JP2024011130A - Cell culture device, cell culture system, and cell culture method - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a cell culture device that can efficiently detach adherent cells from the culture surface to form single cells when culturing adherent cells with a bag-shaped culture container.

SOLUTION: Provided is a cell culture device 20 for culturing cells using a bag-shaped culture container 10 made of soft packaging material, having: a pedestal 21 on which the culture container 10 is placed; a pressing member 24 that presses the culture container 10 placed on the pedestal 21 against the pedestal 21; and an impact applying mechanism 25 that applies an impact to the pressing member 24, the impact applying mechanism 25 applying an impact to the end of the pressing member 24 in the condition that the pressing member 24 is provided so as to be tiltable with respect to the pedestal 21, and the culture container 10 filled with the liquid content is held between the pedestal 21 and the pressing member 24. It is preferable that the thickness of the liquid inside the culture container 10 is 10 mm or less in the condition that the culture container 10 filled with a content liquid is held in parallel between the pedestal 21 and the pressing member 24.

SELECTED DRAWING: Figure 4

COPYRIGHT: (C)2024,JPO&INPIT

Description

The present invention relates to cell culture technology, and particularly to a cell culture device for culturing adherent cells in a bag-shaped culture container.

In recent years, in fields such as pharmaceutical production, gene therapy, regenerative medicine, and immunotherapy, there has been a need to efficiently culture cells, tissues, etc. in large quantities in an artificial environment.
Under these circumstances, cells are cultured in large quantities in a closed system using bag-shaped culture containers made of soft packaging material.

By the way, when adherent cells are cultured in a bag-shaped culture container, as the number of cells increases, the space on the culture surface to which the cells can adhere gradually disappears, resulting in a decrease in cell proliferation efficiency.
Therefore, it was necessary to collect the cells and subculture them before the proliferation efficiency decreased.
Specifically, cells are detached from the culture surface to become single cells, seeded in a new culture container, and then cultured for the second generation, and then cultured for the third generation. It was necessary to culture the cells repeatedly.

Patent No. 6601713 Patent No. 4225924 Japanese Patent Application Publication No. 2018-19631

However, when subculturing cells, etc., there is a problem in that it is not easy to separate cells from the culture surface to form single cells.
That is, adherent cells not only adhere to the culture surface but also adhere to adjacent cells.

For this reason, it is not easy to disintegrate the cell cluster into pieces. For example, if you try to separate the cells from the culture surface using a cell scraper or the like to make single cells, the cells may be pressed too hard. Alternatively, the adhesion proteins on the culture surface and the proteins on the cell surface were forcibly separated and damaged, often resulting in an increase in the number of dead cells.

On the other hand, it is also common practice to detach cells using a detachment solution, but if the cells are immersed in the detachment solution for a long time, the cells will be damaged.
Therefore, after adding a detachment solution and immersing the culture container for an appropriate amount of time, the cells are detached from the culture surface by manually hitting the culture container from the outside, and then adhering to each other. The existing cells were peeled off to form single cells.

However, such methods are not suitable for automatically culturing cells in large quantities.
Therefore, the present inventors conducted intensive research and succeeded in developing a cell culture device capable of efficiently peeling adherent cells from a culture surface to form single cells, thereby completing the present invention.

Specifically, the cell culture device is equipped with a pressing member capable of pressing a bag-shaped culture container filled with a liquid to reduce the thickness of the liquid in the container, and applying an impact to the pressing member. By providing an impact applying mechanism that repeatedly tilts the pressing member, it is possible to efficiently peel adherent cells from the culture surface to form single cells.

Here, Patent Document 1 describes a method of filling a bag-shaped culture container with a medium while draining the stripping solution until at least a portion of the inner surface of the container comes into contact with the container, detaching the cells, and disintegrating the cell mass using a tube pump. is listed.
However, this method has a problem in that a cleaning step cannot be performed appropriately after the stripping solution is discharged. Furthermore, since a means for pressing the bag-shaped culture container is not provided, there is also a problem that even if a cleaning step is carried out, the inside of the culture container cannot be sufficiently cleaned as it is.

Further, Patent Document 2 discloses an automatic subculturing device for subculturing adherent cells. However, this method could not be used in bag-shaped culture containers. Additionally, a special filter material was required.

Further, Patent Document 3 discloses a cell culture device capable of stirring the content liquid in a bag-shaped culture container. However, this cell culture device is for culturing floating cells such as mononuclear cells, and is not capable of efficiently peeling adherent cells from the culture surface to form single cells.

The present invention has been made in view of the above-mentioned circumstances, and provides a cell culture system in which adherent cells can be efficiently detached from the culture surface to form single cells when the adherent cells are cultured in a bag-shaped culture container. The purpose is to provide devices, cell culture systems, and cell culture methods.

In order to achieve the above object, the cell culture device of the present invention is a cell culture device for culturing cells using a bag-shaped culture container made of a flexible packaging material, and includes a pedestal on which the culture container is placed; It has a pressing member that presses the culture container placed on the pedestal against the pedestal, and an impact applying mechanism that applies an impact to the pressing member, and the pressing member is tiltable with respect to the pedestal. The impact applying mechanism applies an impact to the end of the pressing member in a state where the culture container filled with the content liquid is held between the pedestal and the pressing member.

Further, in the cell culture device of the present invention, the two impact applying mechanisms are provided so as to apply impact to opposing ends of the pressing member, and the culture container filled with the liquid content is connected to the pedestal. It is preferable that the two impact applying mechanisms alternately apply impact to opposing ends of the pressing member while being held between the pressing members.
Further, the cell culture device of the present invention may be configured such that the thickness of the liquid in the culture container is 10 mm or less when the culture container filled with the content liquid is held in parallel between the pedestal and the pressing member. It is also preferable to do so.
Further, it is also preferable that the cell culture device of the present invention is configured such that application of an impact by the impact applying mechanism can be stopped in a state where the pressing member is inclined with respect to the pedestal.

Moreover, it is also preferable that the cell culture apparatus of the present invention is configured to use a cam, a solenoid cylinder, an air cylinder, or a linear actuator as the impact applying mechanism.
Moreover, it is also preferable that the cell culture device of the present invention is configured such that the cells are adherent cells.
Furthermore, it is also preferable that the cell culture device of this embodiment is a combination of various configurations of the above-described cell culture devices.

Further, the cell culture system of the present invention includes a plurality of any of the cell culture devices described above, and has a configuration in which the culture vessels placed in each cell culture device are connected in parallel through ports.

Further, the cell culture method of the present invention is a cell culture method using any of the cell culture devices described above, wherein the culture container is filled with adherent cells and a medium, and cell culture is performed, and the culture container is placed on the mount. and the pressing member, and applying an impact to the end of the pressing member using the impact applying mechanism, thereby peeling the adhered cells from the culture container.

Further, the cell culture method of the present invention may include a method of applying an impact to the end of the pressing member using the impact applying mechanism to peel off the adhered cells from the culture container and disassemble the cell mass into single cells. It is preferable to do so.
Moreover, it is also preferable that the cell culture method of the present invention is a method in which the culture container is used for subculturing cells or inducing differentiation of cells.

According to the present invention, there is provided a cell culture device, a cell culture system, and a cell culture system capable of efficiently peeling adherent cells from a culture surface to form single cells when culturing adherent cells in a bag-shaped culture container. It becomes possible to provide a method.

FIG. 2 is a schematic diagram showing a plan view and a front view of a bag-shaped culture container used in a cell culture device according to an embodiment of the present invention. 1 is a schematic diagram showing a plan view and a front view of a cell culture device according to an embodiment of the present invention. FIG. BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a schematic diagram showing a plan view and a front view of a cell culture apparatus equipped with an impact applying mechanism (solenoid cylinder type) according to an embodiment of the present invention. FIG. 3 is a schematic diagram showing the operation of the impact applying mechanism (solenoid cylinder type) according to the embodiment of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS It is a schematic diagram which shows the structure of the cell culture apparatus and its control apparatus provided with the impact applying mechanism (solenoid cylinder type) based on embodiment of this invention. FIG. 1 is a schematic diagram showing a front view of a cell culture device equipped with an impact applying mechanism (cam type) according to an embodiment of the present invention. FIG. 1 is a schematic diagram showing the configuration of a cell culture system including bag-shaped culture vessels arranged in parallel and communicating with each other in a cell culture apparatus according to an embodiment of the present invention. FIG. 1 is a schematic diagram showing the configuration of a cell culture system including bag-shaped culture containers connected in series in a cell culture apparatus according to an embodiment of the present invention.

EMBODIMENT OF THE INVENTION Hereinafter, embodiments of the cell culture device, cell culture system, and cell culture method of the present invention will be described in detail with reference to the drawings. However, the present invention is not limited to the specific contents of the embodiments and examples described below.

[Cell culture device]
The cell culture device of this embodiment is a cell culture device for culturing cells using a bag-shaped culture container made of a flexible packaging material, and includes a pedestal on which the culture container is placed, and a culture container placed on the pedestal. It has a pressing member that presses the container against the pedestal, and an impact applying mechanism that applies an impact to the pressing member, the pressing member is tiltable with respect to the pedestal, and the culture container filled with the content liquid is placed on the pedestal. The impact applying mechanism applies an impact to the end of the pressing member while being held between the pressing member and the pressing member.

Further, the cell culture device of this embodiment is equipped with two impact applying mechanisms so as to apply impact to opposing ends of the pressing member, and the culture container filled with the liquid content is placed between the pedestal and the pressing member. It is preferable that the two impact applying mechanisms alternately apply impact to opposing ends of the pressing member while being sandwiched therebetween.

First, with reference to FIG. 1, a bag-shaped culture container used in the cell culture apparatus of this embodiment will be described. FIG. 1 is a schematic diagram showing a plan view and a front view of a bag-shaped culture container used in the cell culture apparatus of this embodiment.
As shown in FIG. 1, the bag-shaped culture container 10 used in the cell culture device of this embodiment is made by pasting the peripheral edges of two films together by heat sealing or the like, and culturing by adhering adherent cells inside. A culture chamber having a culture surface 11 for the purpose of culturing is formed.

Furthermore, the culture container 10 is equipped with a port 12 for injecting and discharging the contents. Although two ports 12 are provided in the example of FIG. 1, the number is not particularly limited and may be one or three or more.
A tube is connected to the port 12, and the liquid content is supplied to the culture container 10 and the liquid content is discharged from the culture container 10 via the port 12 by a liquid feeding means such as a pump installed in the tube. .

Further, in FIG. 1, a bulge shape extending outward is formed in the peripheral area of the culture chamber of the culture container 10. This makes it easier to apply a shock to the upper surface of the culture container 10 using the pressing member, as will be described later.
Note that the shape of the culture container 10 is not limited to this, and may not have a bulging shape.

As the material for the culture container 10, olefin resins such as polyethylene and polypropylene can be suitably used. Other materials that can be used include polymethylpentene, cyclic olefin polymer, cyclic olefin copolymer, polyvinyl chloride, polyurethane, polymethyl methacrylate, polyester, polyamide, ionomer, ethylene-alpha olefin copolymer, ethylene-vinyl acetate copolymer, etc. Polymer, ethylene-vinyl alcohol copolymer, ethylene-acrylic acid copolymer, ethylene-methacrylic acid copolymer, ethylene-methyl methacrylate copolymer, polyacrylic acid, polymethacrylic acid, polymethyl acrylate, poly Examples include methyl methacrylate, polydimethylsiloxane, fluororesin, silicone resin, polybutadiene resin, and chlorinated polyethylene. Further, thermoplastic elastomers such as olefin thermoplastic elastomer, vinyl chloride thermoplastic elastomer, styrene thermoplastic elastomer, urethane thermoplastic elastomer, ester thermoplastic elastomer, and nylon thermoplastic elastomer can also be used. In addition, thermosetting elastomers such as urethane rubber, silicone rubber, and fluororubber, and thermosetting materials such as phenolic resin, epoxy resin, melamine resin, urea resin, unsaturated polyester resin, alkyd resin, urethane resin, and thermosetting polyimide are also available. Resins can also be used.

Further, as the material for the port 12, for example, thermoplastic resin such as polyethylene, polypropylene, vinyl chloride, polystyrene elastomer, FEP (perfluoroethylene propene copolymer), etc. can be used.
The content liquid filled in the culture container 10 is not particularly limited, but a cell growth medium, a differentiation induction medium, a washing liquid, a stripping liquid, a cell suspension, etc. are used.

Next, with reference to FIG. 2, the cell culture device of this embodiment will be described. FIG. 2 is a schematic diagram showing a plan view and a front view of the cell culture device of this embodiment.
As shown in FIG. 2, the cell culture device 20 of this embodiment includes a pedestal 21 on which the culture container 10 is placed. Further, a top plate support part 22 that supports a top plate part 23 is provided at an end of the pedestal 21 in an upright manner.

Magnets 231 are provided at the four corners of the lower surface of the top plate portion 23, and guide pins 232 are attached.
Further, the top plate portion 23 is equipped with a length measurement sensor 233 that detects the liquid thickness within the culture container 10.

Further, the cell culture device 20 of this embodiment is equipped with a pressing member 24 that presses the culture container 10 against the pedestal 21.
Magnets 241 are provided at the four corners of the upper surface of the pressing member 24 so as to face the magnets 231 of the top plate portion 23. The magnets 231 and 241 repel each other, so that the pressing member 24 can press the culture container 10 against the pedestal 21.
Note that the means for pressing the culture container 10 by the pressing member 24 is not limited to this, but it is also possible to use a biasing means such as a spring, or to press the culture container 10 by the own weight of the pressing member 24. good.

In addition, guide holes 242 (through holes) are formed at the four corners of the pressing member 24 to allow the guide pins 232 of the top plate portion 23 to be movably inserted, and the pressing member 24 can be moved up and down along the guide pins 232. It is possible to move to.
Furthermore, by setting the diameter of the guide hole 242 to be wider than the diameter of the guide pin 232, the pressing member 24 can be tilted with respect to the pedestal 21, as will be described later, and a shock can be applied to the culture container 10. is now possible.

Further, a metal member 243 is provided on the upper surface side of the pressing member 24 so as to face the length measurement sensor 233 of the top plate portion 23 . The length measurement sensor 233 measures the distance to this metal member 243, and based on the measurement result, the liquid thickness of the culture container 10 can be calculated by a control device connected to the length measurement sensor 233.
Further, the pressing member 24 is equipped with a pressing plate 244 that contacts the culture container 10.

Next, with reference to FIG. 3, the impact applying mechanism in the cell culture apparatus of this embodiment will be described. FIG. 3 is a schematic diagram showing a plan view and a front view of a cell culture apparatus equipped with an impact applying mechanism (solenoid cylinder type).
As shown in FIG. 3, the cell culture device 20 of this embodiment further includes an impact applying mechanism 25.
This impact applying mechanism 25 is configured using a solenoid cylinder, is provided with a rod 251, and is fixed to the top plate portion 23 via a bracket 252.

The impact applying mechanism 25 can tilt the pressing member 24 with respect to the pedestal 21 by hitting the pressing member 24 with the rod 251. As a result, a strong impact can be applied to the culture container 10 which is held and pressed between the pressing member 24 and the pedestal 21, and the adherent cells are peeled off from the culture surface 11 of the culture container 10 and broken up into single cells. is now possible.

In FIG. 3, two impact applying mechanisms 25 are provided, but it is also possible to use only one of them.
In addition, when two impact applying mechanisms 25 are provided in the cell culture apparatus of this embodiment, it is preferable that the impact is applied alternately to opposing end regions of the pressing member 24, as shown in FIG. .
In this way, a stronger impact can be applied to the culture container 10, so that adherent cells can be more efficiently peeled off into single cells.

Further, the impact by the impact applying mechanism 25 is applied at the timing when the impact applying mechanism 25 is turned ON (the pressing member 24 is hit by the rod 251). It may also be configured to apply at the timing of turning off (pulling the rod 251 from the pressing member 24) after tilting with respect to the pedestal 21.

Although not shown, in the cell culture device 20 of this embodiment, it is also preferable that the pressing members 24 are provided on the two port sides, and that each pressing member 24 is provided with two impact applying mechanisms 25. At this time, in order to make each pressing member 24 tiltable with respect to the pedestal 21, it is preferable to form a gap between the two pressing members 24.

If the cell culture device 20 of this embodiment is configured in this way, it is possible to apply an impact to the culture container 10 using the two pressing members 24 on the left and right, so that adhesive cells can be more efficiently peeled off. It becomes possible to make it into a single cell. In particular, with such a configuration, even adherent cells present near the center of the culture container 10 can be more efficiently peeled off into single cells. Moreover, it is also possible to apply such a configuration to the case where other impact applying mechanisms described below are provided.

In addition, in the cell culture apparatus of this embodiment, the culture container 10 is pressed from below by the pressing member 24, and it is also possible to apply an impact to the culture container 10 from the bottom surface side by the impact applying mechanism 25. .

Next, with reference to FIG. 5, the control mechanism of the cell culture device of this embodiment will be described. FIG. 5 is a schematic diagram showing the configuration of the cell culture device and its control device of this embodiment.
As shown in FIG. 5, the control device 30 (control unit) may include an input/output section 31, a control section 32, an operation section 33, and a power supply section 34.

The input/output unit 31 is connected to the length measurement sensor 231 and the impact applying mechanism 25 in the cell culture device 20 . Then, input information from the length measurement sensor 231 is output to the control section 32. Further, input information from the control unit 32 is output to the impact applying mechanism 25 .
The control unit 32 is constituted by a PLC (programmable logic controller) or the like, and can program and store desired control contents in advance and control the operations of each section based on this.

That is, the control unit 32 can transmit information for controlling the impact applying mechanism 25 to the impact applying mechanism 25 via the input/output unit 31 at a predetermined timing to control its operation.
Further, the control unit 32 can calculate the liquid thickness within the culture container 10 based on input information from the length measurement sensor 231a.

The operation unit 33 includes a display unit such as a touch panel, and transmits information input by the user to the control unit 32 to perform PLC settings and the like. Additionally, input information from the control unit 32 is displayed.
The power supply section 34 (such as a stabilized power supply) supplies electricity to each section within the control device 30.
Further, although not shown in the drawings, the control device 30 may be configured to further include a relay section (relay) and a wiring interrupting section (breaker). Further, a part or all of the control unit 32, operation unit 33, etc. in the control device 30 may be realized by an information processing device such as a microcomputer or a computer.

Next, with reference to FIG. 6, a cell culture device of this embodiment including another impact applying mechanism will be described. FIG. 6 is a schematic diagram showing a front view of a cell culture device equipped with an impact applying mechanism (cam type).
As shown in FIG. 6, it is also preferable that the cell culture device 20' of this embodiment includes an impact applying mechanism 26 configured using a cam.

In the impact applying mechanism 26, a cam 262 is attached to a rotating shaft 261, and by rotating the rotating shaft 261 with a motor 263, the cam 262 can apply an impact to the pressing member 24. The motor 263 is supported by a support section 264 erected on the pedestal 21'. In addition, in FIG. 6, the same code|symbol is shown for the structure similar to the cell culture apparatus 20 of this embodiment shown in FIG.

Further, although two cams 262 are provided in FIG. 6, only one of them may be provided.
Furthermore, when the cell culture device 20 of this embodiment is provided with two cams 262, it is possible to alternately apply impacts to opposing end regions of the pressing member 24, thereby more efficiently peeling off adhered cells. It is possible to make it into a single cell.

Furthermore, the impact applying mechanism in the cell culture apparatus of this embodiment is not limited to one using a solenoid cylinder or a cam, but may be configured with other mechanisms such as an air cylinder or a linear actuator whose shaft can be expanded and contracted by motor operation. Of course, it is also possible to do so.
Similarly to the cell culture apparatus 20 equipped with a solenoid cylinder-type impact applying mechanism 25 shown in FIG. It is possible to control the impact on the

Next, the liquid thickness of the culture container 10 in the cell culture device 20 of this embodiment will be explained.
In the cell culture device 20 of this embodiment, the thickness of the liquid inside the culture container 10 is set to 10 mm or less when the culture container 10 filled with the content liquid is sandwiched in parallel between the pedestal 21 and the pressing member 24. is preferred.
That is, by applying an impact to the culture vessel 10 while pressing the culture vessel 10 with the pressing member 24 so that the liquid thickness within the culture vessel 10 is 10 mm or less, adhered cells can be removed from the culture surface 11 more efficiently. It is possible to separate the cells into single cells.

Here, in the cell culture device of this embodiment, as the liquid thickness within the culture container 10 increases, it becomes difficult to peel adhered cells from the culture surface 11.
In addition, the cell culture device of this embodiment is configured to apply an impact to the end region of the pressing member 24 by the impact applying mechanism 25 and the impact applying mechanism 26, but the pressing member 24 is tilted with respect to the pedestal 21. Even if the temperature is increased, the liquid thickness in the central region within the culture container 10 does not change much. Therefore, it is difficult to apply a shock to the central region within the culture container 10.

However, as described above, in the cell culture device of the present embodiment, the liquid thickness in the culture container 10 is set to 10 mm or less while the culture container 10 is held in parallel between the pedestal 21 and the pressing member 24, and from this state By applying an impact to the end region of the pressing member 24, it is possible to efficiently separate adherent cells from the culture surface 11 into single cells.
Moreover, at this time, it is more preferable that the liquid thickness in the culture container 10 be 5 mm or less, it is even more preferable that the liquid thickness be 2 mm or less, and it is particularly preferable that the liquid thickness be 1 mm or less.

In the cell culture device 20 of this embodiment, it is also preferable that the impact applying mechanisms 25 and 26 stop applying the impact while the pressing member 24 is tilted with respect to the pedestal 21.
The control device 30 connected to the impact applying mechanisms 25 and 26 can stop or start applying the impact by the impact applying mechanisms 25 and 26.

For example, in FIG. 4, by turning on the left impact applying mechanism 25 to extend the rod 251, and by turning off the right impact applying mechanism 25 to retract the rod 251, the pressing member 24 It is possible to stop applying an impact by the impact applying mechanisms 25 and 26 in a state in which the impact applying mechanisms 25 and 26 are tilted so that the right side is upward with respect to the pedestal 21.

If the cell culture device 20 of the present embodiment is configured in this way, the pressing member 24 can be kept in an inclined state with respect to the pedestal 21, so that, for example, when injecting the content liquid into the culture container 10, In addition, when air in the tube or the like flows into the culture container 10, it becomes possible to collect the air to one port 12 side in the culture container 10 and make it easier to remove.

[Cell culture system]
The cell culture system of this embodiment includes a plurality of cell culture devices 20 of this embodiment described above, and the culture vessels 10 placed on each cell culture device 20 are communicated in parallel via ports 12. It is characterized by
Specifically, as shown in FIG. 7, for example, a first culture container 10-1 is placed on a first cell culture device 20-1, and a second culture container 10-1 is placed on a second cell culture device 20-2. Place the container 10-2, place the third culture container 10-3 on the third cell culture device 20-3, and connect the ports provided in each culture container in parallel using tubes. It is preferable to have a configuration in which: Further, it is preferable that a liquid feeding means such as a pump is provided in each tube connected to the port of each culture container.

In addition, in FIG. 7, the first culture container 10-1, the second culture container 10-2, and the third culture container 10-3 of the same size are shown, but the culture areas are It is also preferable to have a large value.

The cell culture system of this embodiment includes a medium A container 41, a medium B container 42, a washing liquid container 43, and a stripping liquid container 44, each of which is communicated with each culture container 10 via a valve. ing. Further, the waste liquid container 45 is also communicated with each culture container 10 via a valve.

Moreover, in the cell culture system of this embodiment, it is preferable to include the control device 30 described above.
Preferably, the input/output section 31 of the control device 30 is connected to each pump and valve, and the operations of these pumps and valves are controlled based on information from the control section 32.
Although the pump is not particularly limited, for example, a tube pump or the like can be suitably used. Further, the valve is not particularly limited, but a pinch valve or the like can be suitably used, for example.

Thereby, the operation of the pump and valve can be controlled based on the input information from the length measurement sensor 231, and each culture container filled with the liquid content is held between the pedestal 21 and the pressing member 24 in parallel. In this state, it is possible to transfer the content liquid between containers while keeping the liquid thickness in the culture container constant.

The cell culture system of this embodiment can be used, for example, as follows.
First, a medium is supplied from the medium A container 41 to the first culture vessel 10-1, and adherent cells are supplied to the first culture vessel 10-1, and the cells are cultured on the culture surface 11-1 of the first culture vessel 10-1. As a result, a cell mass of adherent cells is formed on the culture surface 11-1.
Next, before detaching the cells from the culture surface 11-1, a cleaning solution is supplied from the cleaning solution container 43 to the first culture container 10-1, and a waste solution container 45 is supplied from the first culture container 10-1. Then, drain the cleaning solution to clean the inside of the first culture container 10-1.

Next, a stripping solution is supplied from the stripping solution container 44 to the first culture container 10-1, and the container is left at 37° C. for 10 minutes, so that the adhered cells are easily peeled off from the culture surface.
Then, the impact applying mechanisms 25 and 26 in the first cell culture device 20-1 are activated to peel the adherent cells in the culture container 10-1 from the culture surface 11-1 and separate adjacent cells. Disassemble cell clumps into single cells.

Next, the cells in the first culture container 10-1 are transferred to the second culture container 10-2, and the same process is performed.
That is, the adherent cells cultured in the second culture vessel 10-2 are detached from the culture surface 11-2 and decomposed into single cells (hereinafter, this may be simply referred to as detachment). Then, the cells in the second culture container 10-2 are transferred to the third culture container 10-3, and the same process is performed.

Next, detailed procedures for the above-described cell detachment and decomposition into single cells will be specifically explained.
(1) Detachment of cells in a state filled with a medium and decomposition into single cells Adhesive cells can be detached by the following procedure in a state filled with a culture medium in a culture container.
(i) Cell culture in the first culture container 10-1 → (ii) Discharging the medium → (iii) Washing with phosphate buffered saline (hereinafter referred to as PBS washing) → (iv) Filling with stripping solution → ( v) Leave to stand (37℃ for 10 minutes) → (vi) Discharge of detachment solution → (vii) PBS washing → (viii) Filling of medium → (ix) Detach cells by applying impact → (x) Transfer to second culture container 10-2 Cell migration → (xi) Add medium to second culture container 10-2

(2) Detachment of cells in a state filled with PBS and decomposition into single cells Adhesive cells can be detached in a state filled with PBS in a culture container by the following procedure.
(i) Cell culture in the first culture container 10-1 → (ii) Discharging the medium → (iii) Washing with PBS → (iv) Filling with stripping solution → (v) Leaving (37°C for 10 minutes) → (vi) Stripping solution Discharge → (vii) PBS filling → (viii) Cell detachment by impact application → (ix) Cell movement to second culture container 10-2 → (x) Addition of medium to second culture container 10-2 → (xi) After a few hours, replace the entire amount of the medium in the second culture container 10-2.

(3) Detachment of cells and decomposition into single cells in a state filled with a detachment solution Adhesive cells can be detached by the following procedure while a culture container is filled with a detachment solution.
(i) Cell culture in the first culture container 10-1 → (ii) Discharging the medium → (iii) Washing with PBS → (iv) Filling with stripping solution → (v) Leaving (37°C for 10 minutes) → (vi) Applying shock Cell detachment → (vii) Cell migration to the second culture vessel 10-2 → (viii) Addition of medium to the second culture vessel 10-2 → (ix) After several hours, transfer of the medium to the second culture vessel 10-2 full amount exchange

Furthermore, the cell culture system of this embodiment can be used not only for subculture but also for inducing cell differentiation.
For example, after iPS cells are adherently cultured and proliferated in the first culture container 10-1, they are peeled off from the culture surface 11-1 and transferred to the second culture container 10-2, and the cells necessary for differentiation induction are It is possible to supply the medium from the medium B container 42 and perform cell culture in the second culture container 10-2. Furthermore, it goes without saying that it is possible to further induce differentiation by transferring cells from the second culture vessel 10-2 to the third culture vessel 10-3, if necessary.

Further, the cell culture system of this embodiment is provided with a plurality of cell culture devices 20 of this embodiment described above, and the culture vessels 10 placed on each cell culture device 20 are connected in series via the port 12. It can also be configured.
Specifically, as shown in FIG. 8, for example, the first culture vessel 10-1 is connected to the second culture vessel 10-2 via a port, and the second culture vessel 10-2 is connected to the third culture vessel 10-2. The culture container 10-3 is communicated with each other through a port, and a medium A container 41, a medium B container 42, a cleaning liquid container 43, a stripping liquid container 44, and a waste liquid container 45 are connected to each culture container through valves. It can also be communicated.

Here, in the cell culture system of this embodiment, when cells are transferred between culture containers, it is preferable to transfer the cells while keeping the liquid thickness in the culture container constant. For this reason, as shown in Figure 8, when culture vessels are connected in series through ports, two pumps are required between each culture vessel, and on-off valves are required at various locations. There is.

As explained above, according to the cell culture system of this embodiment, adherent cells can be automatically detached from the culture surface to form single cells, and subculture and differentiation can be efficiently performed. It is possible. In addition, since the inside of the culture container can be automatically cleaned both before filling the culture container with detachment solution and after draining it, it is possible to perform cell detachment efficiently and also to passage the cells to the next generation. It has become possible to safely perform culture and differentiation induction.

In addition, in the cell culture system of this embodiment, instead of the cell culture device of this embodiment, a cell culture device not equipped with a shock imparting mechanism is used so that the culture vessels placed in each cell culture device have ports. It is also possible to have a configuration in which they are connected in parallel or in series.
Such a cell culture system can be used, for example, as follows.

First, a medium is supplied from the medium A container 41 to the first culture vessel 10-1, and adherent cells are supplied to the first culture vessel 10-1, and the cells are cultured on the culture surface 11-1 of the first culture vessel 10-1. As a result, a cell mass of adherent cells is formed on the culture surface 11-1.
Next, before detaching the cells from the culture surface 11-1, a cleaning solution is supplied from the cleaning solution container 43 to the first culture container 10-1, and a waste solution container 45 is supplied from the first culture container 10-1. Then, drain the cleaning solution to clean the inside of the first culture container 10-1.

Next, a stripping solution is supplied from the stripping solution container 44 to the first culture container 10-1, and the container is left at 37° C. for 10 minutes, so that the adhered cells are easily peeled off from the culture surface.
Then, the first culture container 10-1 is struck from the outside to peel the adherent cells in the culture container 10-1 from the culture surface 11-1, separate adjacent cells, and break down the cell cluster into single cells. .

Next, the cells in the first culture container 10-1 are transferred to the second culture container 10-2, and the same process is performed.
That is, the adherent cells cultured in the second culture container 10-2 are peeled off from the culture surface 11-2 and decomposed into single cells. Then, the cells in the second culture container 10-2 are transferred to the third culture container 10-3, and the same process is performed.
In such a cell culture system, it is possible to manually detach adherent cells from the culture surface into single cells, and perform subculture and differentiation induction.

[Cell culture method]
The cell culture method of this embodiment is a cell culture method using the cell culture device 20 of this embodiment described above, in which a culture container 10 is filled with adherent cells and a medium, cell culture is performed, and the culture container 10 is placed on a mount. 21 and the pressing member 24, an impact is applied to the end of the pressing member 24 by the impact applying mechanisms 25 and 26, thereby peeling the adhered cells from the culture container 10.

Further, in the cell culture method of the present embodiment, an impact is applied to the end of the pressing member 24 by the impact applying mechanisms 25 and 26 to peel off the adherent cells from the culture container 10 and break down the cell mass into single cells. A method is preferred.
Furthermore, it is also preferable that the cell culture method of this embodiment is a method in which the culture container 10 is used for subculturing cells or inducing differentiation of cells.

By carrying out the cell culture method of this embodiment in this manner, when culturing adherent cells in a bag-shaped culture container, the adherent cells can be efficiently peeled off from the culture surface to form single cells, and the cells can be passed on. It can be suitably used for subculture and cell differentiation induction.

Tests conducted to confirm the effects of the cell culture device according to the embodiment of the present invention will be described below.
First, a bag-shaped culture container was created and adherent cells were cultured.
Specifically, a polyethylene sheet (Umerit 125FN, Ube Maruzen Polyethylene Co., Ltd.) was surface-treated, and a film was created using the surface-treated area as a culture surface. Then, the two films were stacked and the peripheral portions were bonded together by heat sealing to form a culture bag. As a result, culture bags for samples 1 to 6 were obtained.

At this time, ports were sandwiched between two opposing sides of the culture bag and the two ports were pasted together to form a culture bag with two ports. The area of the culture part of the culture bag excluding the outer peripheral seal part and the port installation part was about 100 cm ² .
For surface treatment, an excimer irradiation device (manufactured by M.D.Com Co., Ltd.) was used to make the culture surface hydrophilic. Excimer irradiation was performed under the conditions of a voltage of 12 V, an irradiation distance of 4 mm, and an irradiation speed of 2 mm/s x 3 times.

Next, StemFit® (AK-02N, Ajinomoto Healthy Supply Co., Ltd.) was sealed as a medium in each culture bag, and adherent cells (iPS cell 1231A3 strain, Kyoto University iPS Cell Research Institute) were seeded for 7 days. Cultured.
Thereafter, the medium was drained, and the culture bag was washed with PBS (phosphate buffered saline), and then the PBS was drained. Next, a 2-fold dilution of cell dissociation enzyme (TrypLE ^™ Select, Thermo Fisher Scientific) + EDTA in PBS was added, and the mixture was left at 37°C for 10 minutes. After the cell dissociation enzyme was discharged, the cells were washed with PBS and a medium was added.

Next, for the culture bag of Sample 1, the adherent cells were peeled off by manually hitting the bag from the outside. Then, the detached cells were collected with a syringe, and the number of cells was counted using a hemocytometer. This number of cells was used as the reference value for calculating the recovery rate.
Further, the culture bag of sample 2 was installed in the cell culture device (solenoid cylinder type) of this embodiment and pressed, and the amount of medium in the culture bag was 10 ml and the liquid thickness was 1 mm.
Then, the culture bag was shocked 50 times using a shock applying mechanism to peel off the adherent cells. The detached cells were collected with a syringe, the number of cells was counted using a hemocytometer, and the recovery rate relative to the reference value was calculated.

Next, the culture bags of Samples 3 to 6 are similarly placed in the cell culture device (solenoid cylinder type) of this embodiment and pressed, and the amount of medium in the culture bags is adjusted to 20 ml, 50 ml, 100 ml, 120 ml, respectively. The liquid thickness was set to 2 mm, 5 mm, 10 mm, and 12 mm, respectively.
Then, each culture bag was shocked 50 times using a shock applying mechanism to peel off the adherent cells. The detached cells were collected with a syringe, the number of cells was counted using a hemocytometer, and the recovery rate relative to the reference value was calculated. The results are shown in Table 1.

As shown in Table 1, cells are collected by placing a culture bag in the cell culture device of this embodiment and pressing it, applying an impact using the impact mechanism with the liquid thickness in the culture bag set to 1 mm to 12 mm. has been done. The recovery rate is 100% when the liquid thickness is 1 mm, 96% when the liquid thickness is 2 mm, 96% when the liquid thickness is 5 mm, 88% when the liquid thickness is 10 mm, and 88% when the liquid thickness is 12 mm. At that time, it was 48%.
From this result, it was found that by reducing the liquid thickness in the culture bag to 1 mm to 10 mm or less and applying an impact using the impact mechanism, it was possible to obtain a cell recovery rate comparable to that of manually tapping the bag. .

It goes without saying that the present invention is not limited to the above-described embodiments and examples, and that various modifications can be made within the scope of the present invention. For example, it is possible to make appropriate changes such as using other means as the impact applying mechanism or changing the number of impacts to be different from that in the embodiment.

INDUSTRIAL APPLICATION This invention can be suitably utilized when performing cell collection, subculture, differentiation induction, etc. when culturing adherent cells using a bag-shaped culture container.

10, 10-1 to 10-3 Culture container 11 Culture surface 12 Port 20, 20', 20-1 to 20-3 Cell culture device 21, 21' Frame 22 Top plate support part 23 Top plate part 231 Magnet 232 Guide pin 233 Length measurement sensor 24 Pressing member 241 Magnet 242 Guide hole 243 Metal member 244 Pressing plate 25 Impact applying mechanism (solenoid cylinder type)
251 Rod 252 Bracket 26 Impact applying mechanism (cam type)
261 Rotating shaft 262 Cam 263 Motor 264 Support part 30 Control device 31 Input/output part 32 Control part 33 Operation part 34 Power supply part 41 Medium A container 42 Medium B container 43 Washing liquid container 44 Stripping liquid container 45 Waste liquid container

Claims (10)

A cell culture device for culturing cells using a bag-shaped culture container made of soft packaging material,
It has a pedestal on which the culture container is placed, a pressing member that presses the culture container placed on the pedestal against the pedestal, and an impact applying mechanism that applies an impact to the pressing member,
The pressing member is provided to be tiltable with respect to the pedestal,
A cell culture device, wherein the impact applying mechanism applies an impact to an end of the pressing member in a state where the culture container filled with a liquid content is held between the pedestal and the pressing member. The two impact applying mechanisms are provided to apply impact to opposing ends of the pressing member, respectively,
The two impact applying mechanisms alternately apply impact to opposing ends of the pressing member in a state where the culture container filled with the content liquid is held between the pedestal and the pressing member. The cell culture device according to claim 1. 3. The culture container according to claim 1 or 2, wherein the culture container filled with the liquid content has a liquid thickness of 10 mm or less in a state where the culture container is held in parallel between the pedestal and the pressing member. Cell culture equipment. 3. The cell culture device according to claim 1 or 2, wherein application of an impact by the impact applying mechanism can be stopped in a state where the pressing member is inclined with respect to the pedestal. 3. The cell culture apparatus according to claim 1, wherein a cam, a solenoid cylinder, an air cylinder, or a linear actuator is used as the impact applying mechanism. 3. The cell culture device according to claim 1, wherein the cells are adherent cells. A cell culture system comprising a plurality of cell culture devices according to claim 1 or 2, wherein the culture vessels placed in each cell culture device are connected in parallel through ports. A cell culture method using the cell culture device according to claim 1 or 2,
Filling the culture container with adherent cells and a medium to perform cell culture,
With the culture container being held between the pedestal and the pressing member, an impact is applied to an end of the pressing member by the impact applying mechanism to detach the adherent cells from the culture container. cell culture method. 9. The cell culture according to claim 8, wherein the impact applying mechanism applies an impact to the end of the pressing member to peel off the adherent cells from the culture container and break down the cell mass into single cells. Method. 9. The cell culture method according to claim 8, wherein the culture container is used for subculturing cells or inducing differentiation of cells.

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