Cell culture system, method of using the cell culture system, and cell culture unit

Abstract

To provide a cell culture system that can be installed a culture bag and culture cells, can be easily carried in the state that various tools necessary for the culture are bundled, and can subject the culture bag to an observation with a general-purpose microscope.SOLUTION: Provided is a cell culture system for culturing cells in a culture bag made of soft packaging material, comprising: a culture unit having a notch portion for mounting a bag container comprising a pressing member for pressing the bag; a bag disposed on the unit in a bag container; a medium container for supplying medium to the bag; a waste liquid container to which the medium is discharged from the bag; a liquid delivery means disposed on a member that connects the bag and the culture medium container; and a liquid sending means disposed on a member connecting the bag and the waste liquid container, the distance from the bottom of the unit to the culture surface being less than 20 mm, the liquid thickness of the culture medium being uniformed by pressing the bag with the pressing member, and the surface of the unit, the upper and lower surfaces of the pressing member, and the upper and culture surfaces of the bag being substantially parallel.SELECTED DRAWING: Figure 12

Description

Technical Field

[0006]

[0001] The present invention relates to cell culture technology, and particularly to a cell culture system for culturing and observing cells.

Background Art

[0002] When culturing cells using a culture vessel, observing the state of the cells being cultured in the culture vessel with a microscope or a camera is very important for the culturing operation. Also, when performing large-scale cell culture, it is preferable to use a culture bag made of a flexible packaging material that can be easily manufactured and has a large culture area.

[0003] As a technique for making it possible to observe the state of cells being cultured, there is a culture device in which a dedicated observation mechanism is installed in a dedicated incubator. There is also a small culture device that can be placed on a general-purpose microscope.

Prior Art Documents

Patent Documents

[0004]

Patent Document 1

Patent Document 2

Summary of the Invention

Problems to be Solved by the Invention

[0005] The cell culture device described in Patent Document 1 is a culture device in which a dedicated observation mechanism is installed in a dedicated incubator. In such a culture device, since the observation operation cannot be performed manually, for example, even an operation as simple as focusing, which can be easily performed manually by a person, requires an observation mechanism that can operate automatically. For this reason, there is a problem that the device becomes large-scale and the observation mechanism becomes very expensive.

[0006] Furthermore, in order to perform cell culture using multiple culture vessels and observe the cells within each vessel, it is necessary to either install an individual observation mechanism in each vessel or a mechanism to automatically move the culture vessel or microscope, which leads to the problem of increasing costs. Furthermore, when using culture bags made of soft-seal material in such culture devices, there was a problem in that light scattering was prone to occur, making it difficult to observe the cells inside the culture bag.

[0007] Furthermore, the petri dish described in Patent Document 2 is a small culture device that can be placed on a general-purpose microscope. While such culture devices allow for observation of cells being cultured in a petri dish, they require a pipe to be fixed to the lid of the petri dish for draining and refilling the culture medium. Therefore, if the tip of the pipe is far from the culture surface, a large amount of culture medium remains after draining, resulting in a problem where the culture medium exchange rate cannot be increased.

[0008] Furthermore, if the tip of the pipe is close to the culture surface, there is a risk of aspirating cells when the culture medium is discharged. Also, when culturing sphere cells on a culture surface with irregularities, there was a problem of the sphere cells moving when the culture medium was filled. Furthermore, this culture device had the problem of not being applicable when performing cell culture using culture bags made of soft-seal material.

[0009] Therefore, the inventors diligently conducted research and succeeded in developing a cell culture system that can solve these problems. In other words, the present invention aims to provide a cell culture system, a method for using the cell culture system, and a cell culture unit that allows cells to be cultured by setting up a culture bag, allows various tools necessary for the culture to be easily carried together, and allows the culture bag to be observed using a general-purpose microscope. [Means for solving the problem]

[0010] To achieve the above objective, the cell culture system of the present invention is a cell culture system for culturing cells in a culture bag made of soft packaging material, comprising a culture unit having a notch or a transparent mounting portion on which a bag holder equipped with a pressing member for pressing the culture bag is placed, and further comprising on the culture unit a culture bag placed in the bag holder, a culture medium container for supplying culture medium to the culture bag, a waste liquid container from which the culture medium is discharged from the culture bag, a first liquid delivery means disposed on a first tubular member connecting the culture bag and the culture medium container, and a second liquid delivery means disposed on a second tubular member connecting the culture bag and the waste liquid container, wherein the distance from the bottom surface of the culture unit to the culture surface of the culture bag is less than 20 mm, and the pressing member presses the culture bag filled with culture medium, thereby uniformizing the liquid thickness of the culture medium in the culture bag, and the surface of the culture unit, the upper and lower surfaces of the pressing member, and the upper surface and culture surface of the culture bag are substantially parallel.

[0011] Furthermore, it is preferable that the cell culture system of the present invention is configured such that the culture unit has a first liquid delivery means housing section that houses the first liquid delivery means and a second liquid delivery means housing section that houses the second liquid delivery means. Furthermore, it is preferable that the cell culture system of the present invention be configured such that the distance from the bottom surface of the culture unit to the top surface of the first liquid delivery means housing and / or the top surface of the second liquid delivery means housing is 65 mm or less.

[0012] Furthermore, it is preferable that the cell culture system of the present invention is configured such that the culture medium container has two ports, and the culture medium in the culture medium container can be replaced through the other port while the culture medium container is connected to the culture bag via one port.

[0013] Furthermore, it is preferable that the cell culture system of the present invention is configured such that the bag containing the cell culture system is equipped with a liquid thickness measuring unit, the liquid thickness measuring unit is connected to a control device, and the liquid thickness of the culture medium in the culture bag is calculated by the control device based on the value measured by the liquid thickness measuring unit. Furthermore, it is preferable that the cell culture system of the present invention is configured such that the control device is connected to the first liquid delivery means and the second liquid delivery means, and the first liquid delivery means and the second liquid delivery means are controlled by the control device based on the liquid thickness of the culture medium in the culture bag.

[0014] Furthermore, a method for using the cell culture system of the present invention is a method for using the cell culture system described in any of the above, wherein the cell culture system is placed in an incubator, the culture medium is supplied from the culture medium container to the culture bag by the first liquid supply means to perform cell culture, the cell culture system is carried out of the incubator and placed under a microscope, the culture bag is pressed with the pressing member to make the liquid thickness of the culture medium in the culture bag uniform, and the cells in the culture bag are observed using the microscope through the notch or the transparent mounting part.

[0015] Furthermore, it is preferable that the method of using the cell culture system of the present invention involves transferring the culture medium from the culture medium container to the culture bag, and then transferring the culture medium from the culture bag to the waste liquid container, while observing the cells in the culture bag using the microscope.

[0016] Furthermore, the cell culture unit of the present invention is a cell culture unit in which cell culture is performed by installing a culture bag, and the cell culture unit comprises a main body having a notch or transparent mounting portion on which a culture bag holder for accommodating the culture bag is placed, and two liquid delivery means accommodating portions on which liquid delivery means connected to the culture bag via a tubular member can be installed, and a mounting plate on which a culture medium supply bag connected to one of the liquid delivery means via a tubular member can be tilted, and a waste liquid bag connected to the other liquid delivery means via a tubular member can be placed below the mounting plate.

[0017] In addition, it is preferable that the cell culture unit of the present invention is configured to be portable while maintaining the arrangement of the culture bag storage device that houses the culture bag, the medium supply bag, and the waste liquid bag.

Advantages of the Invention

[0018] According to the present invention, it is possible to install a culture bag and culture cells, and it is possible to easily carry the various tools necessary for the culture in a bundled state, and to provide a cell culture system, a method of using the cell culture system, and a cell culture unit that can be used for observation with a general-purpose microscope.

Brief Description of the Drawings

[0019] [Figure 1] It is a schematic diagram (perspective view) showing the configuration of a cell culture unit according to an embodiment of the present invention. [Figure 2] It is a schematic diagram (front view) showing the configuration of a cell culture unit according to an embodiment of the present invention. [Figure 3] It is a schematic diagram (rear view) showing the configuration of a cell culture unit according to an embodiment of the present invention. [Figure 4] It is a schematic diagram (plan view) showing the configuration of a cell culture unit according to an embodiment of the present invention. [Figure 5] It is a schematic diagram (bottom view) showing the configuration of a cell culture unit according to an embodiment of the present invention. [Figure 6] It is a schematic diagram (right side view) showing the configuration of a cell culture unit according to an embodiment of the present invention. [Figure 7] It is a schematic diagram (left side view) showing the configuration of a cell culture unit according to an embodiment of the present invention. [Figure 8] It is a schematic diagram (view with the culture bag storage device placed) showing the configuration of a cell culture unit according to an embodiment of the present invention. [Figure 9] It is a schematic diagram showing the usage state of a cell culture unit according to an embodiment of the present invention. [Figure 10]This is a schematic diagram showing a culture bag holder that can be placed on a cell culture unit according to an embodiment of the present invention. [Figure 11] This is a schematic diagram showing the control mechanism of a cell culture unit according to an embodiment of the present invention. [Figure 12] This is a schematic diagram showing the configuration of a cell culture system according to an embodiment of the present invention. [Figure 13] This is a schematic diagram showing the distance (L1) from the bottom surface of the culture unit to the culture surface of the culture bag in a cell culture system according to an embodiment of the present invention. [Figure 14] This is a schematic diagram showing the distance (L2) from the bottom surface of the culture unit to the top surface of the liquid delivery means housing in a cell culture system according to an embodiment of the present invention. [Figure 15] This is a schematic diagram showing a cell culture system according to an embodiment of the present invention placed under a microscope. [Figure 16] This is a schematic diagram showing the control mechanism of a cell culture system according to an embodiment of the present invention. [Modes for carrying out the invention]

[0020] The following describes in detail embodiments of the cell culture system, method of using the cell culture system, and cell culture unit of the present invention. However, the present invention is not limited to the specific details of the following embodiments.

[0021] [Cell Culture Unit] First, the cell culture unit of this embodiment will be described. The cell culture unit of this embodiment is a jig in which a culture bag is placed and cell culture is performed. As shown in Figures 1 to 7, the cell culture unit 1 of this embodiment comprises a main body 11 and a mounting plate 12. The main body 11 has a notch 111 on which a culture bag holder for accommodating culture bags is placed, and two liquid delivery means accommodating sections 112 on which liquid delivery means such as a pump 20 connected to the culture bag via a tubular member such as a tube can be installed. A support groove capable of supporting a culture bag container is formed around the periphery of the notch 111.

[0022] A culture medium supply bag, connected to one of the pumps 20 via a tubular member, can be placed on the mounting plate 12 in a tiltable manner. In other words, as shown in Figure 2, the mounting plate 12 is equipped with a mounting plate support part 121, which allows the tilt angle of the mounting plate 12 to be adjusted from a horizontal state to a tilted state.

[0023] Furthermore, by placing the culture medium supply bag on the inclined mounting plate 12, air bubbles in the culture medium supply bag can be collected upwards, preventing them from being sent to the culture bag. A waste liquid bag, which is connected to the other pump 20 via a tubular member, can be placed on the underside of the mounting plate 12.

[0024] Furthermore, two liquid delivery means housing sections 112, on which pumps 20 can be installed, are provided at both ends of the rear side of the main body 11, and a culture bag housing section 30 can be placed in the center of the front side of the main body 11, with a mounting plate 12 provided between the two liquid delivery means housing sections 112. As a result, by placing the culture medium supply bag 50 on the mounting plate 12 and the waste liquid bag 60 on the underside of the mounting plate 12, these bags can be compactly arranged in the cell culture unit, and various tools necessary for cell culture can be carried in a balanced manner.

[0025] Furthermore, the main body 11 is provided with sensor code holes 113 and 114. Codes (such as signal lines) extending from the liquid thickness measurement unit 31, such as a length measuring sensor, which will be provided in the culture bag storage unit 30 described later, are connected to the control device 70 through these code holes, and signals from the liquid thickness measurement unit 31 are transmitted to the control device 70.

[0026] Furthermore, the main body 11 is provided with a connector connection section 115, and a cord (such as a power line) extending from the pump 20 is connected to the control device 70 via a connector connected to the connector connection section 115, allowing the control device 70 to control the pump 20. As the pump 20, it is preferable to use a pump capable of low-speed, high-precision fluid delivery, such as a peristaltic pump or a syringe pump.

[0027] As shown in Figure 8, a culture bag storage section 30 for accommodating culture bags can be placed in the notch 111 of the main body 11. By placing the culture bag housing section 30 on the notch 111 in this manner, the cells inside the culture bag can be observed with a microscope from below the culture bag housing section 30. Alternatively, instead of the notch 111, a transparent mounting section can be provided on the main body 11, and the culture bag storage section 30 can be installed on this mounting section, allowing the cells inside the culture bag to be observed from below the culture bag storage section 30.

[0028] Figure 9 shows the cell culture unit 1 of this embodiment with a culture bag holder 30 containing a culture bag 40, a culture medium supply bag 50, and a waste liquid bag 60 placed on it, with the culture bag 40 and the culture medium supply bag 50 connected via a pump 20 and a tube 21, and the culture bag 40 and the waste liquid bag 60 connected via a pump 20 and a tube 21. The culture bag holder 30 is equipped with a liquid thickness measuring unit 31, and a sensor cord 32 extends from this liquid thickness measuring unit 31. With this type of cell culture unit, it is possible to transport the unit while maintaining the arrangement of the culture bag 40, the culture medium supply bag 50, and the waste liquid bag 60.

[0029] Furthermore, it is also preferable to perform cell culture by stacking cell culture units, each containing various tools necessary for such culture, within an incubator. In this way, by stacking the cell culture units of this embodiment inside an incubator and performing cell culture, it is possible to easily expand the culture area.

[0030] The culture bag 40 is a closed-system culture container in the shape of a bag, made of a gas-permeable soft packaging material, in which cells and culture medium are filled and cell culture is carried out. Suitable materials for the culture bag 40 include resin films, and polyolefin resins such as polyethylene and polypropylene can be used. Examples include polyethylene, copolymers of ethylene and α-olefin, copolymers of ethylene and vinyl acetate, and ionomers using copolymers of ethylene and acrylic acid or methacrylic acid with metal ions. Polyolefins, styrene elastomers, and polyester thermoplastic elastomers can also be used. Furthermore, flexible polyvinyl chloride resin, polybutadiene resin, ethylene-vinyl acetate copolymer, chlorinated polyethylene resin, polyurethane thermoplastic elastomer, polyester thermoplastic elastomer, silicone thermoplastic elastomer, styrene elastomer, for example, SBS (styrene-butadiene-styrene), SIS (styrene-isoprene-styrene), SEBS (styrene-ethylene-butylene-styrene), SEPS (styrene-ethylene-propylene-styrene), polyolefin resin, and fluororesin may also be used.

[0031] The cells cultured using the culture bag 40 are not particularly limited and may be suspension cells, adherent cells, spheres (cell aggregates), or organoids created by aggregating several types of cells. Specifically, examples include nerve cells, pancreatic islet cells, kidney cells, hepatocytes, muscle cells, cardiomyocytes, corneal endothelial cells, vascular endothelial cells, mesenchymal stem cells, and lymphocytes. Additionally, induced pluripotent stem cells (iPS cells), embryonic stem cells (ES cells), and cells differentiated from these stem cells into the various types mentioned above can also be included. Furthermore, it is possible to culture intermediate cells, such as progenitor cells, that develop during the differentiation process.

[0032] In this embodiment, the culture bag 40 is equipped with a port 41 used for supplying culture medium and a port 41 used for discharging culture medium. The culture bag 40 may also be equipped with three or more ports.

[0033] The culture medium supply bag 50 is a container filled with culture medium for delivery to the culture bag 40, and is connected to the culture bag 40 via a tube 21 and a pump 20. The waste liquid bag 60 is a container that receives used culture medium from the culture bag 40, and is connected to the culture bag 40 via a tube 21 and a pump 20. The same materials as those used for the culture bag 40 can be used for the culture medium supply bag 50 and the waste liquid bag 60. Furthermore, the culture medium supply bag 50 and the waste liquid bag 60 may also be gas-barrier bags with low gas permeability, and these materials are not particularly limited.

[0034] According to this embodiment, such a cell culture unit can be placed inside a general-purpose incubator, and the cords extending from the liquid thickness measuring unit 31 and the cords extending from the pump 20 can be connected to the control device 70. By operating the pump 20, it is possible to supply culture medium from the culture medium supply bag 50 to the culture bag 40 and perform cell culture. Furthermore, the culture medium in the culture bag 40 can be discharged into the waste liquid bag 60.

[0035] Then, the cord is removed from the cell culture unit in which cell culture has been performed in the culture bag 40, and the cell culture unit is removed from the incubator while maintaining the arrangement of the culture bag 40, culture medium supply bag 50, and waste liquid bag 60. The unit is then moved to a general-purpose microscope while maintaining the closed system, making it possible to observe the cells in the culture bag through the notch 111 using the microscope.

[0036] In other words, with this cell culture unit, cells can be cultured by setting up a culture bag 40 made of soft-seal material without requiring a large-scale and expensive observation mechanism. All the necessary tools for the culture can be easily transported while maintaining a closed system, and the culture bag 40 can be used for microscopic observation.

[0037] Next, with reference to Figure 10, the configuration of the culture bag housing section 30 containing the culture bag 40 will be described. The culture bag holder 30 is equipped with a pressing member 33, which presses the culture bag 40 filled with culture medium, thereby making it possible to equalize the liquid thickness of the culture medium inside the culture bag 40 (the distance from the inner surface of the upper film of the culture bag 40 to the inner surface of the lower film (culture surface)).

[0038] In Figure 10, the pressing member 33 consists of a pressing portion 331 and a mounting portion 332. A guide pin 333 is provided around the periphery of the top plate portion, and the guide pin 333 passes through a guide hole provided in the mounting portion 332, allowing the pressing member 33 to move vertically along the guide pin 333.

[0039] This pressing member 33 is movable vertically while being pressed perpendicularly against the upper film of the culture bag 40 by means of a permanent magnet or the weight of the pressing member 33 itself. In other words, when the culture bag 40 is filled with culture medium, the pressing member 33 is pushed up by the culture bag 40, and when the culture medium is discharged from the culture bag 40, the liquid thickness of the culture medium in the culture bag 40 decreases accordingly, and the pressing member 33 descends as a result.

[0040] By using such a culture bag containment section 30, the pressing member 33 can follow the upper film of the culture bag 40 and apply pressure according to the amount of liquid culture medium in the culture bag 40, making it possible to observe the cells in the culture bag 40 even while the culture medium is being dispensed. Furthermore, by using such a culture bag containment section 30, it is possible to prevent the upper and lower films of the culture bag 40 from coming into contact even when the liquid thickness of the culture medium inside the culture bag 40 is reduced to less than 1 mm.

[0041] The pressing member 33 may be made of a synthetic resin such as polycarbonate, or of glass. Furthermore, the pressing member 33 may be partially or entirely transparent. The top of the culture bag storage section 30 has a top opening, which allows the inside of the culture bag storage section 30 to be seen through the top opening.

[0042] Furthermore, in the cell culture unit of this embodiment, the surface of the main body 11, the upper and lower surfaces of the pressing member 33, and the upper surface and culture surface of the culture bag 40 are substantially parallel. Therefore, when irradiating the culture bag 40 with light from above and observing the cells inside the culture bag 40 with a microscope from below, it is possible to prevent light from scattering and to observe them favorably.

[0043] Furthermore, in the cell culture unit of this embodiment, the distance from the bottom surface of the main body 11 to the culture surface inside the culture bag 40 (thickness of the stand of the main body 11 + thickness of the mounting platform of the culture bag storage section 30 + thickness of the bottom film of the culture bag 40) is preferably less than 20 mm, more preferably less than 10 mm, and particularly preferably about 5 to 6 mm. By configuring the cell culture unit of this embodiment in this way, it becomes possible to suitably observe the cells in the culture bag 40 using a general-purpose phase-contrast microscope or the like.

[0044] Furthermore, a liquid thickness measuring unit 31 is fixed to the top plate of the culture bag storage unit 30, and a metal member 3321 is attached to the mounting part 332 of the pressing member 33 so as to face it. The liquid thickness measurement unit 31 measures the distance to the metal member 3321 and outputs it to the control device 70, which then calculates the liquid thickness of the culture medium in the culture bag 40.

[0045] Furthermore, the pump 20 is controlled by the control device 70 based on the liquid thickness of the culture medium in the culture bag 40. In other words, the control device 70 calculates the liquid thickness of the culture medium in the culture bag 40 based on the value (output voltage or output current) measured by the liquid thickness measuring unit 31, and controls the start and stop of the operation of the pump 20, as well as the rotation speed, etc., based on the liquid thickness, thereby enabling control of the liquid thickness to a desired size.

[0046] As shown in Figure 11, the control device 70 may include an input / output unit 71, a control unit 72, an operation unit 73, and a power supply unit 74. The input / output unit 71 is connected to the pump 20 and controls the operation of the pump 20 based on input information from the control unit 72. This allows culture medium to be supplied from the culture medium supply bag 50 to the culture bag 40, and used culture medium to be discharged from the culture bag 40 to the waste liquid bag 60.

[0047] The control unit 72 is composed of a PLC (programmable logic controller) or the like, and can store pre-programmed control content, thereby controlling the operation of each part. Specifically, the control unit 72 transmits information for controlling the pump to the input / output unit 71 at a predetermined timing.

[0048] The operation unit 73 is equipped with a display unit such as a touch panel and transmits user input information to the control unit 72 to perform PLC settings and other operations. It also displays input information from the control unit 72. The power supply unit 74 (such as a stabilized power supply) supplies electricity to each part of the control device 60. Furthermore, although not shown in the figures, the control device 60 may be configured to include a relay unit and a circuit breaker. In addition, some or all of the components of the control unit 72 and the operation unit 73 in the control device 60 may be implemented by a microcontroller or computer.

[0049] [How to use the cell culture unit] The method of using the cell culture unit of this embodiment involves placing the cell culture unit described above inside an incubator and supplying culture medium from the culture medium supply bag 50 to the culture bag 40 via the tube 21 using one of the pumps 20 to perform cell culture. Furthermore, the other pump 20 can also be used to discharge the culture medium from the culture bag 40 through the tube 21 into the waste liquid bag 60. Then, the cell culture unit is carried out of the incubator while maintaining the arrangement of the culture bag 40, culture medium supply bag 50, and waste liquid bag 60, and the culture bag 40 is subjected to microscopic observation of the cells through the notch 111 or the transparent mounting section.

[0050] This method of using cell culture units eliminates the need for large-scale and expensive observation mechanisms, allowing cell culture to be performed by placing the cell culture unit inside a general-purpose incubator. Furthermore, the cell culture unit can be easily removed from the incubator and transported while maintaining a closed system, and can be placed on a general-purpose microscope to observe the cells in the culture bag.

[0051] [Cell Culture System] Next, the cell culture system of this embodiment will be described. The cell culture system of this embodiment consists of the cell culture unit described above and various tools necessary for cell culture, and has the features that it can be carried around while cells are being cultured in a culture bag, and can be suitably used for microscopic observation of cells in the culture bag.

[0052] Specifically, as shown in Figure 12, the cell culture system of this embodiment is a cell culture system that cultures cells in a culture bag 40 made of soft packaging material, and includes a culture unit 1 having a notch 111 or a transparent mounting part on which a bag holder 30 equipped with a pressing member for pressing the culture bag 40 is placed.

[0053] Furthermore, the culture unit 1 includes a culture bag 40 placed in a bag holder 30, a culture medium container 50 for supplying culture medium to the culture bag 40, a waste liquid container 60 from which the culture medium is discharged from the culture bag 40, a first liquid supply means 20-1 disposed on a first tubular member 21-1 that connects (communicates) the culture bag 40 and the culture medium container 50, and a second liquid supply means 20-2 disposed on a second tubular member 21-2 that connects the culture bag 40 and the waste liquid container 60. The culture medium container 50 and the waste liquid container 60 may be bags made of flexible packaging material or rigid containers.

[0054] Furthermore, in the cell culture system of this embodiment, the distance L1 (thickness of the stand of the main body 11 + thickness of the mounting platform of the culture bag 30 + thickness of the bottom film of the culture bag 40) from the bottom surface (11b) of the culture unit 1 shown in Figure 13 to the culture surface (40b) of the culture bag 40 is preferably less than 20 mm, more preferably less than 10 mm, even more preferably 7 to 8 mm, and particularly preferably 5 to 6 mm. By configuring the cell culture system of this embodiment in this way, the focal length can be easily and appropriately adjusted when the cell culture system is placed on a general-purpose phase-contrast microscope or the like, making it possible to suitably observe the cells in the culture bag 40 using a microscope.

[0055] Furthermore, in the cell culture system of this embodiment, as shown in Figure 13, the pressing member 33 presses the culture bag 40 filled with culture medium, which equalizes the liquid thickness of the culture medium inside the culture bag 40, so that the surface (11a) of the culture unit 1, the upper surface (33a) and lower surface (33b) of the pressing member 33, and the upper surface (40a) and culture surface (40b) of the culture bag 40 become substantially parallel. Therefore, when irradiating the culture bag 40 with light from above and observing the cells inside the culture bag 40 with a microscope from below, it is possible to prevent light from scattering and to observe them favorably.

[0056] Furthermore, as shown in Figure 14, the cell culture system of this embodiment has a culture unit 1 which includes a first liquid delivery means housing section 112-1 that houses the first liquid delivery means 20-1 and a second liquid delivery means housing section 112-2 that houses the second liquid delivery means 20-2. Furthermore, the distance L2 from the bottom surface of the culture unit 1 to the top surface of the first liquid delivery means housing and / or the top surface of the second liquid delivery means housing is preferably 65 mm or less, more preferably 60 mm or less, and even more preferably 55 mm or less.

[0057] In other words, general-purpose phase-contrast microscopes typically have a lens and light source in the light source unit, positioned so that the light is focused on the object being observed. Therefore, the light source unit is fixed and cannot be moved vertically. When installing a cell culture system in a microscope, it is necessary to pass the system under the light source unit, so it is desirable to make the height of at least one of the liquid delivery means housings smaller than the distance between the light source unit and the observation table. Therefore, with the above configuration, the cell culture system of this embodiment can be easily and appropriately placed on the observation stand of a general-purpose phase-contrast microscope or the like without being obstructed by the light source of the microscope, and the cells in the culture bag 40 can be suitably observed using the microscope.

[0058] Figure 15 shows the cell culture system of this embodiment placed in a general-purpose phase-contrast microscope. This cell culture system allows for easy observation of cells in the culture bag 40 by transporting the system while it is still in the culture state and placing it on the observation stand M10 of a general-purpose phase-contrast microscope M1. Furthermore, the cell culture system can be easily placed on the observation stand M10 without being obstructed by the light source M11 of the general-purpose phase-contrast microscope M1.

[0059] On the other hand, conventionally, when observing cells in a culture bag, it was necessary to place the bag holder containing the culture bag, the culture medium container, the waste liquid container, two liquid delivery means, and the tubes connecting them onto a stand, carry them to the microscope, and then observe the cells in the culture bag, which was extremely cumbersome.

[0060] In contrast, the cell culture system of this embodiment makes it possible to significantly reduce the effort required to observe the state of cells when culturing cells using culture bags. Such a reduction in effort is extremely important in culture settings where the same operations need to be repeated.

[0061] Furthermore, the cell culture system of this embodiment allows for direct observation of the state of cells during culture, and as described later in the section on how to use the cell culture system, it is also possible to observe the state of cells while supplying the culture medium. Thus, the cell culture system of this embodiment makes it possible to easily realize observation methods that were not easily achievable in the past.

[0062] Furthermore, in the cell culture system of this embodiment, as shown in Figure 12, it is preferable that the culture medium container 50 has two ports, and that the culture medium in the culture medium container 50 can be replaced through the other port while the culture medium container 50 is connected to the culture bag 40 via one port.

[0063] In other words, when using the cell culture system of this embodiment to induce differentiation of iPS cell spheres, it becomes necessary to frequently change the type of culture medium. Depending on the type of differentiation induction, the culture medium may even need to be changed daily. At this time, the cell culture system will be moved to a clean bench or similar location, and the entire contents of the culture medium in the culture medium container 50 will be replaced. However, although the air inside the clean bench is clean, there is a risk of contamination if objects are touched, so touching is prohibited, and if it is touched, the work will have to be redone.

[0064] In the cell culture system of this embodiment, as shown in Figure 12, the culture medium container 50 is equipped with ports 51 and 52. Therefore, the culture medium can be replaced by opening port 52 on the opposite side of port 51, which is connected to the culture bag 40, and connecting a syringe or the like. Thus, with the cell culture system of this embodiment, only one port 52 of the culture medium container 50 needs to be opened, making operation easier and reducing the risk of contamination.

[0065] On the other hand, in the cell culture system of this embodiment, if a culture medium container 50 equipped with only one port 51 for connecting to the culture bag 40 is used, it becomes necessary to disconnect the tube near the pump 20-1 to change the culture medium, which presents the problem that the tube is prone to coming into contact with objects.

[0066] Furthermore, since both the tube on the culture bag 40 and the port 51 on the culture medium container 50 were open, and it was necessary to handle both the tube and port 51 while working, there was a problem of increased contamination risk. In addition, the work was very complicated due to the intricate working conditions. In contrast, in the cell culture system of this embodiment, where the culture medium container 50 has two ports, it is possible to simplify the culture process because it is easy to change the culture medium inside the culture medium container 50.

[0067] Furthermore, in the cell culture system of this embodiment, it is preferable to provide two ports on the waste liquid container 60 from the viewpoint of reducing the risk of contamination. Of course, by using a larger waste liquid container 60, it is also possible to omit the discharge of the culture medium from the waste liquid container 60.

[0068] Furthermore, the cell culture system of this embodiment preferably includes a control device, similar to the cell culture unit described above. In other words, as shown in Figure 16, in the cell culture system of this embodiment, the bag holder 30 is equipped with a liquid thickness measuring unit 31, the liquid thickness measuring unit 31 is connected to a control device 70, and the liquid thickness of the culture medium in the culture bag 40 is calculated by the control device 70 based on the value measured by the liquid thickness measuring unit 31.

[0069] Furthermore, the control device 70 is connected to pumps 20-1 and 20-2, and pumps 20-1 and 20-2 are controlled by the control device 70 based on the liquid thickness of the culture medium in the culture bag 40. The specific configuration of the control device 70 is omitted here, as it has been described above in the explanation of the cell culture unit.

[0070] [How to use the cell culture system] The method of using the cell culture system of this embodiment is the method of using the cell culture system described above, characterized in that the cell culture system is placed in an incubator, culture medium is supplied from the culture medium container 50 to the culture bag 40 by the pump 20-1 to perform cell culture, the cell culture system is carried out of the incubator and placed on the microscope M1, the culture bag 40 is pressed with the pressing member 33 to make the liquid thickness of the culture medium in the culture bag 40 uniform, and the cells in the culture bag 40 are observed using the microscope M1 through the notch 111 or the transparent mounting part.

[0071] Furthermore, a preferred method of using the cell culture system of this embodiment is to transfer the culture medium from the culture medium container 50 to the culture bag 40, and then transfer the culture medium from the culture bag 40 to the waste liquid container 50, while observing the cells in the culture bag 40 using a microscope M1.

[0072] When transporting the cell culture system out of the incubator, disconnect the cords extending from the liquid thickness measuring unit 31 and the cords extending from pumps 20-1 and 20-2, which are connected to the culture unit 1. After placing the cell culture system on the microscope M1, reconnect these cords to the culture unit 1. Then, by operating pumps 20-1 and 20-2, the state of the cells during fluid delivery can be observed using microscope M1.

[0073] According to this method of using the cell culture system of this embodiment, it is possible to observe behaviors such as whether or not spheres move due to fluid delivery. This not only allows for understanding the state of cells during culture, but also enables the development of better culture bags and the identification of fluid delivery conditions that do not cause sphere movement.

[0074] As described above, the cell culture system, method of using the cell culture system, and cell culture unit of this embodiment do not require a large-scale and expensive observation mechanism, and cell culture can be performed by placing the cell culture unit inside a general-purpose incubator. Furthermore, the cell culture unit can be easily removed from the incubator and transported while maintaining a closed system, and it can be placed on a general-purpose microscope to observe the cells in the culture bag.

[0075] Furthermore, when the cell culture system is placed on a general-purpose microscope, the focal length can be easily and appropriately adjusted, preventing light scattering, and it can be easily and appropriately positioned on the observation stand, making it possible to observe the cells in the culture bag effectively. Furthermore, it significantly reduces the effort required to observe the state of cells, simplifies the process of changing the culture medium, and minimizes the risk of contamination. Furthermore, it is possible to observe the state of the cells during culture while delivering the culture medium, allowing for observation of behavior such as the movement of spheres due to the delivery. This not only allows for understanding the state of the cells during culture but also enables the development of better culture bags.

[0076] Furthermore, in addition to the configuration described above, the cell culture unit of the present invention may also have the following configuration. In other words, it is also preferable that the cell culture unit of the present invention be configured such that the cells in the culture bag can be observed through the notched portion or the transparent mounting portion.

[0077] Furthermore, it is also preferable that the cell culture unit of the present invention be configured such that a pressing member is provided in the culture bag holder, and the pressing member presses the culture bag filled with culture medium, thereby making the liquid thickness of the culture medium within the culture bag uniform. Furthermore, it is also preferable that the cell culture unit of the present invention be configured such that the surface of the main body, the upper and lower surfaces of the pressing member, and the upper surface and culture surface of the culture bag are substantially parallel. Furthermore, it is also preferable that the cell culture unit of the present invention be configured such that the distance from the bottom surface of the main body to the culture surface inside the culture bag is less than 20 mm.

[0078] Furthermore, it is also preferable that the cell culture unit of the present invention be configured such that the culture bag containment is equipped with a liquid thickness measuring unit, the liquid thickness measuring unit is connected to a control device, and the liquid thickness of the culture medium in the culture bag is calculated by the control device based on the value measured by the liquid thickness measuring unit. Furthermore, it is also preferable that the cell culture unit of the present invention be configured such that the control device is connected to the liquid delivery means, and the liquid delivery means is controlled by the control device based on the liquid thickness of the culture medium in the culture bag.

[0079] Furthermore, the method of using the cell culture unit of the present invention is the method of using the cell culture unit described in any of the above, wherein the cell culture unit is placed in an incubator, the culture medium is supplied from the culture medium supply bag to the culture bag via a tubular member by one of the liquid supply means to perform cell culture, the culture medium can be discharged from the culture bag to the waste liquid bag via a tubular member by the other liquid supply means, the cell culture unit is carried out of the incubator while the arrangement of the culture bag, the culture medium supply bag, and the waste liquid bag is maintained, and the culture bag is subjected to microscopic observation of cells via the notch or the transparent mounting part.

[0080] The present invention is not limited to the embodiments described above, and it goes without saying that various modifications can be made within the scope of the present invention. For example, the arrangement of the liquid delivery means housing, mounting plate, and notches in the cell culture unit can be appropriately changed. [Industrial applicability]

[0081] This invention is suitable for use when culturing cells in a compact, integrated state, transporting the various tools necessary for cell culture, and observing cultured cells under a microscope. [Explanation of Symbols]

[0082] 1 Cell culture unit 11 Main body 111 Notch 112, 112-1, 112-2 Liquid delivery means housing 113 Sensor cord hole 114 Sensor cord holes 115 Connector connection part 12 Mounting plate 121 Mounting plate support section 20, 20-1, 20-2 Pump (liquid delivery means) 21, 21-1, 21-2 Tube (tubular member) 30. Culture bag container (bag container) 31 Liquid Thickness Measurement Section 32 Sensor Codes 33 Pressing member 331 Pressing part 332 Mounting part 3321 Metal components 333 Guide pins 40 culture bags 41 ports 50 culture medium supply bags (culture medium containers) Ports 51 and 52 60 waste liquid bags (waste liquid containers) 70 Control device 71 Input / output section 72 Control Unit 73 Operation section 74 Power supply section

Claims

1. A cell culture system for culturing cells using a culture bag made of soft-dispensing material, The culture unit is provided with a notch or a transparent mounting section for mounting a bag holder equipped with a pressing member for pressing the culture bag, The culture unit comprises a culture bag placed in the bag holder, a culture medium container for supplying culture medium to the culture bag, a waste liquid container from which the culture medium is discharged from the culture bag, a first liquid delivery means disposed on a first tubular member connecting the culture bag and the culture medium container, and a second liquid delivery means disposed on a second tubular member connecting the culture bag and the waste liquid container. The distance from the bottom surface of the culture unit to the culture surface of the culture bag is less than 20 mm. When the pressing member presses the culture bag filled with culture medium, the liquid thickness of the culture medium inside the culture bag becomes uniform, and the surface of the culture unit, the upper and lower surfaces of the pressing member, and the upper surface and culture surface of the culture bag become substantially parallel. A cell culture system characterized by the following features.

2. The culture unit has a first liquid delivery means housing section that houses the first liquid delivery means and a second liquid delivery means housing section that houses the second liquid delivery means. The cell culture system according to claim 1, characterized in that it is a cell culture system.

3. The cell culture system according to claim 2, characterized in that the distance from the bottom surface of the culture unit to the top surface of the first liquid delivery means housing and / or the top surface of the second liquid delivery means housing is 65 mm or less.

4. The cell culture system according to any one of claims 1 to 3, characterized in that the culture medium container has two ports, and the culture medium in the culture medium container can be replaced through the other port while the culture medium container is connected to the culture bag via one port.

5. The cell culture system according to any one of claims 1 to 4, characterized in that the bag containing the cell is equipped with a liquid thickness measuring unit, the liquid thickness measuring unit is connected to a control device, and the liquid thickness of the culture medium in the culture bag is calculated by the control device based on the value measured by the liquid thickness measuring unit.

6. The control device is connected to the first liquid delivery means and the second liquid delivery means, The cell culture system according to claim 5, characterized in that the first liquid delivery means and the second liquid delivery means are controlled by the control device based on the liquid thickness of the culture medium in the culture bag.

7. A method for using the cell culture system according to any one of claims 1 to 6, The cell culture system is placed inside the incubator, and the culture medium is supplied from the culture medium container to the culture bag by the first liquid delivery means to perform cell culture. The cell culture system is removed from the incubator and placed under a microscope. The culture bag is then pressed with the pressing member to equalize the thickness of the culture medium inside the bag. The cells inside the culture bag are then observed using the microscope through the notch or the transparent mounting section. A method for using a cell culture system characterized by the features described herein.

8. A method for using the cell culture system according to claim 7, characterized in that the culture medium is transferred from the culture medium container to the culture bag, and the culture medium is transferred from the culture bag to the waste liquid container, and the cells in the culture bag are observed using the microscope.

9. A cell culture unit in which a culture bag is installed and cell culture is performed, The cell culture unit comprises a main body having a notch or transparent mounting section on which a culture bag holder for containing the culture bag is placed, and two mounting sections for mounting liquid delivery means connected to the culture bag via a tubular member; and a mounting plate on which a culture medium supply bag connected to one of the liquid delivery means via a tubular member can be tilted, and a waste liquid bag connected to the other liquid delivery means via a tubular member can be placed below the mounting plate. A cell culture unit characterized by the following features.

10. The system includes a culture bag container containing the culture bag, a culture medium supply bag, and a waste liquid bag. The culture bag, the culture medium supply bag, and the waste liquid bag can be carried while maintaining their arrangement. The cell culture unit according to feature 9.

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