WO2022065400A1

WO2022065400A1 - Cell culture apparatus and method for producing cell groups

Info

Publication number: WO2022065400A1
Application number: PCT/JP2021/034943
Authority: WO
Inventors: 悠午鈴木; 雅弘岡野定; 亮介高橋
Original assignee: 昭和電工マテリアルズ株式会社
Priority date: 2020-09-25
Filing date: 2021-09-24
Publication date: 2022-03-31
Also published as: JP2023165684A

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

Cell culture device and method for producing cell population

The present disclosure relates to a cell culture apparatus and a method for producing a cell group.

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. ..

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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. ..

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.

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.

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.

It is a schematic diagram schematically showing the structure of the cell culture apparatus which concerns on one Embodiment. It is a schematic diagram schematically showing the structure in the case where the 1st measuring apparatus is used as a measuring apparatus in the cell culture apparatus which concerns on 1 Embodiment. 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. It is a figure which shows typically an example of the relationship between the determination result by the determination unit of the control device which concerns on one Embodiment, and the rocking speed of a drive device 20 determined by an output unit. It is a figure which shows typically an example of the relationship between the determination result by the determination unit of the control device which concerns on one Embodiment, and the rocking speed of a drive device 20 determined by an output unit. It is a flow chart which shows an example of a part of the operation performed in the cell culture apparatus which concerns on one Embodiment. 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 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.

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. 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 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. 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 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.

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.

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).

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 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. 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. 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. ..

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.

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".

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.

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.

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.

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. 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.

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.

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.

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.

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.

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.

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. Drive device 20
The drive 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.

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.

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. Measuring device 30
The measuring device 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).

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.

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.

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.

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.

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. 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).

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.

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. 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.

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.

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. 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).

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.

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.

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.

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).

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).

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).

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.

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).

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.

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.

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. 2. Operation of Cell 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).

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.

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.

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).

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.

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.

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).

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.

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.

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".

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.

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. 3. Modification Example Next, a modification of the cell culture apparatus 1 according to the embodiment described above will be described.

3-1. Modification 1
The cell 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.

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. 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. ..

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.

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.

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.

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.

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.

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.

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. Modification 2
Next, the cell 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.

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.

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.

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.

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.

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.

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.

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.

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. Modification 3
Next, the cell 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.

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.

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.

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. ..

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".

All documents, patent applications, and technical standards described in this disclosure are incorporated herein by reference in their entirety.

Claims

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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.