is set to 1 hour (60 minutes), the cell distribution information value at the start of the 1 hour and the end time (that is, 60 minutes after the start). By comparing with the cell distribution information value of the above, the rate of change of the cell distribution information value in the one hour can be calculated. Further, for example, when the predetermined unit time is set to 1 hour (60 minutes), a total of 7 times (that is, at the start time, 10 minutes later, 20 minutes later, 30 minutes later) every 10 minutes from the start of the 1 hour. , 40 minutes, 50 minutes, and end) Acquire cell distribution information value, change rate of cell distribution information value from start to 10 minutes, cell distribution information from 10 minutes to 20 minutes Rate of change in value, rate of change in cell distribution information value from 20 minutes to 30 minutes, rate of change in cell distribution information value from 30 minutes to 40 minutes, cell distribution from 40 minutes to 50 minutes The rate of change of the information value and the rate of change of the cell distribution information value from 50 minutes to the end are calculated, and the average value of these 7 times of change is used as the rate of change of the cell distribution information value per predetermined unit time. May be good. As described above, since the capacitance and the number of living cells have a proportional relationship, the rate of change in capacitance per predetermined unit time may be used instead of the rate of change in the cell distribution information value per predetermined unit time. .. In this case, the measuring device 30 uses the measured capacitance information (capacitance value) together with the cell distribution information value calculated by the main body 330 or in place of the cell distribution information value calculated by the main body 330. It may be transmitted to the control device 40 described later at a predetermined time interval (for example, an interval of a predetermined unit time) (hence, in the present disclosure, the “change rate of cell distribution information value” also includes a “capacitance change rate”. It shall be included).
変化率に関する所定単位時間は、特に制限はないが、例えば、10分~24時間の間で適宜に設定すればよい。但し、駆動装置20の揺動速度を細かく制御して、培養される細胞(培養される細胞の集合体を、本開示において「細胞群」ともいう。)の収率性と効率性を最大化する観点でいえば、所定単位時間を、例えば、30分~1時間とすることが好ましい。
The predetermined unit time regarding the rate of change is not particularly limited, but may be appropriately set, for example, between 10 minutes and 24 hours. However, the rocking speed of the drive device 20 is finely controlled to maximize the yield and efficiency of the cells to be cultured (the aggregate of cells to be cultured is also referred to as "cell group" in the present disclosure). From this point of view, it is preferable that the predetermined unit time is, for example, 30 minutes to 1 hour.
前述の閾値としては、例えば、前述の変化率に対して、「0%」に1つ設定される(この場合における閾値を、「第1閾値」ともいう。)。変化率が0%とは、測定対象となる前述の細胞分布情報値が所定単位時間において(所定単位時間の開始時と終了時で)一定となる場合を意味するものであって、変化率が0%より大きい場合は、細胞分布情報値が増加していることを意味し、変化率が0%未満の場合は、細胞分布情報値が減少していることを意味する。前述の判定部43は、例えば、変化率が0%以上(第1閾値以上)の場合に細胞の増殖が「正常」と判定し、変化率が0%未満(第1閾値未満)の場合に細胞の増殖が「異常」と判定することができる。なお、「正常」及び「異常」との具体的な判定内容については、適宜に変更すればよい。
As the above-mentioned threshold value, for example, one is set to "0%" with respect to the above-mentioned change rate (the threshold value in this case is also referred to as "first threshold value"). The rate of change of 0% means that the above-mentioned cell distribution information value to be measured is constant at a predetermined unit time (at the start and end of the predetermined unit time), and the rate of change is If it is larger than 0%, it means that the cell distribution information value is increasing, and if the rate of change is less than 0%, it means that the cell distribution information value is decreasing. The determination unit 43 described above determines, for example, that cell proliferation is "normal" when the rate of change is 0% or more (first threshold value or higher), and when the rate of change is less than 0% (less than the first threshold value). Cell proliferation can be determined to be "abnormal". The specific contents of the determination of "normal" and "abnormal" may be changed as appropriate.
なお、閾値として、前述の第1閾値に加えて、0%よりも大きい値に別の閾値(この場合における別の閾値を、「第2閾値」ともいう。)をさらに設定してもよい。第2閾値としては、例えば、変化率が10%~90%の範囲のいずれかの値に対して設定することができる。このように、第1閾値に加えて第2閾値を設定することにより、判定部43による判定のバリエーションを増やし、駆動装置20の揺動速度を細かく制御することが可能となる。つまり、判定部43は、例えば、変化率が第2閾値以上の場合に細胞の増殖が「正常」と判定し、変化率が第1閾値以上第2閾値未満の場合に細胞の増殖が「やや低調」と判定し、変化率が第1閾値未満の場合に細胞の増殖が「異常」と判定することができる。なお、この場合における「正常」、「やや低調」、及び「異常」との具体的な判定内容についても、適宜に変更すればよい。
As the threshold value, in addition to the above-mentioned first threshold value, another threshold value (another threshold value in this case is also referred to as a "second threshold value") may be further set to a value larger than 0%. The second threshold value can be set, for example, for any value in the range of 10% to 90% of the rate of change. By setting the second threshold value in addition to the first threshold value in this way, it is possible to increase the variation of the determination by the determination unit 43 and finely control the swing speed of the drive device 20. That is, for example, the determination unit 43 determines that the cell proliferation is "normal" when the rate of change is equal to or higher than the second threshold value, and the cell proliferation is "slightly" when the rate of change is equal to or greater than the first threshold value and less than the second threshold value. When it is determined to be "low" and the rate of change is less than the first threshold value, cell proliferation can be determined to be "abnormal". In this case, the specific determination contents of "normal", "slightly low", and "abnormal" may be changed as appropriate.
出力部44は、前述の判定部43の判定結果に基づいて、駆動装置20の揺動速度(駆動装置20として撹拌機が用いられる場合においては、撹拌速度)の値を決定する。判定部43の判定結果と出力部44が決定する駆動装置20の揺動速度との関係について、図6及び図7を参照しつつ説明する。図6及び図7において、所定単位時間(図6においては、所定単位時間A~所定単位時間J、図7においては、所定単位時間A~所定単位時間I)の長さが全て一定(例えば、前述のとおり1時間)とし、隣接する所定単位時間の終了時と開始時は、実質的に同じと理解されたい(つまり、所定単位時間Aの終了時と、所定単位時間Bの開始時は、実質的に同じ)。
The output unit 44 determines the value of the swing speed of the drive device 20 (in the case where the stirrer is used as the drive device 20, the stirring speed) based on the determination result of the determination unit 43 described above. The relationship between the determination result of the determination unit 43 and the swing speed of the drive device 20 determined by the output unit 44 will be described with reference to FIGS. 6 and 7. In FIGS. 6 and 7, the length of the predetermined unit time (predetermined unit time A to predetermined unit time J in FIG. 6 and predetermined unit time A to predetermined unit time I in FIG. 7) is all constant (for example,). It should be understood that the end time and the start time of the adjacent predetermined unit time are substantially the same (that is, the end time of the predetermined unit time A and the start time of the predetermined unit time B). Substantially the same).
まず、変化率(Xt)に対する閾値として、前述の第1閾値のみが設定されている場合について、図6を参照しつつ説明する。この場合において出力部44は、或る所定単位時間(例えば、図6における所定単位時間A)の終了時において、判定部43が前述のとおり「正常」(変化率が第1閾値「0」以上)と判定すると、該判定時(つまり、所定単位時間Aの時)の駆動装置20の揺動速度を増加させるように、新たな揺動速度を出力する(なお、新たな揺動速度が出力された時点が、次の所定単位時間Bの開始時となる)。
First, a case where only the above-mentioned first threshold value is set as the threshold value for the rate of change (Xt) will be described with reference to FIG. In this case, at the end of a certain predetermined unit time (for example, the predetermined unit time A in FIG. 6), the output unit 44 is "normal" (the rate of change is the first threshold value "0" or more" as described above. ), A new rocking speed is output so as to increase the rocking speed of the drive device 20 at the time of the judgment (that is, when the predetermined unit time A) (note that the new rocking speed is output). The time point is the start time of the next predetermined unit time B).
一方、出力部44は、或る所定単位時間(例えば、図6における所定単位時間E)の終了時において、判定部43が前述のとおり「異常」(変化率が第1閾値「0」未満)と判定すると、該判定時(つまり、所定単位時間Eの時)の駆動装置20の揺動速度を減少させるように、新たな揺動速度を出力する(なお、新たな揺動速度が出力された時点が、次の所定単位時間Fの開始時となる)。この揺動速度の減少を実行した結果、再び変化率が第1閾値(変化率が第1閾値「0」)以上となる場合には、判定部43は「正常」と判定するため、上記の場合と同様に、駆動装置20の揺動速度を増加させる新たな揺動速度が出力されることとなる(図6における、所定単位時間F~所定単位時間G参照)。
On the other hand, in the output unit 44, at the end of a certain predetermined unit time (for example, the predetermined unit time E in FIG. 6), the determination unit 43 is "abnormal" as described above (the rate of change is less than the first threshold value "0"). If it is determined, a new swing speed is output so as to reduce the swing speed of the drive device 20 at the time of the determination (that is, at the time of the predetermined unit time E) (note that a new swing speed is output). The time point is the start time of the next predetermined unit time F). When the rate of change becomes equal to or higher than the first threshold value (the rate of change is the first threshold value "0") as a result of executing the reduction of the swing speed, the determination unit 43 determines that it is "normal". As in the case, a new swing speed that increases the swing speed of the drive device 20 is output (see the predetermined unit time F to the predetermined unit time G in FIG. 6).
なお、出力部44は、判定部43による所定単位時間毎の判定が複数回連続で「異常」(つまり、変化率が第1閾値未満)と判定された場合、駆動装置20の駆動を停止すべく、揺動速度0を出力する(図6における所定単位時間G~所定単位時間J参照)。所定単位時間の判定が複数回連続で「異常」と判定される場合には、培養容器10内に雑菌等のコンタミネーションの発生、駆動装置20の揺動速度が速すぎることに起因する生細胞へのダメージ等による問題が生じている可能性が考えられるため、即座に細胞の培養を停止する必要がある。ここで、前述の「複数回連続」とは、所定単位時間毎の判定が2回連続であってもよいし、3回連続であってもよいし、4回連続以上であってもよく、細胞の種類や培養条件に応じて適宜に設定すればよい。一実施形態においては、導電率測定部340によって測定される懸濁液中の導電率、及びpH測定部341によって測定される懸濁液のpHの少なくともいずれか一方の測定値に基づいて、前述のような問題を把握するようにしてもよい。
The output unit 44 stops driving the drive device 20 when the determination unit 43 determines that the determination for each predetermined unit time is "abnormal" (that is, the rate of change is less than the first threshold value) a plurality of times in a row. Therefore, the swing speed 0 is output (see the predetermined unit time G to the predetermined unit time J in FIG. 6). When the determination of the predetermined unit time is determined to be "abnormal" a plurality of times in a row, the living cells are caused by the occurrence of contamination such as germs in the culture vessel 10 and the shaking speed of the driving device 20 being too fast. It is necessary to stop the cell culture immediately because there is a possibility that a problem may occur due to damage to the cells. Here, the above-mentioned "multiple times in a row" means that the determination for each predetermined unit time may be two times in a row, three times in a row, or four or more times in a row. It may be appropriately set according to the cell type and the culture conditions. In one embodiment, the above-mentioned is based on the measured value of at least one of the conductivity in the suspension measured by the conductivity measuring unit 340 and the pH of the suspension measured by the pH measuring unit 341. You may try to grasp the problem such as.
次に、変化率(Xt)に対する閾値として、前述の第1閾値に加えて第2閾値Yも設定されている場合について、図7を参照しつつ説明する。この場合の出力部44は、或る所定単位時間(例えば、図7における所定単位時間A)の終了時において、判定部43が前述のとおり「やや低調」(変化率Xtが「Y>Xt≧0」)と判定すると、該判定時(該所定単位時間Aの時)の駆動装置20の揺動速度を増加させるように、新たな揺動速度を出力する(なお、新たな揺動速度が出力された時点が、次の所定単位時間Bの開始時となる)。
Next, a case where a second threshold value Y is set in addition to the above-mentioned first threshold value as a threshold value for the rate of change (Xt) will be described with reference to FIG. 7. In this case, at the end of a predetermined unit time (for example, the predetermined unit time A in FIG. 7), the determination unit 43 is "slightly low" as described above (change rate Xt is "Y> Xt ≧"). If it is determined to be "0"), a new rocking speed is output so as to increase the rocking speed of the drive device 20 at the time of the judgment (at the time of the predetermined unit time A) (note that the new rocking speed is calculated). The output time is the start time of the next predetermined unit time B).
次に、出力部44は、或る所定単位時間(例えば、図7における所定単位時間D)の終了時において、判定部43が前述のとおり「正常」(変化率Xtが「Y≦Xt」)と判定すると、該判定時(該所定単位時間Dの時)の揺動速度を維持する。
Next, in the output unit 44, at the end of a certain predetermined unit time (for example, the predetermined unit time D in FIG. 7), the determination unit 43 is “normal” as described above (change rate Xt is “Y ≦ Xt”). If it is determined, the rocking speed at the time of the determination (at the time of the predetermined unit time D) is maintained.
さらに、出力部44は、或る所定単位時間(例えば、図7における所定単位時間F)の終了時において、判定部43が前述のとおり「異常」(変化率Xtが「Xt<0」)と判定すると、該判定時(該所定単位時間Fの時)の揺動速度を減少させるように、新たな揺動速度を出力する(なお、新たな揺動速度が出力された時点が、次の所定単位時間Gの開始時となる)。なお、図7に示される場合においても、図6の場合と同様、所定期間にわたって複数回連続で「異常」と判定された場合、駆動装置20を停止すべく、出力部44は揺動速度0を出力する。
Further, at the end of a predetermined unit time (for example, the predetermined unit time F in FIG. 7), the output unit 44 determines that the determination unit 43 is “abnormal” (change rate Xt is “Xt <0”) as described above. When the determination is made, a new rocking speed is output so as to reduce the rocking speed at the time of the judgment (at the time of the predetermined unit time F) (note that the time when the new rocking speed is output is the next time. It is the start of the predetermined unit time G). Even in the case shown in FIG. 7, as in the case of FIG. 6, when the "abnormality" is continuously determined a plurality of times over a predetermined period, the output unit 44 has a swing speed of 0 in order to stop the drive device 20. Is output.
ところで、図6及び図7に示される場合における、駆動装置20の具体的な揺動速度、及び揺動速度の増減の程度は、細胞の種類、培養容器10の容量等に応じて適宜に設定すればよい。但し、駆動装置20の揺動速度が速すぎると、細胞を傷つけてしまう恐れがあるため、具体的な揺動速度としては、10mm/s~100mm/s、好ましくは20mm/s~80mm/s、より好ましくは30mm/s~60mm/s、の範囲内で設定されうる。なお、この際の揺動速度は、駆動装置20として撹拌機が用いられる場合には、撹拌翼23の回転速度を意味し、上記の揺動速度を、撹拌翼23の外周端の移動距離として回転速度に変換すればよい。
By the way, in the cases shown in FIGS. 6 and 7, the specific rocking speed of the driving device 20 and the degree of increase / decrease in the rocking speed are appropriately set according to the cell type, the capacity of the culture vessel 10, and the like. do it. However, if the rocking speed of the drive device 20 is too high, there is a risk of damaging the cells. Therefore, the specific rocking speed is 10 mm / s to 100 mm / s, preferably 20 mm / s to 80 mm / s. , More preferably in the range of 30 mm / s to 60 mm / s. The swing speed at this time means the rotation speed of the stirring blade 23 when the stirrer is used as the drive device 20, and the above swing speed is used as the moving distance of the outer peripheral end of the stirring blade 23. It may be converted into a rotation speed.
2.一実施形態に係る細胞培養装置1の動作
前述のとおり説明した細胞培養装置1における一連の動作について、図8乃至図10を参照しつつ、さらに詳細に説明する。図8は、一実施形態に係る細胞培養装置1において行われる動作の一部の一例を示すフロー図である。図9及び図10は、一実施形態に係る細胞培養装置1による細胞(例えば、間葉系幹細胞)の増殖状況に関し、時間経過に対するキャパシタンス及び生細胞数の変化を表す図である。なお、以下に説明する一連の動作において、閾値は、前述にて説明した、第1閾値及び第2閾値の両方が予め設定されているものとする。また、図9及び図10中、点線は、キャパシタンス値(pF/cm)を示し、黒丸は生細胞数(個/mL)を示す。 2. 2. Operation ofCell Culture Device 1 According to One Embodiment A series of operations in the cell culture device 1 described above will be described in more detail with reference to FIGS. 8 to 10. FIG. 8 is a flow chart showing an example of a part of the operation performed in the cell culture apparatus 1 according to the embodiment. 9 and 10 are diagrams showing changes in capacitance and the number of living cells over time with respect to the proliferation status of cells (for example, mesenchymal stem cells) by the cell culture apparatus 1 according to the embodiment. In the series of operations described below, it is assumed that both the first threshold value and the second threshold value described above are preset as the threshold value. Further, in FIGS. 9 and 10, the dotted line indicates the capacitance value (pF / cm), and the black circle indicates the number of living cells (cells / mL).
前述のとおり説明した細胞培養装置1における一連の動作について、図8乃至図10を参照しつつ、さらに詳細に説明する。図8は、一実施形態に係る細胞培養装置1において行われる動作の一部の一例を示すフロー図である。図9及び図10は、一実施形態に係る細胞培養装置1による細胞(例えば、間葉系幹細胞)の増殖状況に関し、時間経過に対するキャパシタンス及び生細胞数の変化を表す図である。なお、以下に説明する一連の動作において、閾値は、前述にて説明した、第1閾値及び第2閾値の両方が予め設定されているものとする。また、図9及び図10中、点線は、キャパシタンス値(pF/cm)を示し、黒丸は生細胞数(個/mL)を示す。 2. 2. Operation of
図8に示されるように、まず、培地を含む培養容器10内に、培養に供される細胞が収容されて浮遊培養が開始される。その後、所定時間経過すると、ステップ(以下、「ST」という。)500において、複数の細胞の凝集体である細胞塊を含む懸濁液が培養容器10内で形成(収容)される(収容工程)。なお、細胞の種類に応じ、培養容器10内に培養担体が予め収容される場合において、浮遊培養開始後所定時間経過すると、細胞と培養担体との複合体である細胞複合体が培養容器10内で形成(収容)される。
As shown in FIG. 8, first, the cells to be cultured are housed in the culture vessel 10 containing the medium, and the suspension culture is started. Then, after a lapse of a predetermined time, in step (hereinafter referred to as “ST”) 500, a suspension containing a cell mass which is an aggregate of a plurality of cells is formed (contained) in the culture vessel 10 (containment step). ). When the culture carrier is preliminarily housed in the culture vessel 10 according to the type of cells, a cell complex, which is a complex of the cells and the culture carrier, is contained in the culture vessel 10 after a predetermined time has passed after the start of suspension culture. Is formed (contained) in.
次に、ST501において、駆動装置20が、培養容器10内の懸濁液を予め決められた駆動方法(一定時間停止、間欠駆動、連続駆動、等)で揺動する(揺動工程)。なお、駆動装置20による懸濁液の揺動は、ST500の前から開始されていてもよい。
Next, in ST501, the drive device 20 swings the suspension in the culture vessel 10 by a predetermined drive method (stop for a certain period of time, intermittent drive, continuous drive, etc.) (rocking step). The swing of the suspension by the drive device 20 may be started before the ST500.
次に、ST502において、測定装置30(第1測定装置300)が、懸濁液中の少なくとも一部の領域(領域P)における細胞分布情報値(生細胞数又は生細胞濃度)を測定する(測定工程)。なお、このST502に係る測定工程においては、後述の変形例にて詳述される担体分布情報値が、測定装置30(第2測定装置350)によって測定されてもよい。
Next, in ST502, the measuring device 30 (first measuring device 300) measures the cell distribution information value (number of living cells or living cell concentration) in at least a part of the region (region P) in the suspension (the number of living cells or the concentration of living cells). Measurement process). In the measuring step according to ST502, the carrier distribution information value described in detail in the modification described later may be measured by the measuring device 30 (second measuring device 350).
次に、ST503において、測定装置30(第1測定装置300)が、測定した細胞分布情報値(生細胞数又は生細胞濃度)を制御装置40へ送信する。なお、ST502において担体分布情報値が測定される場合、このST503においては、測定装置30(第2測定装置350)が、測定した担体分布情報値(培養担体数又は培養担体濃度)を制御装置40へと送信してもよい。
Next, in ST503, the measuring device 30 (first measuring device 300) transmits the measured cell distribution information value (live cell number or live cell concentration) to the control device 40. When the carrier distribution information value is measured in ST502, in this ST503, the measuring device 30 (second measuring device 350) controls the measured carrier distribution information value (number of cultured carriers or culture carrier concentration) 40. May be sent to.
次に、ST504において、制御装置40の判定部43が、細胞分布情報値の所定単位時間あたりの変化率と、予め設定される閾値(例えば、前述の第1閾値及び第2閾値)との関係を所定単位時間毎に判定し、その判定結果を出力部44に送信する。なお、ST502において担体分布情報値が測定される場合、このST504においては、制御装置40の判定部43は、担体分布情報値の所定単位時間あたりの変化率と、予め設定される閾値(例えば、後述の第3閾値及び第4閾値)との関係を所定単位時間毎に判定し、その判定結果を出力部44に送信してもよい。
Next, in ST504, the determination unit 43 of the control device 40 relates the rate of change of the cell distribution information value per predetermined unit time to a preset threshold value (for example, the above-mentioned first threshold value and second threshold value). Is determined every predetermined unit time, and the determination result is transmitted to the output unit 44. When the carrier distribution information value is measured in ST502, in this ST504, the determination unit 43 of the control device 40 determines the rate of change of the carrier distribution information value per predetermined unit time and a preset threshold value (for example,). The relationship with the third threshold value and the fourth threshold value described later) may be determined every predetermined unit time, and the determination result may be transmitted to the output unit 44.
次に、ST505において、判定部43の判定結果に基づいて、制御装置40の出力部44が駆動装置20の揺動速度を決定する(揺動速度決定工程)。
Next, in ST505, the output unit 44 of the control device 40 determines the swing speed of the drive device 20 based on the judgment result of the determination unit 43 (swing speed determination step).
そして、ST506において、制御装置40の通信部41が、出力部44によって決定された駆動装置20の揺動速度を、駆動装置20への制御指令として、該駆動装置20へ出力する(出力工程)。これにより、駆動装置20は、当該制御指令に従った揺動速度を出力する。
Then, in ST506, the communication unit 41 of the control device 40 outputs the swing speed of the drive device 20 determined by the output unit 44 to the drive device 20 as a control command to the drive device 20 (output step). .. As a result, the drive device 20 outputs the swing speed according to the control command.
以降、細胞培養装置1は、細胞の培養プロセスを続行する限り、ST502~ST506に関する各動作を繰り返す(ST507)。生細胞数(生細胞濃度)が目標の値に到達した場合、駆動装置20の揺動速度に係わらず、細胞分布情報値の変化率が所定期間「0」で一定となった場合、又は、培養途中において、判定部43による所定単位時間毎の判定が複数回連続で「異常」となった場合等には、一連の動作は終了する(停止される)。なお、前述の「生細胞数(生細胞濃度)の目標の値」は、培養対象となる細胞の種類、培養スケール、用途等によって異なるが、例えば、培養開始時の細胞濃度の5倍~100倍、5倍~50倍、又は10~30倍の生細胞数(生細胞濃度)とすることができる。
After that, the cell culture apparatus 1 repeats each operation related to ST502 to ST506 as long as the cell culture process is continued (ST507). When the number of living cells (living cell concentration) reaches the target value, the rate of change of the cell distribution information value becomes constant at "0" for a predetermined period regardless of the shaking speed of the driving device 20, or In the middle of culturing, when the determination by the determination unit 43 for each predetermined unit time becomes "abnormal" a plurality of times in succession, a series of operations is terminated (stopped). The above-mentioned "target value of the number of living cells (living cell concentration)" varies depending on the type of cells to be cultured, the culture scale, the intended use, etc., but is, for example, 5 to 100 times the cell concentration at the start of culture. The number of viable cells (live cell concentration) can be doubled, 5 times to 50 times, or 10 to 30 times.
次に、ST500~ST506の一連の動作に基づく細胞の増殖状況について、図9及び図10を参照しつつ説明する。図9及び図10に関し、所定単位時間は1時間(60分)で設定される。また、図9及び図10に関し、判定部43の判定に基づく前述の「変化率」には、「細胞分布情報値の変化率」の代わりに「キャパシタンスの変化率」が用いられている。
Next, the state of cell proliferation based on the series of actions of ST500 to ST506 will be described with reference to FIGS. 9 and 10. With respect to FIGS. 9 and 10, the predetermined unit time is set to 1 hour (60 minutes). Further, with respect to FIGS. 9 and 10, in the above-mentioned "change rate" based on the determination of the determination unit 43, the "capacitance change rate" is used instead of the "cell distribution information value change rate".
図9を参照すると、特に、時間t1~t2にかけて、所定単位時間における変化率(キャパシタンスの変化率)が予め設定された第2閾値(前述のY)以上となっていることから、この間、駆動装置20の揺動速度(撹拌翼の回転速度)は、所定単位時間毎(1時間毎)に増加する。逆にいえば、所定単位時間毎に駆動装置20の揺動速度を増加させることで、変化率も第2閾値以上に増加している。このことから、培養容器10内において、細胞塊、細胞複合体等の沈降は抑制されて、細胞の増殖(細胞群の生産)が効率的に実行されていることが理解される。時間t2以降においては、駆動装置20の揺動速度に係わらず、キャパシタンスの変化率が一定期間に渡って「0」であることから、細胞の増殖(細胞群の生産)が実質的に終了(生細胞数が目標の値に到達)したものと理解される。なお、図9において、一時的にキャパシタンスの値が降下している箇所が示されているが、これは培地交換に由来するキャパシタンス値の一時的なブレであり、駆動装置20の揺動速度の制御に影響を与えるものではない点を付言する。
Referring to FIG. 9, in particular, since the rate of change (rate of change in capacitance) in a predetermined unit time is equal to or higher than the preset second threshold value (Y described above) over the time t1 to t2, it is driven during this period. The rocking speed (rotational speed of the stirring blade) of the device 20 increases every predetermined unit time (every hour). Conversely, by increasing the swing speed of the drive device 20 every predetermined unit time, the rate of change is also increased to the second threshold value or higher. From this, it is understood that the sedimentation of cell masses, cell complexes and the like is suppressed in the culture vessel 10, and cell proliferation (production of cell groups) is efficiently executed. After the time t2, the rate of change of the capacitance is "0" over a certain period regardless of the swing speed of the drive device 20, so that the cell proliferation (production of the cell group) is substantially completed (the production of the cell group). It is understood that the number of living cells has reached the target value). Note that FIG. 9 shows a place where the capacitance value temporarily drops, but this is a temporary fluctuation in the capacitance value due to the culture medium exchange, and the swing speed of the drive device 20 is increased. It should be added that it does not affect the control.
一方、図10を参照すると、図10に示される場合は、基本的に図9に示される場合と同様であるものの、時間t3付近において、キャパシタンスの変化率が急激に低下し、時間t3以降における変化率は、所定単位時間毎の判定が複数回連続で第1閾値(0)未満(異常)となる。したがって、図10に示される場合においては、制御装置40は、最終的に、駆動装置20の揺動速度を停止すべく0とする。なお、図10に示されるように、変化率が急激に低下する場合を想定して、変化率が0未満となる値(例えば、-10%等)に対して別途の閾値をさらに設けてもよい。このように、一実施形態に係る細胞培養装置1は、異常と判定された場合に適切に培養を停止することができるので、異常と判定された原因の究明、不必要な培養進行の防止等の観点で時間効率性上有用である。
On the other hand, referring to FIG. 10, the case shown in FIG. 10 is basically the same as the case shown in FIG. 9, but the rate of change of the capacitance sharply decreases near the time t3, and after the time t3. The rate of change is less than the first threshold value (0) (abnormal) when the determination for each predetermined unit time is performed a plurality of times in succession. Therefore, in the case shown in FIG. 10, the control device 40 is finally set to 0 in order to stop the swing speed of the drive device 20. As shown in FIG. 10, assuming a case where the rate of change drops sharply, a separate threshold value may be further set for a value (for example, -10%) in which the rate of change is less than 0. good. As described above, the cell culture apparatus 1 according to the embodiment can appropriately stop the culture when it is determined to be abnormal, so that the cause of the determination of abnormality can be investigated, unnecessary progress of culture can be prevented, and the like. It is useful in terms of time efficiency from the viewpoint of.
3.変形例
次に、前述にて説明した一実施形態に係る細胞培養装置1の変形例について説明する。 3. 3. Modification Example Next, a modification of thecell culture apparatus 1 according to the embodiment described above will be described.
次に、前述にて説明した一実施形態に係る細胞培養装置1の変形例について説明する。 3. 3. Modification Example Next, a modification of the
3-1.変形例1
変形例1に係る細胞培養装置1について、図11を参照しつつ説明する。図11は、変形例1に係る細胞培養装置1であって、測定装置30として第2測定装置350が用いられる場合の構成を模式的に示す概略図である。 3-1.Modification 1
Thecell culture apparatus 1 according to the modified example 1 will be described with reference to FIG. FIG. 11 is a schematic diagram schematically showing the configuration of the cell culture device 1 according to the modified example 1 in the case where the second measuring device 350 is used as the measuring device 30.
変形例1に係る細胞培養装置1について、図11を参照しつつ説明する。図11は、変形例1に係る細胞培養装置1であって、測定装置30として第2測定装置350が用いられる場合の構成を模式的に示す概略図である。 3-1.
The
変形例1に係る細胞培養装置1は、前述にて説明した一実施形態に係る細胞培養装置1とほぼ同様であるが、第1測定装置300に代わって第2測定装置350を適用するものである。したがって、変形例1に係る細胞培養装置1においては、第2測定装置350について以下のとおり詳細を説明し、その他の構成要素の詳細な説明は省略する。
The cell culture device 1 according to the modified example 1 is substantially the same as the cell culture device 1 according to the embodiment described above, but the second measurement device 350 is applied instead of the first measurement device 300. be. Therefore, in the cell culture device 1 according to the modified example 1, the details of the second measuring device 350 will be described as follows, and the detailed description of other components will be omitted.
変形例1に係る細胞培養装置1に適用される第2測定装置350は、培養容器10内の少なくとも一部の領域の懸濁液中に含まれる培養担体(図11における参照符号MC)を撮像して画像(動画であってもよい)を取得する撮像部360と、撮像部360によって撮像された画像に基づいて、当該領域における担体分布情報値を測定する本体部370と、を有することができる。なお、第2測定装置350による担体分布情報値の「測定」とは、画像に基づいて担体分布情報値を「算出」することを含む。ところで、懸濁液中に培養担体MCが混入される場合、細胞は主に培養担体MC上で増殖するため、細胞と培養担体MCとの複合体である細胞複合体が形成されることとなる。
The second measuring device 350 applied to the cell culture device 1 according to the first modification captures an image of the culture carrier (reference numeral MC in FIG. 11) contained in the suspension of at least a part of the region in the culture vessel 10. It may have an image pickup unit 360 for acquiring an image (which may be a moving image), and a main body unit 370 for measuring a carrier distribution information value in the region based on the image captured by the image pickup unit 360. can. The "measurement" of the carrier distribution information value by the second measuring device 350 includes "calculating" the carrier distribution information value based on the image. By the way, when the culture carrier MC is mixed in the suspension, the cells proliferate mainly on the culture carrier MC, so that a cell complex which is a complex of the cells and the culture carrier MC is formed. ..
撮像部360は、一般的に知られる画像センサ等、被対象物を所定間隔で連続的に撮像して、二次元又は三次元の画像を取得することが可能なものを用いることができる。また、撮像部360は、培養容器10内を深さ方向にみて、一様に撮像することができる大型のものを用いてもよいし、図11に示すように、複数(例えば、図11においては4つ)の小型の撮像部361乃至364を、培養容器10に対して深さ方向に並べて配置してもよいし、これら4つの撮像部361乃至364を、培養容器10の外周面において、異なる周位置(例えば、撮像部361を「12時の位置」、撮像部362を「3時の位置」、撮像部363を「6時の位置」、撮像部364を「9時の位置」)に配してもよい。また、場合によっては、図11に示される撮像部361のみを設けるようにしてもよい。
As the image pickup unit 360, a generally known image sensor or the like that can continuously image an object at predetermined intervals and acquire a two-dimensional or three-dimensional image can be used. Further, the imaging unit 360 may be a large one capable of uniformly imaging the inside of the culture vessel 10 in the depth direction, or as shown in FIG. 11, a plurality of (for example, in FIG. 11) may be used. 4) small imaging units 361 to 364 may be arranged side by side with respect to the culture vessel 10 in the depth direction, or these four imaging units 361 to 364 may be arranged on the outer peripheral surface of the culture vessel 10. Different peripheral positions (for example, the imaging unit 361 is the "12 o'clock position", the imaging unit 362 is the "3 o'clock position", the imaging unit 363 is the "6 o'clock position", and the imaging unit 364 is the "9 o'clock position"). You may arrange it in. Further, in some cases, only the image pickup unit 361 shown in FIG. 11 may be provided.
本体部370は、撮像部360が撮像することにより取得した画像を連続的に入手して、該画像に含まれる培養担体MCを画像認識したうえで、該画像に含まれる培養担体MCの数を、担体分布情報値としてカウントできる機能を有するものである。具体的には、本体部370による担体分布情報値のカウントは、例えば、撮像部361乃至364のいずれか一つが撮像することにより取得した画像を連続的に入手して、その画像をもとに実行されてもよい。或いは、本体部370による担体分布情報値のカウントは、例えば、撮像部361乃至364の少なくとも2つが撮像することにより取得した画像を各々連続的に入手して、各撮像部から取得した画像に基づく担体分布情報値を各々算出したうえで、それらの平均値を最終的な担体分布情報値として算出するようにしてもよい。また、本体部370は、撮像部360(撮像部361乃至364)によって、懸濁液中の細胞塊の数、及び細胞複合体上の細胞の数(生細胞数)を、細胞分布情報値としてカウントできる機能を有していてもよい。
The main body unit 370 continuously obtains the images acquired by the image pickup unit 360, recognizes the culture carrier MC contained in the image, and then determines the number of the culture carrier MC included in the image. , It has a function of being able to be counted as a carrier distribution information value. Specifically, the counting of the carrier distribution information value by the main body unit 370 is based on, for example, continuously obtaining an image acquired by any one of the image pickup units 361 to 364 and taking the image. It may be executed. Alternatively, the counting of the carrier distribution information values by the main body unit 370 is based on, for example, the images acquired by at least two of the image pickup units 361 to 364 continuously obtaining the images acquired from each image pickup unit. After calculating each carrier distribution information value, the average value thereof may be calculated as the final carrier distribution information value. Further, in the main body portion 370, the number of cell clusters in the suspension and the number of cells on the cell complex (number of viable cells) are used as cell distribution information values by the imaging unit 360 (imaging units 361 to 364). It may have a function that can be counted.
また、本体部370は、培養担体MCを直接画像認識してカウントする機能を有する代替として、画像の色彩や濃淡に基づいて、画像に含まれる培養担体濃度を、担体分布情報値として演算できる機能を有するものであってもよい。このような場合には、本体部370を、予め様々な培養担体濃度に対応する色彩や濃淡を大量に学習させて、色彩又は濃淡と培養担体濃度とを関連付けたデータを記憶させておき、このデータに基づいて、撮像部360が取得した画像に含まれる培養担体濃度を算出するようにしてもよい。
Further, as an alternative to having a function of directly recognizing and counting the culture carrier MC as an image, the main body portion 370 has a function of calculating the concentration of the culture carrier contained in the image as a carrier distribution information value based on the color and shading of the image. It may have. In such a case, the main body 370 is trained in advance to learn a large amount of colors and shades corresponding to various culture carrier concentrations, and stores data relating the colors or shades to the culture carrier concentration. Based on the data, the concentration of the culture carrier contained in the image acquired by the imaging unit 360 may be calculated.
本体部370は、以上のとおり算出した培養担体MCの数又は培養担体濃度を、制御装置40へと送信し、制御装置40は、一実施形態と同様の処理及び制御を実行する。なお、本体部370は、前述のとおり、細胞塊の数(及び細胞複合体上の細胞の数)をカウントする場合においては、その細胞塊の数(及び細胞複合体上の細胞の数)を、培養担体MCの数とともに、又は培養担体MCの数に代えて、制御装置40へと送信することもできる。
The main body unit 370 transmits the number of culture carrier MCs or the culture carrier concentration calculated as described above to the control device 40, and the control device 40 executes the same processing and control as in one embodiment. As described above, when counting the number of cell clusters (and the number of cells on the cell complex), the main body portion 370 counts the number of cell clusters (and the number of cells on the cell complex). , With the number of culture carrier MCs, or in place of the number of culture carrier MCs, can also be transmitted to the control device 40.
ここで、変形例1において、第2測定装置350が、担体分布情報値としての培養担体MCの数(又は培養担体濃度)を演算する場合には、所定単位時間あたりの担体分布情報値の変化率に対する第3閾値(一実施形態における第1閾値に相当)が設定されることとなる。さらに、必要に応じて、所定単位時間あたりの担体分布情報値の変化率に対する第4閾値(一実施形態における第2閾値に相当)が、さらに予め設定されていてもよい。したがって、変形例1における判定部43による判定は、所定単位時間あたりの担体分布情報値の変化率をもとに実行されることとなる。この場合の第3閾値及び第4閾値は、一実施形態における第1閾値及び第2閾値と同じであってもよいし異なっていてもよい。
Here, in the first modification, when the second measuring device 350 calculates the number of culture carrier MCs (or the culture carrier concentration) as the carrier distribution information value, the change in the carrier distribution information value per predetermined unit time. A third threshold value for the rate (corresponding to the first threshold value in one embodiment) will be set. Further, if necessary, a fourth threshold value (corresponding to the second threshold value in one embodiment) for the rate of change of the carrier distribution information value per predetermined unit time may be further set in advance. Therefore, the determination by the determination unit 43 in the modification 1 is executed based on the rate of change of the carrier distribution information value per predetermined unit time. In this case, the third threshold value and the fourth threshold value may be the same as or different from the first threshold value and the second threshold value in one embodiment.
また、変形例1において、第2測定装置350が、担体分布情報値としての培養担体MCの数(又は培養担体濃度)と、細胞分布情報値としての細胞塊の数(及び細胞の数)の両方を演算する場合、第2測定装置350は、細胞分布情報値及び担体分布情報値の両方を制御装置40に送信してもよい。この場合、変形例1における制御装置40の判定部43による判定バリエーションは増加し、例えば、以下表1のように設定することも可能となる。なお、以下表1のように設定される場合における細胞分布情報値及び担体分布情報値は、例えば、図11における上から2つ目の撮像部363又は上から3つ目の撮像部362が撮像する画像に基づいて取得されるものとする。
Further, in the first modification, the second measuring device 350 determines the number of culture carrier MCs (or the culture carrier concentration) as the carrier distribution information value and the number of cell clusters (and the number of cells) as the cell distribution information value. When calculating both, the second measuring device 350 may transmit both the cell distribution information value and the carrier distribution information value to the control device 40. In this case, the determination variation by the determination unit 43 of the control device 40 in the modification 1 increases, and for example, it is possible to set as shown in Table 1 below. The cell distribution information value and the carrier distribution information value when set as shown in Table 1 below are, for example, captured by the second image pickup unit 363 from the top or the third image pickup unit 362 from the top in FIG. It shall be acquired based on the image to be used.
第2測定装置350が細胞分布情報値及び担体分布情報値の両方を演算する場合、表1に示すように、例えば、状況1~状況5に関する各判定に対応して駆動装置20の揺動速度を予め設定することができるので、駆動装置20の揺動速度をきめ細かく制御できる。したがって、培養容器10内での細胞塊、細胞複合体等の沈降を効率的に抑制して、細胞群の生産に関し、収率性と時間効率性をさらに向上させることが可能となる。
When the second measuring device 350 calculates both the cell distribution information value and the carrier distribution information value, as shown in Table 1, for example, the rocking speed of the drive device 20 corresponding to each determination regarding the situations 1 to 5. Can be set in advance, so that the swing speed of the drive device 20 can be finely controlled. Therefore, it is possible to efficiently suppress the sedimentation of cell masses, cell complexes and the like in the culture vessel 10, and further improve the yield and time efficiency of the production of cell groups.
3-2.変形例2
次に、変形例2に係る細胞培養装置1について、図12を参照しつつ説明する。図12は、変形例2に係る細胞培養装置1における第3測定装置351の撮像装置360(撮像部360)によって撮像される画像の一例を示す図である。 3-2.Modification 2
Next, thecell culture apparatus 1 according to the modified example 2 will be described with reference to FIG. 12. FIG. 12 is a diagram showing an example of an image captured by the imaging device 360 (imaging unit 360) of the third measuring device 351 in the cell culture device 1 according to the modified example 2.
次に、変形例2に係る細胞培養装置1について、図12を参照しつつ説明する。図12は、変形例2に係る細胞培養装置1における第3測定装置351の撮像装置360(撮像部360)によって撮像される画像の一例を示す図である。 3-2.
Next, the
変形例2に係る細胞培養装置1は、前述にて説明した図11に示される変形例1に係る細胞培養装置1とほぼ同一であるが、第2測定装置350に代わって第3測定装置351を適用するものである。したがって、変形例2に係る細胞培養装置1においては、第3測定装置351について以下のとおり詳細を説明し、その他の構成要素に関する詳細な説明は省略する。なお、第3測定装置351の物理的構成は、図11に示される第2測定装置350と同じである。
The cell culture device 1 according to the modified example 2 is substantially the same as the cell culture device 1 according to the modified example 1 shown in FIG. 11 described above, but instead of the second measuring device 350, the third measuring device 351 Is applied. Therefore, in the cell culture device 1 according to the modified example 2, the details of the third measuring device 351 will be described as follows, and the detailed description of other components will be omitted. The physical configuration of the third measuring device 351 is the same as that of the second measuring device 350 shown in FIG.
変形例2に係る第3測定装置351は、変形例1に係る撮像部360と同じものを撮像装置360として用いることができる。そのうえで、第3測定装置351は、培養容器10内の懸濁液中の複数の領域における担体分布情報値を各々測定し、該複数の領域のうちの第1の領域における第1担体分布情報値と、該複数の領域のうちの第2の領域における第2担体分布情報値とを比較演算することで、担体分布情報相違値を演算する。そして、当該担体分布情報相違値と、これに対して予め設定される少なくとも1つ以上の閾値との関係に基づいて、駆動装置20の揺動速度を制御するものである。
As the third measuring device 351 according to the modified example 2, the same as the image pickup unit 360 according to the modified example 1 can be used as the image pickup device 360. Then, the third measuring device 351 measures the carrier distribution information value in each of the plurality of regions in the suspension in the culture vessel 10, and the first carrier distribution information value in the first region of the plurality of regions. And the second carrier distribution information value in the second region of the plurality of regions are compared and calculated to calculate the carrier distribution information difference value. Then, the rocking speed of the drive device 20 is controlled based on the relationship between the carrier distribution information difference value and at least one or more threshold values set in advance for the difference value.
ここで、第1担体分布情報値とは、第1の領域における担体分布情報値(具体的には、培養担体の数又は培養担体の濃度)であり、第2担体分布情報値とは、第2の領域における担体分布情報値を意味する。
Here, the first carrier distribution information value is a carrier distribution information value in the first region (specifically, the number of culture carriers or the concentration of the culture carrier), and the second carrier distribution information value is a second carrier distribution information value. It means the carrier distribution information value in the region 2.
さらに詳細を説明すると、変形例2に係る撮像装置360は、図11及び図12に示すように、複数(例えば、図11では4つ)の小型のものが用いられてもよいし、1つの大型の撮像装置360が用いられてもよい。そのうえで、例えば、撮像装置360が、図11及び図12に示すように、4つの小型の撮像装置361乃至364(又はこれらのうちの少なくとも2つの撮像装置)である場合、これらの各撮像装置361乃至364は、対応する懸濁液の領域Q1乃至Q4(又は領域Q1乃至Q4のうちの少なくとも2つの領域)を撮像する。
More specifically, as the image pickup apparatus 360 according to the second modification, as shown in FIGS. 11 and 12, a plurality of small ones (for example, four in FIG. 11) may be used, or one image pickup apparatus 360 may be used. A large image pickup device 360 may be used. Then, for example, when the image pickup device 360 is four small image pickup devices 361 to 364 (or at least two of these image pickup devices) as shown in FIGS. 11 and 12, each of these image pickup devices 361 To 364 image the regions Q1 to Q4 (or at least two regions of the regions Q1 to Q4) of the corresponding suspension.
そして、各撮像装置361乃至364は、各領域で撮像して取得した画像を本体部371へ送信する。これにより、本体部371は、領域Q1乃至Q4(又はこれらの領域のうちの少なくとも2つの領域)における各々の担体分布情報値を測定し、測定された各々の担体分布情報値を制御装置40へ送信する。
Then, each of the image pickup devices 361 to 364 transmits the image captured and acquired in each area to the main body unit 371. As a result, the main body 371 measures each carrier distribution information value in the regions Q1 to Q4 (or at least two regions of these regions), and transfers each measured carrier distribution information value to the control device 40. Send.
本体部371から領域Q1乃至Q4(又はこれらの領域のうちの少なくとも2つの領域)における各々の担体分布情報値を取得する制御装置40は、例えば、領域Q3(第1の領域に相当)における担体分布情報値(第1担体分布情報値)と、領域Q1(第2の領域に相当)における担体分布情報値(第2担体分布情報値)とを比較演算して、担体分布情報相違値を演算する。担体分布情報相違値とは、例えば、第1担体分布情報値から第2担体分布情報値を減算して得られる。
The control device 40 for acquiring each carrier distribution information value in the regions Q1 to Q4 (or at least two regions of these regions) from the main body portion 371 is, for example, a carrier in the region Q3 (corresponding to the first region). The distribution information value (first carrier distribution information value) and the carrier distribution information value (second carrier distribution information value) in the region Q1 (corresponding to the second region) are compared and calculated to calculate the carrier distribution information difference value. do. The carrier distribution information difference value is obtained, for example, by subtracting the second carrier distribution information value from the first carrier distribution information value.
さらに制御装置40の判定部43は、このように演算された担体分布情報相違値をモニタリングし、この担体分布情報相違値に対して予め設定される閾値との関係に基づいて、一実施形態と同様に、「正常」、「異常」等の判定を実行する。
Further, the determination unit 43 of the control device 40 monitors the carrier distribution information difference value calculated in this way, and based on the relationship with the threshold value set in advance for the carrier distribution information difference value, the present embodiment Similarly, the determination of "normal", "abnormal" and the like is executed.
例えば、領域Q3における担体分布情報値(第1担体分布情報値)から領域Q1における担体分布情報値(第2担体分布情報値)を減算した値が略0であれば、培養担体は、懸濁液中に均一に分散していると考えられるため「正常」と判定することができ、減算した値が0を大きく下回る場合には、培養担体が沈降等していると考えられるため「異常」と判定することができる。したがって、培養容器10内での細胞塊、細胞複合体等の沈降を効率的に抑制して、細胞群の生産に関し、収率性と時間効率性をさらに向上させることが可能となる。
For example, if the value obtained by subtracting the carrier distribution information value (second carrier distribution information value) in the region Q1 from the carrier distribution information value (first carrier distribution information value) in the region Q3 is approximately 0, the cultured carrier is suspended. Since it is considered to be uniformly dispersed in the liquid, it can be judged as "normal", and if the subtracted value is significantly lower than 0, it is considered that the culture carrier has settled, etc., so it is "abnormal". Can be determined. Therefore, it is possible to efficiently suppress the sedimentation of cell masses, cell complexes and the like in the culture vessel 10, and further improve the yield and time efficiency of the production of cell groups.
3-3.変形例3
次に、変形例3に係る細胞培養装置1について、図13を参照しつつ説明する。図13は、変形例3に係る細胞培養装置1の構成を模式的に示す概略図である。 3-3. Modification 3
Next, thecell culture apparatus 1 according to the modified example 3 will be described with reference to FIG. FIG. 13 is a schematic view schematically showing the configuration of the cell culture apparatus 1 according to the modified example 3.
次に、変形例3に係る細胞培養装置1について、図13を参照しつつ説明する。図13は、変形例3に係る細胞培養装置1の構成を模式的に示す概略図である。 3-3. Modification 3
Next, the
変形例3に係る細胞培養装置1は、一実施形態に係る細胞培養装置1に適用される第1測定装置300と、変形例1(及び変形例2)に係る細胞培養装置1に適用される第2測定装置350(及び第3測定装置351)の両方が適用されるものである。したがって、変形例3に係る制御装置40は、第1測定装置300と第2測定装置350(及び第3測定装置351)から、細胞分布情報値及び担体分布情報値(場合によっては、細胞分布情報値だけでもよい)に関する多数の値を受信することができる。したがって、制御装置40の判定部43の判定バリエーションを最も多くすることができる。その結果、駆動装置20の揺動速度をさらにきめ細かく制御できる。これにより、培養容器10内での細胞塊、細胞複合体等の沈降をさらに効率的に抑制して、細胞群の生産に関し、収率性と時間効率性をさらに向上させることが可能となる。
The cell culture device 1 according to the modified example 3 is applied to the first measuring device 300 applied to the cell culture device 1 according to the embodiment and the cell culture device 1 according to the modified example 1 (and the modified example 2). Both the second measuring device 350 (and the third measuring device 351) are applied. Therefore, the control device 40 according to the modified example 3 has a cell distribution information value and a carrier distribution information value (in some cases, cell distribution information) from the first measuring device 300 and the second measuring device 350 (and the third measuring device 351). You can receive a large number of values for). Therefore, the determination variation of the determination unit 43 of the control device 40 can be maximized. As a result, the swing speed of the drive device 20 can be controlled more finely. This makes it possible to more efficiently suppress the sedimentation of cell masses, cell complexes and the like in the culture vessel 10, and further improve the yield and time efficiency of the production of cell groups.
以上、前述の通り、様々な実施形態を例示したが、上記実施形態はあくまで一例であって、発明の範囲を限定することは意図していない。上記実施形態は、その他の様々な形態で実施されることが可能であり、発明の要旨を逸脱しない範囲で、種々の省略、置換、変更を行うことができる。また、各構成や、形状、大きさ、長さ、幅、厚さ、高さ、数等は適宜変更して実施することができる。また、本開示において説明した様々な実施形態においては、高い収率性と時間効率性で細胞を培養することを可能とする範囲で、所定の手作業のプロセスを介在させてもよい。
As described above, various embodiments have been exemplified, but the above embodiments are merely examples and are not intended to limit the scope of the invention. The above embodiment can be implemented in various other embodiments, and various omissions, substitutions, and changes can be made without departing from the gist of the invention. In addition, each configuration, shape, size, length, width, thickness, height, number, and the like can be appropriately changed. In addition, in the various embodiments described in the present disclosure, predetermined manual processes may be intervened to the extent that cells can be cultured with high yield and time efficiency.
本開示において、「~」を用いて示された数値範囲は、「~」の前後に記載される数値をそれぞれ最小値および最大値として含む範囲を示す。本開示に段階的に記載されている数値範囲において、ある段階の数値範囲の上限値または下限値は、他の段階の数値範囲の上限値または下限値と任意に組み合わせることができる。本明細書において「工程」との語は、独立した工程だけではなく、他の工程と明確に区別できない場合であってもその工程の所期の作用が達成されれば、本用語に含まれる。
In the present disclosure, the numerical range indicated by using "-" indicates a range including the numerical values before and after "-" as the minimum value and the maximum value, respectively. Within the numerical range described stepwise in the present disclosure, the upper or lower limit of the numerical range at one stage may be optionally combined with the upper or lower limit of the numerical range at another stage. In the present specification, the term "process" is included in this term not only as an independent process but also as long as the intended action of the process is achieved even if it cannot be clearly distinguished from other processes. ..
本開示は、以下の日本国特許出願に基づくものであって、当該日本国特許出願による優先権の利益を享受するものである。また、以下の日本国特許出願の全体の内容が参照により本開示に組み入れられる。
(1)「細胞培養装置、及び、細胞群を生産する方法」と題して2020年9月25日に提出された日本国特許出願第2020-161196 This disclosure is based on the following Japanese patent application and enjoys the priority of the Japanese patent application. In addition, the entire contents of the following Japanese patent application are incorporated into this disclosure by reference.
(1) Japanese Patent Application No. 2020-161196 filed on September 25, 2020, entitled "Cell Culture Device and Method for Producing Cell Group".
(1)「細胞培養装置、及び、細胞群を生産する方法」と題して2020年9月25日に提出された日本国特許出願第2020-161196 This disclosure is based on the following Japanese patent application and enjoys the priority of the Japanese patent application. In addition, the entire contents of the following Japanese patent application are incorporated into this disclosure by reference.
(1) Japanese Patent Application No. 2020-161196 filed on September 25, 2020, entitled "Cell Culture Device and Method for Producing Cell Group".
本開示に記載されたすべての文献、特許出願、および技術規格は、参照によりその全体が本開示に組み入れられる。
All documents, patent applications, and technical standards described in this disclosure are incorporated herein by reference in their entirety.
Claims (24)
- 細胞と、前記細胞が接着する被接着体との塊体を少なくとも含む懸濁液を収容する培養容器と、
前記懸濁液を揺動させる駆動装置と、
前記懸濁液中の少なくとも一部の領域における、前記塊体に関する塊体分布情報値を測定する測定装置と、
前記塊体分布情報値の所定単位時間あたりの変化率と、前記変化率に対して設定される少なくとも1つ以上の閾値との関係に基づいて、前記駆動装置の揺動速度を制御する制御装置と、
を具備する、細胞培養装置。 A culture vessel containing a suspension containing at least a mass of cells and an adherend to which the cells adhere.
The driving device that shakes the suspension and
A measuring device for measuring a mass distribution information value for the mass in at least a part of the region of the suspension, and a measuring device.
A control device that controls the swing speed of the drive device based on the relationship between the rate of change of the mass distribution information value per predetermined unit time and at least one or more threshold values set for the rate of change. When,
A cell culture device. - 複数の細胞の凝集体である細胞塊、及び前記細胞と培養担体との複合体である細胞複合体の少なくとも一方を含む懸濁液を収容する培養容器と、
前記懸濁液を揺動させる駆動装置と、
前記懸濁液中の少なくとも一部の領域における、前記細胞に関する細胞分布情報値、及び前記培養担体に関する担体分布情報値の少なくとも一方を測定する測定装置と、
前記細胞分布情報値及び前記担体分布情報値の少なくとも一方の所定単位時間あたりの変化率と、前記変化率に対して設定される少なくとも1つ以上の閾値との関係に基づいて、前記駆動装置の揺動速度を制御する制御装置と、
を具備する、請求項1に記載の細胞培養装置。 A culture vessel containing a suspension containing at least one of a cell mass, which is an aggregate of a plurality of cells, and a cell complex, which is a complex of the cells and a culture carrier.
The driving device that shakes the suspension and
A measuring device for measuring at least one of the cell distribution information value for the cell and the carrier distribution information value for the culture carrier in at least a part of the region in the suspension.
The driving device of the driving device is based on the relationship between the rate of change of at least one of the cell distribution information value and the carrier distribution information value per predetermined unit time and at least one or more threshold values set for the rate of change. A control device that controls the rocking speed and
The cell culture apparatus according to claim 1. - 前記測定装置は、
前記少なくとも一部の領域において電場を形成させる電極と、前記電場内のキャパシタンスを測定するキャパシタンス測定部と、を有する第1測定装置を含み、
前記第1測定装置は、前記キャパシタンスに基づいて、前記少なくとも一部の領域における前記細胞分布情報値を測定する、請求項2に記載の細胞培養装置。 The measuring device is
A first measuring device including an electrode for forming an electric field in at least a part of the region and a capacitance measuring unit for measuring the capacitance in the electric field is included.
The cell culture device according to claim 2, wherein the first measuring device measures the cell distribution information value in at least a part of the region based on the capacitance. - 前記第1測定装置は、前記少なくとも一部の領域における導電率を測定する導電率測定部、をさらに有する、請求項3に記載の細胞培養装置。 The cell culture apparatus according to claim 3, further comprising a conductivity measuring unit for measuring conductivity in at least a part of the region.
- 前記第1測定装置は、前記少なくとも一部の領域におけるpHを測定するpH測定部、をさらに有する、請求項3又は4に記載の細胞培養装置。 The cell culture device according to claim 3 or 4, further comprising a pH measuring unit for measuring pH in at least a part of the region.
- 前記測定装置は、
前記少なくとも一部の領域を撮像して1つ以上の画像を取得する撮像部、を有する第2測定装置を含み、
前記第2測定装置は、前記画像に基づいて、前記少なくとも一部の領域における前記細胞分布情報値及び前記担体分布情報値の少なくとも一方を測定する、請求項2乃至5のいずれか一項に記載の細胞培養装置。 The measuring device is
A second measuring device including an image pickup unit, which captures an image of at least a part of the area and acquires one or more images.
The second measuring device according to any one of claims 2 to 5, wherein the second measuring device measures at least one of the cell distribution information value and the carrier distribution information value in the at least a part of the region based on the image. Cell culture device. - 前記細胞分布情報値は、前記少なくとも一部の領域における、前記懸濁液中の前記細胞のうちの生細胞数又は生細胞濃度である、請求項2乃至6のいずれか一項に記載の細胞培養装置。 The cell according to any one of claims 2 to 6, wherein the cell distribution information value is the number of living cells or the concentration of living cells among the cells in the suspension in the at least a part of the region. Incubator.
- 前記担体分布情報値は、前記少なくとも一部の領域における、前記懸濁液中の前記培養担体の数又は前記培養担体の濃度である、請求項2乃至7のいずれか一項に記載の細胞培養装置。 The cell culture according to any one of claims 2 to 7, wherein the carrier distribution information value is the number of the culture carriers in the suspension or the concentration of the culture carriers in at least a part of the region. Device.
- 前記閾値は、前記変化率に対して、0%に設定される第1閾値を含む、請求項2乃至8のいずれか一項に記載の細胞培養装置。 The cell culture apparatus according to any one of claims 2 to 8, wherein the threshold value includes a first threshold value set to 0% with respect to the rate of change.
- 前記閾値は、前記変化率に対して、0%よりも大きい値に設定される第2閾値をさらに含む、請求項9に記載の細胞培養装置。 The cell culture apparatus according to claim 9, wherein the threshold value further includes a second threshold value set to a value larger than 0% with respect to the rate of change.
- 前記制御装置は、
前記変化率が前記第1閾値以上と判定された場合、前記駆動装置の前記揺動速度を増加させ、
前記変化率が前記第1閾値未満と判定された場合、前記駆動装置の前記揺動速度を減少させる、請求項9又は10に記載の細胞培養装置。 The control device is
When the rate of change is determined to be equal to or higher than the first threshold value, the rocking speed of the driving device is increased.
The cell culture apparatus according to claim 9 or 10, wherein when the rate of change is determined to be less than the first threshold value, the rocking speed of the driving device is reduced. - 前記制御装置は、
前記変化率が、複数の前記所定単位時間に渡って連続で前記第1閾値未満と判定された場合、前記駆動装置の前記揺動速度を0とする、請求項11に記載の細胞培養装置。 The control device is
The cell culture apparatus according to claim 11, wherein when the rate of change is continuously determined to be less than the first threshold value over a plurality of predetermined unit times, the rocking speed of the driving device is set to 0. - 前記制御装置は、
前記変化率が前記第2閾値以上と判定された場合、該判定時の前記揺動速度を維持させる、請求項11又は12に記載の細胞培養装置。 The control device is
The cell culture apparatus according to claim 11 or 12, wherein when the rate of change is determined to be equal to or higher than the second threshold value, the rocking rate at the time of the determination is maintained. - 前記駆動装置は、前記懸濁液を撹拌する撹拌機又は振盪機である、請求項2乃至13のいずれか一項に記載の細胞培養装置。 The cell culture device according to any one of claims 2 to 13, wherein the driving device is a stirrer or a shaker that stirs the suspension.
- 前記培養容器の容量は、2.0L以上である、請求項2乃至14のいずれか一項に記載の細胞培養装置。 The cell culture apparatus according to any one of claims 2 to 14, wherein the capacity of the culture container is 2.0 L or more.
- 前記少なくとも一部の領域は、前記懸濁液中であって前記培養容器の深さ方向における中央部分、及び前記懸濁液中であって前記培養容器の底面部分の少なくとも一方を含む、請求項2乃至15のいずれか一項に記載の細胞培養装置。 Claimed that the at least a portion of the region comprises at least one of the central portion of the suspension in the depth direction of the culture vessel and the bottom portion of the suspension in the culture vessel. The cell culture apparatus according to any one of 2 to 15.
- 細胞と、前記細胞が接着する被接着体との塊体を少なくとも含む懸濁液を収容する培養容器と、
前記懸濁液を揺動させる駆動装置と、
前記懸濁液中の複数の領域における、前記塊体に関する塊体分布情報値を各々測定する測定装置と、
前記複数の領域のうちの第1の領域における第1塊体分布情報値と、前記複数の領域のうちの第2の領域における第2塊体分布情報値とを比較演算することにより算出される塊体分布情報相違値と、該塊体分布情報相違値に対して設定される少なくとも1つ以上の閾値との関係に基づいて、前記駆動装置の揺動速度を制御する制御装置と、
を具備する、細胞培養装置。 A culture vessel containing a suspension containing at least a mass of cells and an adherend to which the cells adhere.
The driving device that shakes the suspension and
A measuring device for measuring the mass distribution information value for the mass in a plurality of regions in the suspension, and a measuring device.
It is calculated by comparing the first mass distribution information value in the first region of the plurality of regions with the second mass distribution information value in the second region of the plurality of regions. A control device that controls the swing speed of the drive device based on the relationship between the mass distribution information difference value and at least one or more threshold values set for the mass distribution information difference value.
A cell culture device. - 複数の細胞の凝集体である細胞塊、及び前記細胞と培養担体との複合体である細胞複合体の少なくとも一方を含む懸濁液を収容する培養容器と、
前記懸濁液を揺動させる駆動装置と、
前記懸濁液中の複数の領域における、前記培養担体に関する担体分布情報値を各々測定する測定装置と、
前記複数の領域のうちの第1の領域における第1担体分布情報値と、前記複数の領域のうちの第2の領域における第2担体分布情報値とを比較演算することにより算出される担体分布情報相違値と、該担体分布情報相違値に対して設定される少なくとも1つ以上の閾値との関係に基づいて、前記駆動装置の揺動速度を制御する制御装置と、
を具備する、請求項17に記載の細胞培養装置。 A culture vessel containing a suspension containing at least one of a cell mass, which is an aggregate of a plurality of cells, and a cell complex, which is a complex of the cells and a culture carrier.
The driving device that shakes the suspension and
A measuring device for measuring carrier distribution information values related to the culture carrier in a plurality of regions in the suspension, and a measuring device.
The carrier distribution calculated by comparing the first carrier distribution information value in the first region of the plurality of regions with the second carrier distribution information value in the second region of the plurality of regions. A control device that controls the swing speed of the drive device based on the relationship between the information difference value and at least one or more threshold values set for the carrier distribution information difference value.
17. The cell culture apparatus according to claim 17. - 前記測定装置は、
前記複数の領域を撮像して1つ以上の画像を取得する撮像装置、を有する第3測定装置を含み、
前記第3測定装置は、前記第1の領域における前記画像に基づいて前記第1担体分布情報値を測定し、且つ前記第2の領域における前記画像に基づいて前記第2担体分布情報値を測定する、請求項18に記載の細胞培養装置。 The measuring device is
A third measuring device comprising an imaging device, which captures the plurality of regions and acquires one or more images.
The third measuring device measures the first carrier distribution information value based on the image in the first region, and measures the second carrier distribution information value based on the image in the second region. The cell culture apparatus according to claim 18. - 前記第1の領域は、前記懸濁液中であって前記培養容器の深さ方向における中央部分であり、
前記第2の領域は、前記懸濁液中であって前記培養容器の底面部分である、請求項18又は19に記載の細胞培養装置。 The first region is the central portion of the suspension in the depth direction of the culture vessel.
The cell culture apparatus according to claim 18 or 19, wherein the second region is a bottom portion of the culture vessel in the suspension. - 細胞と、前記細胞が接着する被接着体との塊体を少なくとも含む懸濁液を培養容器内に収容すること、
駆動装置によって前記懸濁液を揺動させること、
前記懸濁液中の少なくとも一部の領域における、前記塊体に関する塊体分布情報値を測定すること、
前記塊体分布情報値の所定単位時間あたりの変化率と、前記変化率に対して設定される少なくとも1つ以上の閾値との関係を判定し、該判定の結果に基づいて前記駆動装置の揺動速度を決定すること、及び、
決定された前記揺動速度を前記駆動装置に出力すること、
を含む培養方法により、細胞群を生産する方法。 A suspension containing at least a mass of cells and an adherend to which the cells adhere is contained in a culture vessel.
Swinging the suspension with a drive,
To measure the mass distribution information value for the mass in at least a part of the region in the suspension.
The relationship between the rate of change of the mass distribution information value per predetermined unit time and at least one or more threshold values set for the rate of change is determined, and the driving device is shaken based on the result of the determination. Determining the moving speed and
To output the determined rocking speed to the driving device,
A method for producing a cell group by a culture method including. - 複数の細胞の凝集体である細胞塊、及び前記細胞と培養担体との複合体である細胞複合体の少なくとも一方を含む懸濁液を培養容器内に収容すること、
駆動装置によって前記懸濁液を揺動させること、
前記懸濁液中の少なくとも一部の領域における、前記細胞に関する細胞分布情報値、及び前記培養担体に関する担体分布情報値の少なくとも一方を測定すること、
前記細胞分布情報値及び前記担体分布情報値の少なくとも一方の所定単位時間あたりの変化率と、前記変化率に対して設定される少なくとも1つ以上の閾値との関係を判定し、該判定の結果に基づいて前記駆動装置の揺動速度を決定すること、及び、
決定された前記揺動速度を前記駆動装置に出力すること、
を含む、請求項21に記載の細胞群を生産する方法。 A suspension containing at least one of a cell mass, which is an aggregate of a plurality of cells, and a cell complex, which is a complex of the cells and a culture carrier, is contained in a culture vessel.
Swinging the suspension with a drive,
To measure at least one of the cell distribution information value for the cell and the carrier distribution information value for the culture carrier in at least a part of the region in the suspension.
The relationship between the rate of change of at least one of the cell distribution information value and the carrier distribution information value per predetermined unit time and at least one or more threshold values set for the rate of change is determined, and the result of the determination is determined. To determine the rocking speed of the drive device based on, and
To output the determined rocking speed to the driving device,
21. The method for producing a cell group according to claim 21. - 細胞と、前記細胞が接着する被接着体との塊体を少なくとも含む懸濁液を培養容器内に収容すること、
駆動装置によって前記懸濁液を揺動させること、
前記懸濁液中の複数の領域における、前記塊体に関する塊体分布情報値を各々測定すること、
前記複数の領域のうちの第1の領域における第1塊体分布情報値と、前記複数の領域のうちの第2の領域における第2塊体分布情報値とを比較演算することにより算出される塊体分布情報相違値と、該塊体分布情報相違値に対して設定される少なくとも1つ以上の閾値との関係を判定し、該判定の結果に基づいて前記駆動装置の揺動速度を決定すること、及び、
決定された前記揺動速度を前記駆動装置に出力すること、
を含む培養方法により、細胞群を生産する方法。 A suspension containing at least a mass of cells and an adherend to which the cells adhere is contained in a culture vessel.
Swinging the suspension with a drive,
To measure the mass distribution information value for the mass in each of the plurality of regions in the suspension.
Calculated by comparing the first mass distribution information value in the first region of the plurality of regions with the second mass distribution information value in the second region of the plurality of regions. The relationship between the mass distribution information difference value and at least one or more threshold values set for the mass distribution information difference value is determined, and the swing speed of the drive device is determined based on the result of the determination. What to do and
To output the determined rocking speed to the driving device,
A method for producing a cell group by a culture method including. - 複数の細胞の凝集体である細胞塊、及び前記細胞と培養担体との複合体である細胞複合体の少なくとも一方を含む懸濁液を培養容器内に収容すること、
駆動装置によって前記懸濁液を揺動させること、
前記懸濁液中の複数の領域における、前記培養担体に関する担体分布情報値を各々測定すること、
前記複数の領域のうちの第1の領域における第1担体分布情報値と、前記複数の領域のうちの第2の領域における第2担体分布情報値とを比較演算することにより算出される担体分布情報相違値と、該担体分布情報相違値に対して設定される少なくとも1つ以上の閾値との関係を判定し、該判定の結果に基づいて前記駆動装置の揺動速度を決定すること、及び、
決定された前記揺動速度を前記駆動装置に出力すること、
を含む培養方法により、細胞群を生産する請求項23に記載の方法。 A suspension containing at least one of a cell mass, which is an aggregate of a plurality of cells, and a cell complex, which is a complex of the cells and a culture carrier, is contained in a culture vessel.
Swinging the suspension with a drive,
To measure the carrier distribution information value for the culture carrier in each of the plurality of regions in the suspension.
The carrier distribution calculated by comparing the first carrier distribution information value in the first region of the plurality of regions with the second carrier distribution information value in the second region of the plurality of regions. The relationship between the information difference value and at least one or more threshold values set for the carrier distribution information difference value is determined, and the rocking speed of the drive device is determined based on the result of the determination. ,
To output the determined rocking speed to the driving device,
23. The method of claim 23, wherein a cell group is produced by a culture method comprising.
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JP2013192477A (en) * | 2012-03-16 | 2013-09-30 | Hitachi Ltd | Method for monitoring number of cell |
JP2019124594A (en) * | 2018-01-17 | 2019-07-25 | 横河電機株式会社 | Cell inspection device, cell inspection method, program and recording medium |
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JP2010099011A (en) * | 2008-10-24 | 2010-05-06 | Panasonic Corp | Cell-culturing device and cell-culturing method |
JP2013192477A (en) * | 2012-03-16 | 2013-09-30 | Hitachi Ltd | Method for monitoring number of cell |
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