JP5995397B2 - Cell culture method and cell culture apparatus - Google Patents

Description

  The present invention relates to a cell culture method and a cell culture apparatus for culturing cells such as cells, tissues, and microorganisms.

In recent years, in the fields of pharmaceutical production, gene therapy, regenerative medicine, immunotherapy, and the like, it has been required to efficiently culture a large amount of cells, tissues, microorganisms, and the like in an artificial environment.
In culturing such cells in large quantities, especially when culturing suspension cells, stirring culture using a culture tank equipped with a stirring blade is generally used. However, in the case of cells that are particularly vulnerable to damage due to external forces or cells that grow while forming aggregates, do not use a stirring blade and enclose the cells in a culture vessel and leave them still (with the cells sinking to the bottom). ) A method of culturing and transferring to a larger container bottom area or increasing the number of containers according to cell growth is widely used. However, in this stationary culture, when the aggregate of cells increases with the proliferation of cells, there is a problem that the supply of oxygen and nutrients to each cell gradually becomes insufficient and the proliferation efficiency decreases.
In addition, when the container is transferred, the culture solution is once agitated, and the unevenness of oxygen and nutrients is resolved. .

On the other hand, shaking culture in which the culture vessel is constantly stirred is also widely performed.
For example, according to the cell culture device described in Patent Document 1, it is possible to move the stage on which the culture container is loaded in various patterns such as rotation and shaking, and to stir the culture solution in the culture container. .
The cell culture apparatus described in Patent Document 2 or Patent Document 3 is a mechanism for supplying oxygen by the movement of waves so that the liquid medium in the culture container is shaken so as not to generate bubbles and the cells are not damaged. It has become.
In such a shaking method using a cell culture device, the entire medium is vigorously stirred, so that the cells are separated individually, and oxygen and nutrients are diffused throughout and can be sufficiently supplied to each cell. It has become.

US Pat. No. 5,057,429 International Publication No. 2000/66706 Pamphlet Special table 2007-511231 gazette

However, depending on the type of cell, cell growth is unlikely to occur when the cells are in pieces, and the growth efficiency is improved when the individual cells adhere to form an appropriately sized aggregate. is there. Specific examples include adhering cells such as neural stem cells, embryonic stem cells, hepatocytes, corneal stem cells, islet cells, and floating cells such as blood cells.
In the case of such cells, in the conventional static culture, since the culture vessel is vigorously stirred at each transfer timing, each time the cells are separated, there is a problem that the proliferation efficiency of the cells is lowered.
In addition, when performing conventional shaking culture, the whole medium is similarly vigorously stirred and floats apart in the medium, so that the cells are less likely to form an appropriately sized aggregate and the cell growth efficiency is improved. There was a problem that it was difficult to optimize.

  The present invention has been considered in view of the above circumstances, and by controlling the formation of aggregates of cells in a culture vessel and controlling the decomposition of aggregates, the aggregates are adjusted to an appropriate size, and cell proliferation An object is to provide a cell culture apparatus and a cell culture method capable of improving efficiency.

  In order to achieve the above object, the cell culturing method of the present invention is a cell culturing method using a culture container, and controls the formation of aggregates of cells in the culture container by applying an external force to the culture container. This is a method for culturing cells by controlling at least one of the degradation control of aggregates.

  Further, the cell culture device of the present invention is a cell culture device for culturing cells using a culture vessel, pressing a culture vessel with a predetermined pushing amount, And a stirring member that can move in the horizontal direction, and by applying an external force to the culture vessel by moving the stirring member, it is configured to control at least one of the formation and decomposition of the cell clumps in the culture vessel .

  ADVANTAGE OF THE INVENTION According to this invention, when performing cell culture of a cell, a structure | tissue, microorganisms, etc., it becomes possible to adjust an aggregate to an appropriate size and to improve the proliferation efficiency of a cell.

It is a figure which shows the structure of the cell culture apparatus of 1st embodiment of this invention. It is a figure which shows the structure of the drive device in the cell culture apparatus of 1st embodiment of this invention. It is a schematic side view of the cell culture device of the first embodiment of the present invention. It is a figure which shows formation of the aggregate of a cell in this invention, and decomposition | disassembly of the aggregate of a cell. It is a figure which shows the structure of the cell culture apparatus of 2nd embodiment of this invention. It is a figure which shows the structure of the cell culture apparatus of 3rd embodiment of this invention. It is a figure which shows the kind of stirring conditions performed using the cell culture apparatus of 1st embodiment of this invention. It is the figure which put together the result of the cell state by each stirring condition performed using the cell culture apparatus of 1st embodiment of this invention.

  Hereinafter, preferred embodiments of the cell culture method and the cell culture apparatus of the present invention will be described with reference to the drawings.

[First embodiment]
First, with reference to FIGS. 1-4, 1st embodiment of this invention is described. FIG. 1 is a diagram showing the configuration of the cell culture device of the present embodiment. FIG. 2 is a diagram illustrating a configuration of a driving device in the cell culture device of the present embodiment. FIG. 3 is a schematic side view of the cell culture device of the present embodiment. FIG. 4 is a diagram showing the formation of cell aggregates and the decomposition of cell aggregates in the present invention.

[Cell culture device 10]
As shown in FIG. 1, the cell culture device 10 of the present embodiment includes a culture vessel 11, a loading table 13, and a stirring member 14. Cells are encapsulated and the tube 12 is connected.

  The culture container 11 is a container formed in a bag shape (back shape) using a soft packaging material. Thus, by using a soft wrapping material as the material of the culture vessel 11, flexibility and softness can be imparted to the culture vessel 11. Examples of the soft packaging material include, for example, Japanese Patent Application Laid-Open No. 2002-255277 (a food packaging body using a soft packaging material film sheet and a method for taking out food), and Japanese Patent Application Laid-Open No. 2004-323077 (a pressure extraction type bag shape). Those described in (Containers) can be used.

  The culture vessel 11 has gas permeability necessary for cell culture, and part or all of the culture vessel 11 is transparent so that the contents can be confirmed. Examples of the material for the culture container satisfying such conditions include polyolefin, ethylene-vinyl acetate copolymer, styrene elastomer, polyester thermoplastic elastomer, silicone thermoplastic elastomer, and silicone rubber.

  The tube 12 is for injecting the culture solution and cells in the culture vessel 11 from the outside or collecting them outside, and each side of the culture vessel 11 is sealed, but at least two or more of them are sealed. A tube 12 is connected. One of these is for injection for injecting cultured cells and medium from outside into the culture container 11, and the other is for recovery for recovering cultured cells and medium from the culture container 11. Further, as shown in FIG. 1, when three tubes 12 are attached, the third tube can be used for sampling for taking out cultured cells and culture media from the culture vessel 11 as samples.

  The material of the tube 12 may be appropriately selected according to the usage environment. For example, silicone rubber, soft vinyl chloride resin, polybutadiene resin, ethylene-vinyl acetate copolymer, chlorinated polyethylene resin, polyurethane-based thermoplastic elastomer, polyester-based thermoplastic elastomer, silicone-based thermoplastic elastomer, styrene-based elastomer, for example, Use SBS (styrene, butadiene, styrene), SIS (styrene, isoprene, styrene), SEBS (styrene, ethylene, butylene, styrene), SEPS (styrene, ethylene, propylene, styrene), polyolefin resins, fluorine resins, etc. Can do.

The loading table 13 is a flat table on which the culture vessel 11 is placed on the upper surface, and the stirring member 14 is disposed on the upper surface of the culture vessel 11.
Stop members 13-1 are erected at the four corners of the portion on which the culture vessel 11 is placed on the upper surface of the loading table 13. On the other hand, at the four corners of the culture vessel 11, holes 11-2 into which the stopper members 13-1 are engaged are formed.
By passing each hole 11-2 of the culture vessel 11 through each of the stopper members 13-1, the culture vessel 11 can be placed on the upper surface of the loading table 13. Further, it is possible to prevent the culture vessel 11 from being displaced with the movement of the stirring member 14.
The stop member is not limited to the above member, and various members can be used as long as they have a mechanism for preventing the culture vessel 11 from shifting.

The stirring member 14 controls the formation of cell aggregates in the culture container 11 and the decomposition of the cell aggregates by applying an external force to the culture container 11.
In the cell culture method of the present embodiment, as shown in FIG. 3, the stirring member 14 is moved in parallel with the loading table 13 at a predetermined speed while pressing the culture vessel 11 with a predetermined pushing amount by the stirring member 14. . This movement is repeatedly executed in a predetermined cycle. As the stirring member 14, for example, a roller can be used.

  As described above, according to the present embodiment, the stirring member 14 is used to apply an external force to the culture vessel, so that the stirring in the culture vessel 11 can be finely adjusted. It is possible to perform agitation suitable for performing and decomposing cell clumps.

  As shown in FIG. 1, the support table 15 connects the bearing units to a bearing unit that is erected upward on each side of the loading table 13 and supports both ends of the stirring member 14 so as to be rotatable. It is formed by the connecting part.

  As shown in FIG. 2, the support base 15 can be moved up and down by a rod-type electric cylinder 17 (vertical operation actuator) attached below the connecting portion, and by the stirring member 14 attached to the support base 15. It is possible to finely adjust the push-in amount with respect to the culture vessel 11 by 0.1 mm.

The rod-type electric cylinder 17 is attached to the moving table 16 on the slider-type electric cylinder 21 (horizontal operation actuator), and moves in the horizontal direction with respect to the loading table 13. Further, the moving speed of the stirring member 14 attached to the support table 15 is adjusted by controlling the moving speed of the moving table 16 in the horizontal direction.
The drive device in the cell culture device 10 of the present embodiment includes a support base 15, a moving base 16, a rod-type electric cylinder 17, a slider-type electric cylinder 21, and the like.

As described above, according to the cell culture device 10 of the present embodiment, the rod-type electric cylinder 17 and the slider-type electric cylinder 21 are used to adjust the pushing amount of the stirring member 14 with respect to the culture container 11 and the moving speed of the stirring member 14. This makes it possible to optimally adjust the size of the cell aggregate by controlling the stirring of the culture solution in the culture vessel 11.
For the operation control of the stirring member, an actuator using air pressure, hydraulic pressure, electromagnetic force, or a motor or cam is used instead of the electric actuator such as the rod type electric cylinder 17 or the slider type electric cylinder 21. It is also possible to make a configuration that was suitable.

<Formation of cell aggregates>
Here, as shown in FIG. 4, in cell culture, there is a size of a cell aggregate suitable for culture depending on the cell type.
That is, a cultured cell has a property that a single cell has a low division rate, and it only starts to divide enough when it adheres to each other to form a certain aggregate.
In normal static culture, when the cell density at the initial stage of culture is low, the cells adhere to each other by diffusion and gradually form aggregates, but the rate is relatively slow.

For this reason, conventionally, at the initial stage of culture, for example, a well plate is used to forcibly collect cells in one place at a high density to facilitate cell adhesion, or a small volume container is used to proliferate cells. Therefore, the culture was performed while preventing a decrease in cell density by using a larger container.
On the other hand, in this embodiment, the cells floating on the bottom surface in the culture vessel 11 are actively moved by stirring, the probability that the cells adhere to each other is increased, and an appropriately sized aggregate can be formed more quickly. ing.

Thereby, according to the cell culture method using the cell culture apparatus 10 of the present embodiment, when the cells are in an individual state at the start of the cell culture, the cells come into contact with each other to form an appropriately sized aggregate. Can be promoted, and cell proliferation efficiency can be improved.
In addition, the control of the formation of the aggregate of cells is not limited to the initial stage of culture (during seeding). For example, as a result of applying an excessive external force to the culture vessel 11 during cell culture, the aggregate Can be suitably performed even when the cells have collapsed and the cells have become individual, and the cell proliferation efficiency can be improved.

<Decomposition of cell clumps>
On the other hand, if the cell aggregate is too large, the inside of the aggregate becomes oxygen deficient and nutrient deficient, and the growth efficiency decreases.
For this reason, when the aggregate is enlarged, it is preferable to cause a strong flow (turbulent flow) in the culture solution to decompose the aggregate.
According to the culture method using the cell culture device 10 of the present embodiment, when the agglomerate is enlarged, the strong amount of the culture solution can be generated by adjusting the pushing amount and the moving speed of the stirring member 14. Agitation can be controlled to break up the agglomerates to an appropriate size.

As described above, according to the cell culture device 10 and the cell culture method using the same according to the present embodiment, the stirring member 14 is pressed against the loading container 14 while the culture container 11 is pressed by the stirring member 14 with a predetermined pushing amount. 13 can be moved at a predetermined speed in parallel with 13, and the strength of the external force applied to the culture vessel can be appropriately controlled.
For this reason, the agitation in the culture vessel 11 can be finely adjusted to perform agitation suitable for the formation of cell agglomerates, and the agglomerates can be decomposed so that the cells do not fall apart. It is possible to adjust the mass to a size suitable for growth.

[Second Embodiment]
Next, a second embodiment of the present invention will be described with reference to FIG. This figure is a diagram showing the configuration of the cell culture device of the present embodiment.
In this embodiment, in the cell culture device 10 in which the culture vessel 11 is divided into a culture part and an expandable part using a partition member, and the culture medium volume can be adjusted to an appropriate size according to cell growth, the culture part Is different from the first embodiment in that it can be stirred by a stirring member (stirring roller) 14. In the present embodiment, both ends of the culture vessel 11 are fixed by the clamp members 23. About another point, it is the same as that of 1st embodiment.

That is, as shown in FIG. 5, the cell culture device 10 of the present embodiment divides the culture vessel 11 using a partition roller (partition member) 22, a culture unit that encloses the culture solution and cells, and the partition roller 22. And an expandable part for expanding the volume of the culture part. The partition roller 22 is provided in parallel with the stirring member (stirring roller) 14 and can move in parallel with the loading table 13.
By using such a partition roller 22, the volume of the culture part can be continuously changed, and the cell density can be adjusted appropriately by moving the partition roller 22 along with cell proliferation and increasing the volume of the culture part. It is possible to maintain within the range.

In the example of the figure, the culture vessel 11 is sandwiched and partitioned from above and below using two partition rollers 22, but the present invention is not limited to this, and the culture vessel 11 is viewed from above by one partition roller 22. It is also possible to have a configuration in which the culture vessel 11 is partitioned into a culture part and an expandable part by pressing against the loading table 13.
The technology for controlling the culture volume and improving the culture efficiency using such a partition member is described in detail in International Publication WO2008 / 136371 and International Publication WO2008 / 136339 by the applicant. Yes.

According to the cell culture apparatus 10 and the cell culture method using the same according to the present embodiment, the size of the culture volume can be controlled using the partition roller 22 so that high cell proliferation efficiency can be obtained, and the culture unit The size of the cell aggregates in can be adjusted so as to obtain high cell growth efficiency.
For this reason, it is possible to further improve the cell proliferation efficiency.

[Third embodiment]
Next, a third embodiment of the present invention will be described with reference to FIG. FIG. 1 is a diagram showing a configuration of the cell culture device 10 of the present embodiment.
This embodiment is the first in that the cells in the culture vessel 11 are photographed to automatically determine whether the size of the aggregate is within a predetermined range, and the aggregate is adjusted to an appropriate size based on the determination result. Different from the embodiment. About another point, it is the same as that of 1st embodiment.

That is, the cell culture device 10 of this embodiment is provided with the imaging device 30 and the control device 40 in addition to the configuration of the first embodiment, as shown in FIG.
When receiving imaging instruction information from the control device 40, the imaging device 30 images the cells in the culture vessel 11 and transmits the obtained image to the control device 40. The instruction information for photographing by the photographing device 30 can be automatically transmitted from the control device 40 at a predetermined timing.
As the photographing apparatus 30, for example, a CCD camera can be attached to a lens barrel of a phase contrast microscope.

The control device 40 is an information processing device that controls the driving device for moving the stirring member 14 in the cell culture device 10 and the imaging device 30. As shown in FIG. 6, the control device 40 includes an imaging device control unit 41, an aggregate size determination unit 42, and a drive device control unit 43.
The imaging device control unit 41 transmits instruction information for causing the imaging device 30 to perform imaging at a predetermined timing, and receives an image captured from the imaging device 30.

When an image is received by the imaging device control unit 41, the aggregate size determination unit 42 determines whether the size of the cell aggregate in the image information is within a predetermined range. As this predetermined range, it can be set as 100 micrometers-600 micrometers, for example. As the size of the aggregate, an average value of the photographed aggregate can be used.
Then, as a result of the determination, when the size of the cell aggregate is smaller or larger than the predetermined range, the determination result is output to the drive device control unit 43.

Based on the determination result input from the agglomerate size determination unit 42, the drive device control unit 43 determines the pushing amount, moving speed, and cycle of movement of the stirring member 14, and the rod-type electric cylinder 17 according to these driving conditions. And the slider type electric cylinder 21 is controlled.
Thereby, when the size of the cell aggregate is smaller than the predetermined range, the cell aggregate in the culture vessel 11 is formed to an appropriate size, and the size of the cell aggregate is larger than the predetermined range. If so, it can be broken down to the appropriate size and adjusted to the optimal size for growth.

  In order to perform such processing, the control device 40 or the drive device control unit 43 has various agglomerate sizes, the pushing amount of the stirring member 14 suitable for each case, the moving speed, and the cycle for moving. It is preferable to have a table or the like stored in association.

  As described above, according to the cell culture device 10 of the present embodiment and the cell culture method using the same, the size of the aggregate in the culture vessel 11 can be automatically adjusted, and the cell proliferation efficiency can be stably performed. Can be improved.

  Next, the Example which performed the cell culture using the cell culture apparatus 10 of 1st embodiment is demonstrated. First, with reference to FIG.7 and FIG.8, various culture conditions and the grade of the stirring in each case are demonstrated. FIG. 7 is a diagram showing the types of stirring conditions performed using the cell culture apparatus 10 of the present embodiment, and FIG. 8 is a diagram summarizing the results of the cell state under each stirring condition.

In the culturing method of the present invention, as shown in FIG. 7, the culture vessel 11 is pressed from above using the stirring member 14, and the stirring member 14 is moved in parallel with respect to the loading table 13. Stir the medium inside.
The pushing amount when the stirring member 14 is pressed against the culture vessel 11 can take various values. For example, as shown in the figure, when the thickness of the culture vessel 11 is 10.5 mm, Values such as 2 mm, 4 mm, 6 mm, and 8 mm can be taken.
And according to each pushing amount, the moving speed of the stirring member 14 is adjusted, respectively, so that the culture solution in the culture vessel 11 can be stirred, which is suitable for the formation of cell aggregates, or cell aggregates Control to one suitable for disassembly.

As the moving speed of the stirring member 14, for example, values such as 2.5 mm / s, 12.5 mm / s, and 50 mm / s can be taken as shown in FIG.
The diameter of the stirring member 14 is not particularly limited, but is preferably 0.5 to 3.0 times the thickness of the culture vessel 11 in order to accurately control stirring.

FIG. 8 shows how much the culture solution in the culture vessel 11 is stirred when stirring is performed under the stirring conditions as described above.
As shown in the figure, when the pushing amount is 2 mm and the moving speed is 2.5 mm / s, 12.5 mm / s, and when the pushing amount is 4 mm and the moving speed is 2.5 mm / s, the culture vessel 11 All of the cells remained submerged.
In the present specification, such stirring is referred to as “weak stirring”. By performing such “weak stirring” stirring, it is possible to promote the formation of cell aggregates.

In FIG. 8, when the pushing amount is 2 mm and the moving speed is 50 mm / s, when the pushing amount is 4 mm, the moving speed is 12.5 mm / s and 50 mm / s, the pushing amount is 6 mm and the moving speed is 2. When 5 mm / s, 12.5 mm / s, and when the pushing amount is 8 mm and the moving speed is 2.5 mm / s, the culture solution in the culture vessel 11 is agitated to some extent, but the cells are almost sunk. It was up to. Furthermore, when the pushing amount is 6 mm and the moving speed is 50 mm / s, and when the pushing amount is 8 mm and the moving speed is 12.5 mm / s, the culture solution in the culture vessel 11 is stirred to some extent, Almost lifted up.
Such agitation under the agitation conditions in the vicinity of the boundary between the state in which the cells are mostly submerged and the state in which the cells are almost lifted is referred to as “medium agitation” in the present specification. By performing such “medium agitation” stirring, it becomes possible to adjust the cell agglomerates to an appropriate size without overly decomposing.

On the other hand, in FIG. 8, when the pushing amount was 8 mm and the moving speed was 50 mm / s, the culture solution in the culture vessel 11 was vigorously stirred and all the cells were lifted. Such agitation where all the cells float is referred to as “strong agitation” in the present specification. In such “strong agitation”, the aggregates of cells are broken apart into individual cells, and the cells are suspended by repeating “strong agitation”, but when the cells fall apart individually, On the other hand, the growth efficiency is lowered.
In the conventional agitation in cell culture, the culture vessel is usually vigorously agitated. As a result, agitation corresponding to “strong agitation” is performed, which causes a decrease in proliferation efficiency. It is possible to solve the problem.

It goes without saying that the present invention is not limited to the above embodiment, and that various modifications can be made within the scope of the present invention.
For example, the culture vessel 11 has a shape with rounded corners so that no corner portion is generated, or the stirring member 14 is not cylindrical, and the cross section shown in FIG. It is possible to make appropriate changes such as obtaining an effect.

  The present invention can be suitably used in the fields of biopharmaceuticals, regenerative medicine, immunotherapy and the like that require culturing a large amount of cells.

DESCRIPTION OF SYMBOLS 10 Cell culture apparatus 11 Culture container 11-1 Storage part 11-2 Hole 12 Tube 13 Loading platform
13-1 Stopping member 14 Stirring member 15 Support base 16 Moving base 17 Rod type electric cylinder (actuator for vertical operation)
18 Ball screw 19 Guide rod 20 Base 21 Slider type electric cylinder (actuator for horizontal operation)
22 partition roller 30 imaging device 40 control device 41 imaging device control unit 42 agglomerate size determination unit 43 drive device control unit

Claims (4)

An apparatus for culturing adherent cells or suspension cells,
A loading table on which a culture vessel formed in a bag shape using a flexible soft packaging material as a material is encapsulated with a culture solution containing a culture medium and adherent cells or floating cells;
A stirring member that is pressed against the upper surface of the culture vessel with a predetermined pushing amount, and is configured to be movable in a predetermined movement cycle in parallel with the loading platform in a horizontal direction at a predetermined speed,
The flexible soft packaging material is selected from the group consisting of polyolefin, ethylene-vinyl acetate copolymer, styrene elastomer, polyester thermoplastic elastomer, silicone thermoplastic elastomer, and silicone rubber,
The stirring member is configured to control the formation of the cell aggregate or the decomposition of the cell aggregate for the cells in the culture container by moving to stir the culture solution in the culture container, The stirring member has a roll shape,
An imaging device configured to image cells in the culture vessel;
An image taken by the imaging device is input, and an aggregate size determination unit that determines whether the size of the aggregate of the cells is a size within a predetermined range;
Based on the determination result input of the agglomerate size determination unit, the pushing amount, the moving speed, and the moving cycle are determined, the stirring member is pressed against the upper surface of the culture vessel placed in a plane direction, and the stirring member is When the size of the cell aggregate is smaller than a predetermined range by moving at a predetermined speed, a predetermined pushing amount, and a predetermined movement cycle in parallel with the loading platform in the horizontal direction, the cell aggregate A culture vessel in which the amount of pushing, moving speed, and moving cycle is determined based on the determination result input of the agglomerate size determining unit, and the stirring member is placed in a plane direction The agitation member is moved in a horizontal direction parallel to the loading platform in a horizontal direction at a predetermined speed, a predetermined pushing amount, and a predetermined movement cycle, so that the size of the aggregate of cells is predetermined. Than range Big case, cell culture apparatus characterized by comprising a driving unit controller that is configured to perform the degradation of the cell clumps. A partition member;
The partition member is configured to divide the culture vessel into two or more chambers including a culture unit and an expandable unit, and is configured to expand the volume of the culture unit in accordance with an increase in the number of cells in the culture unit. The cell culturing apparatus according to claim 1, wherein the stirring member is configured to stir the medium in the culture unit. A method for culturing adherent cells or floating cells using the cell culture device according to claim 1,
Photograph the cells in the culture vessel,
Input an image taken by the photographing device,
Determining whether the size of the aggregate of the cells is within a predetermined range;
Based on the determination result input of the agglomerate size determination unit, determine the pushing amount, moving speed, and moving cycle,
According to the determination of the aggregate size determination unit, an external force is applied to the culture container, and control of cell aggregate formation or cell aggregate decomposition control is performed on the cells in the culture solution in the culture container. And applying the external force to the culture vessel,
When the size of the cell aggregate is smaller than a predetermined range, the stirring member is pressed against the upper surface of the culture vessel placed in a planar direction so as to form the cell aggregate, and the stirring member is Move in a horizontal direction parallel to the loading platform at a predetermined speed, a predetermined push-in amount, and a predetermined movement cycle;
When the size of the cell aggregate is larger than a predetermined range, the stirring member is pressed against the upper surface of the culture vessel placed in a plane direction so as to decompose the cell aggregate, and the stirring member is Moving in a horizontal direction at a predetermined speed, a predetermined pushing amount, and a predetermined moving cycle. 4. The cell culture method according to claim 3, wherein the formation of the cell aggregate is performed on the cells seeded in the culture container at the start of the culture.

Images