Cell observation device, cell culture device, and cell culture method

Abstract

This invention provides a cell observation device that allows for easy observation of cells in a culture vessel, as well as a cell culture device and cell culture method using the same. [Solution] A cell observation device for observing cells 30 in a culture vessel 10 with a camera 20, comprising a camera 20 for observing cells 30 in the culture vessel 10, a channel 15 for discharging culture medium 11 from the culture vessel 10, and a porous material 13 placed between the culture vessel 10 and the channel 15, wherein the porous material 13 allows the liquid 32 in the culture medium 11 to pass through without passing through cell aggregates or scaffolding material 31 to which the cells 30 are attached, and the camera 20 is a camera for observing cells 30 contained in cell aggregates attached to the porous material 13 or cells 30 present on the scaffolding material.

Description

Technical Field

[0001] The present invention relates to a cell observation device, a cell culture device, and a cell culture method.

Background Art

[0002] The culture of microorganisms, insect cells, plant cells, animal cells, etc. is widely used in the production of various substances useful as pharmaceuticals, foods, cosmetics, and raw materials thereof. Further, Patent Document 1 describes a method of monitoring the state of cell culture by continuous imaging while a cell suspension containing cells or cell aggregates flows through a flow path provided with an imaging unit in the middle of the flow path.

Prior Art Documents

Patent Documents

[0003]

Patent Document 1

Summary of the Invention

Problems to be Solved by the Invention

[0004] In the method described in Patent Document 1, since an image of cells is acquired while the cells flow through the flow path, a flow cell having a flat flow path is used. If the thickness of the flat flow path is increased, it is difficult to focus on the target cells or cell aggregates, and if the thickness of the flat flow path is decreased, the size of the cell aggregates is not constant, so the cell aggregates may be clogged.

[0005] The present invention has been made in view of the above circumstances, and an object thereof is to provide a cell observation device capable of easily observing cells in a culture container, a cell culture device using the same, and a cell culture method.

Means for Solving the Problems

[0006] The present invention includes the following aspects. [1] A cell observation device for observing cells in a culture vessel with a camera, comprising: a camera for observing cells in a culture vessel; a channel for discharging culture medium from the culture vessel; and a porous material disposed between the culture vessel and the channel, wherein the porous material allows the liquid in the culture medium to pass through without passing through cell aggregates or scaffolding material to which the cells are attached, and the camera is a camera for observing cells contained in cell aggregates attached to the porous material or cells present on the scaffolding material. [2] The cell observation apparatus according to [1], wherein the camera is positioned in the channel opposite the porous material. [3] The cell observation apparatus according to [1] or [2], wherein the flow path has an inlet and an outlet for the culture vessel, and a pump for circulating the culture medium from the inlet to the outlet when observing cells in the culture vessel using the camera. [4] The cell observation apparatus according to [3], wherein the pump is capable of backflowing the culture medium from the outlet towards the inlet. [5] The cell observation apparatus according to [3] or [4], wherein the flow channel is provided with a porous material at the outlet. [6] The cell observation apparatus according to any one of [1] to [5], wherein the culture vessel comprises a flexible synthetic resin container for containing the culture medium, and the channel is connectable to the synthetic resin container. A cell culture apparatus comprising the cell observation apparatus described in any one of items [1] to [6] and the culture vessel. A cell culture method comprising using a cell observation device described in any one of items [1] to [6], while discharging the culture medium from the culture vessel into the channel, and observing the cells in the culture vessel with the camera. [Effects of the Invention]

[0007] According to the present invention, by using a porous material to drain the culture medium, cells in the culture vessel can be easily observed even if the cells remain inside the culture vessel. [Brief explanation of the drawing]

[0008] [Figure 1] This is an explanatory diagram showing an example of a cell culture apparatus. [Figure 2] This is an explanatory diagram showing an example of a cell observation device. [Modes for carrying out the invention]

[0009] The present invention will be described below based on preferred embodiments.

[0010] Figure 1 illustrates a cell culture apparatus according to the embodiment. Figure 2 also illustrates a cell observation apparatus according to the embodiment. The illustrated cell culture apparatus includes the culture vessel 10 shown in Figure 1, along with the cell observation apparatus shown in Figure 2.

[0011] The illustrated cell observation device is a cell observation device that uses a camera 20 to observe cells 30 contained in cell aggregates in a culture vessel 10 and cells 30 present on a scaffolding material. This cell observation device comprises a camera 20, a channel 15 for discharging culture medium 11 from the culture vessel 10, and porous materials 13 and 17 placed between the culture vessel 10 and the channel 15. If the culture medium 11 discharged from the culture vessel 10 is not to be returned to the culture vessel 10, the porous material 17 on the outlet side may be omitted, and the outlet side of the channel 15 may be connected to an appropriate drainage reservoir (not shown).

[0012] In the illustrated example, porous materials 13 and 17 are placed at the inlet and outlet of the channel 15 to the culture vessel 10, respectively. The porous material 13 at the inlet allows the liquid 32 in the culture medium 11 to pass through without passing through the cell aggregate or scaffold material 31 to which the cells 30 are attached. If a porous material 17 is provided at the outlet of the channel 15, it is preferable that the porous material 17 at the outlet also allows the liquid 32 in the culture medium 11 to pass through without passing through the cell aggregate or scaffold material 31.

[0013] Porous materials 13 and 17 that the cell aggregate or scaffold material 31 cannot pass through are provided at least on the inlet side of the channel 15 in the culture vessel 10 (two locations including the outlet side of the channel 15 in the illustrated example), and the liquid 32 in the culture medium 11 is discharged using a pump 16. As a result, the cell aggregate or scaffold material 31 is sucked in and approaches or comes into contact with the porous material 13 on the inlet side. This allows the cells 30 on the cell aggregate or scaffold material 31 attached to the porous material 13 to be observed with the camera 20, and can even be photographed. The speed and pressure of discharging the liquid 32 from the culture vessel 10 may be changed as appropriate. For example, the cell aggregate or scaffold material 31 may be sucked into the porous material 13 on the inlet side, or the suction may be loosened. The cell aggregate or scaffold material 31 may also be hooked into the holes of the porous material 13 on the inlet side.

[0014] Since the cells 30 on the surface of the cell aggregate or scaffolding material 31 that are attached to or caught on the porous material 13 are observed by the camera 20, the cells 30 to be observed are almost fixed, making it easy to focus. Since the cell aggregate or scaffolding material 31 does not enter the channel 15, clogging of the channel 15 is suppressed. The porous material 13 can capture the cell aggregate or scaffolding material 31 when observing the cells 30 on the cell aggregate or scaffolding material 31 with the camera 20. The camera 20 is a camera for observing cells 30 contained in the cell aggregate attached to the porous material 13 or cells 30 present on the scaffolding material.

[0015] Pump 16 circulates the culture medium 11 from the inlet to the outlet of the channel 15 when observing the cells 30 in the culture vessel 10 using the camera 20. The pump 16 is not particularly limited, but it is preferable to use a tube pump (peristaltic pump) that can transport the liquid 32 in the channel 15 from the outside of the tube. Pump 16 may be electric or manual. If the culture medium 11 discharged from the culture vessel 10 is not to be circulated back into the culture vessel 10, a diaphragm pump or the like may be used as the pump 16, or a suction device such as a syringe may be used. In this case, after the observation of the cells 30 is finished, it is possible to return the culture medium 11 in the diaphragm pump or suction device such as a syringe back into the culture vessel 10. When using a suction device such as a diaphragm pump or a syringe, the volume of the culture medium 11 that can be discharged outside the culture vessel is determined by the size of the diaphragm pump or the suction device, and it is not always possible to fix the cell mass or the scaffold material 31 to the porous material 13 with a single discharge of the culture medium 11. Therefore, it is preferable to circulate the culture medium 11 using the pump 16.

[0016] When the cell mass or the scaffold material 31 is adhered to the porous material 13 on the inlet side, the cell mass or the scaffold material 31 on the porous material 13 can be easily returned into the culture vessel 10 by reversing the pump 16. When reversing the pump 16 from the outlet to the inlet, the porous material 17 on the outlet side of the flow path 15 can block the passage of the cell mass or the scaffold material 31. Therefore, even when the pump 16 is reversed, only the liquid 32 in the culture medium 11 flows backward in the flow path 15.

[0017] The porous materials 13 and 17 are not particularly limited, and examples thereof include films (porous membranes) having a thickness of about 1 to 200 μm and a pore diameter of about 200 μm or less. Examples of the method of perforating the resin film include a method of locally melting the resin with a heated needle, laser, etc., and a method of locally removing the resin by embossing, punching, etc. using a processing jig such as a mold. As the porous materials 13 and 17, a mesh, a wire net, a non-woven fabric, etc. may be used.

[0018] The porous materials 13 and 17 preferably have pores that allow at least water molecules to pass through. The material forming the porous materials 13 and 17 is not particularly limited, and it may be hydrophobic or hydrophilic, but it is preferably not overly water-repellent. Specific examples of the pore diameter include 10 μm, 30 μm, 50 μm, 60 μm, 80 μm, 100 μm, 200 μm, etc., or a range between any two points arbitrarily selected from these numerical groups. Among these, when it is 100 μm or more and 200 μm or less, it is preferable because the cell mass or the scaffold material 31 is particularly likely to be adsorbed or caught by the porous material 13.

[0019] According to the size of the cell mass or scaffold material 31, the pore size range of the porous materials 13 and 17 can be appropriately changed. The pore size of the porous material 17 on the outlet side may be the same as or different from (a larger diameter or a smaller diameter) the pore size of the porous material 13 on the inlet side.

[0020] When observing or photographing the cells 30 on the cell mass or scaffold material 31 adsorbed or caught on the porous material 13 on the inlet side, the position of the camera 20 is not particularly limited as long as it is near the target cells 30. The camera 20 may be arranged near the porous material 13, and is preferably arranged in the flow path 15 facing the porous material 13. The camera 20 may be fixed at a predetermined position, or the position may be changed within a predetermined range (moved outside the flow path 15 or adjusted inside and outside the flow path 15).

[0021] The porous material 17 on the outlet side may be located above or below the height of the liquid surface of the culture solution 11. When the cell mass or scaffold material does not separate from the porous material 13 after observing the cells 30, it is possible to reverse the flow of the culture solution 11 and the gas 12 (such as air) from the outlet side using the pump 16. However, when the porous material 17 is located above the height of the liquid surface of the culture solution 11, it is preferable because it is less likely to occur that the cell mass or scaffold material 31 is fixed to the porous material 17 and does not separate during reverse flow.

[0022] In the illustrated example, a tubular camera installation portion 14 is provided branching from the flow path 15. The camera installation portion 14 in the illustrated example is formed perpendicular and linearly to the porous material 13. When the camera 20 is installed in this camera installation portion 14, the camera 20 is arranged so as to face the porous material 13. The shape of the camera installation portion 14 can be appropriately set according to the shape of the camera 20. The flow path 15 in the illustrated example may be curved between the porous materials 13 and 17.

[0023] In the illustrated example, the camera 20 is located at the end of the cable 21. The cable 21 is routed outside the camera mounting section 14 and connected to the information processing device 22. The transmission and reception method between the camera 20 and the information processing device 22 is not limited to the illustrated example and may be wired or wireless. The information processing device 22 is not particularly limited and may be a general-purpose product such as a personal computer or smartphone.

[0024] A typical information processing device 22 includes an input device, an output device, a storage device, etc. Image data from camera 20 may be stored in the storage device. Camera 20 may create full-color images or monochrome images. Camera 20 may create a series of images such as a video.

[0025] The types of data handled by the information processing device 22 are not particularly limited, but include electronic data, character data, image data, and audio data. Input devices include keyboards, touch panels, scanners, and microphones. Output devices include display devices such as displays and printing devices such as printers. Storage devices include semiconductor storage devices, magnetic storage devices, and optical storage devices.

[0026] The materials of the camera mounting section 14 and the flow path 15 are not particularly limited, but examples include flexible tubes, hoses, rigid pipes, microchips with flow paths, etc. Flexible materials can be used at least in the parts driven by the pump 16, and the camera mounting section 14 may be rigid.

[0027] The camera mounting section 14 or the flow path 15 may be optically transparent or opaque. If at least the area around the camera mounting section 14 is transparent, external illumination light may be used. The camera 20 may be equipped with a light source, or a light source may be installed on the side of the camera mounting section 14 or the flow path 15. The cross-sectional shape of the flow path 15 is not particularly limited, and the cross-section may be circular, polygonal, or the like.

[0028] The image may be obtained as an electrical signal to be transmitted to an external device such as the information processing device 22. In this case, an image sensor such as a CCD (charge-coupled device) sensor or an image sensor included in a camera such as a digital camera can be used to create the image.

[0029] The images obtained by the camera 20 can be processed by the information processing device 22 using signal processing programs such as image processing software. The information processing device 22 can observe the cells 30 from the images obtained by the camera 20 and use them for analysis, etc.

[0030] The scaffolding material is not particularly limited, but a scaffolding material that can float in the culture medium 11 is preferred. It may be a fibrous scaffolding material such as nanofibers, or a particulate scaffolding material such as microbeads. Examples of materials for the scaffolding material include polysaccharides, proteins, synthetic resins, and glass. When a scaffolding material is not needed, such as when culturing cell aggregates suspended in the culture medium 11, the cells 30 attached to the cell aggregates may be observed.

[0031] The cells used are not particularly limited and may include cells from multicellular organisms or unicellular organisms. Specific examples include fungi, bacteria, yeast, algae, insect cells, plant cells, and animal cells. The cultured cells may be cell lines such as CHO cells, HeLa cells, and COS cells, or stem cells such as embryonic stem cells (ES cells), induced pluripotent stem cells (iPS cells), mesenchymal stem cells, and neural stem cells, or differentiated tissue cells.

[0032] The cell observation device of this embodiment can be used for cell culture and biopharmaceutical manufacturing. Biopharmaceuticals are not particularly limited, but include proteins, nucleic acids, enzymes, hormones, vaccines, antibodies, and cells. The cell culture method is not particularly limited, but includes shaking culture and agitation culture.

[0033] The culture medium used for cell culture is not particularly limited, but examples include solutions, emulsions, suspensions, dispersions, and gels. The culture medium may be a homogeneous single-phase composition, or it may consist of two or more phases, such as a liquid containing solid matter. It is preferable that the culture medium is sterilized or the bacterial cells are removed by filtration before culturing. The culture method is not particularly limited, but can be selected from known culture methods such as batch culture, fed-batch culture, and perfusion culture.

[0034] The material of the culture vessel 10 is not particularly limited, but examples include resin, rubber, elastomer, metal, glass, etc. The culture vessel 10 may be a rigid container into which the culture medium 11 is directly introduced.

[0035] The culture vessel 10 may include a flexible synthetic resin container for holding the culture medium 11. When a camera mounting section 14 or a flow channel 15 is attached to this type of culture vessel 10, the material of the camera mounting section 14 or the flow channel 15 may be made such that it can be attached to the synthetic resin container. The culture vessel 10, which includes a flexible synthetic resin container, may further include a rigid container on the outside of the synthetic resin container to house the synthetic resin container.

[0036] The material of the flexible synthetic resin container is not particularly limited, but for example, a film or sheet of thermoplastic resin may be used. Examples of thermoplastic resins are not particularly limited, but include polyolefin resins such as polyethylene (PE) and polypropylene (PP), polyester resins such as polystyrene (PS) and polyethylene terephthalate (PET), polyamide resins such as nylon, and acrylic resins.

[0037] The culture vessel 10 may comprise an inner bag made of a flexible synthetic resin container and an outer container that houses the inner bag. It is preferable to construct the inner bag with multiple layers, such as a double or triple bag, as this makes it less likely for the contents to leak. The outer container is not particularly limited as long as it can accommodate the inner bag, and examples include boxes, bottles, bags, cans, tanks, etc. The material of the outer container is not particularly limited and can be made of resin, rubber, elastomer, glass, metal, wood, fiber-reinforced plastic, etc.

[0038] The shape of the culture vessel 10 is not particularly limited, but examples include cylindrical, conical, prismatic, and pyramidal shapes. When a taper is provided, such as in a conical or pyramidal shape, the upper cross-sectional shape may be larger than the lower cross-sectional shape, or the lower cross-sectional shape may be larger than the upper cross-sectional shape. The size of the culture vessel 10 is not particularly limited, but for example, it can be 0.1 L to 5000 L in size.

[0039] The culture medium 11 in the culture vessel 10 may contain gas 12 at its upper surface. The position and shape of the interface (liquid surface) between the culture medium 11 and the gas 12 may fluctuate depending on shaking, stirring, etc., during cultivation. Filters may be provided at the points where the gas 12 in the culture vessel 10 enters and exits the culture vessel 10. The gas 12 supplied to the culture vessel 10 may be air originating from the outside air, or gas supplied from a gas cylinder or the like.

[0040] The components contained in gas 12 include N2, O2, CO2, NH3, H2O (water vapor), H2S, He, Ar, etc. Gas 12 may contain two or more components. The main components of gas 12 may be N2 and O2 derived from air.

[0041] The culture vessel 10 may be equipped with connecting devices such as ports and connectors for the inflow and outflow (supply or discharge) of the culture medium 11, gas 12, or components thereof. It is preferable to use sterile connection ports, sterile connectors, etc. that enable sterile connection. Examples of conduits used for the inflow and outflow of fluids such as liquids or gases include flexible tubes, hoses, etc., or rigid pipes, etc. Examples of conduit materials include resin, rubber, elastomer, metal, etc. Filters, flow monitors, flow meters, etc. may be provided in the conduits.

[0042] The temperature of the culture medium 11 is not particularly limited, but the temperature can be adjusted using a temperature control function such as a heater in the culture vessel 10. The culture temperature depends on the type of organism being cultured, but for example, in the case of microorganisms, 4 to 40°C is preferred, and 25 to 37°C is particularly preferred.

[0043] When performing shaking culture, the culture can be carried out by controlling one or more control factors selected from, for example, the shaking speed, shaking direction, medium pH, medium temperature, and wave height of the medium. The shaking direction may be a combination of right and left rotations, or forward and counter-forward rotations, repeated in any pattern.

[0044] During shaking culture, it is preferable for the culture vessel to rotate horizontally, and more preferably, the rotation axis should pass vertically through a point (eccentricity) offset from the center of the horizontal cross-section of the culture vessel. The direction of rotation may include both horizontal and vertical components. It is preferable that the magnitude of the vertical component is smaller than the magnitude of the horizontal component (including cases where the vertical component is zero). By incorporating rotation rather than unidirectional shaking, efficient mixing can be achieved. By changing the rotation speed and direction of the shaking, not only horizontal mixing of the liquid but also vertical mixing can be achieved. It is preferable to set the rotation speed, amplitude, etc. according to the culture system and control it with a program.

[0045] The information processing device 22 may include a control unit for the camera 20. The control unit may include, for example, a processor, internal memory, storage device, input / output interface (input / output I / F), communication interface (communication I / F), display unit, etc.

[0046] Examples of processors include CPUs (Central Processing Units) and GPUs (Graphics Processing Units). Internal memory includes RAM (Random Access Memory) and ROM (Read Only Memory). Storage devices include HDDs (Hard Disk Drives) and SSDs (Solid State Drives).

[0047] An input / output interface (I / F) is a type of device used to connect peripheral devices. A communication interface (communication I / F) is a type of device that communicates with external devices such as cameras, pumps, etc., of a cell observation system, or with external equipment for a cell culture system.

[0048] The information processing device 22 can perform various functions, such as cell observation or cell culture control, by, for example, having the processor execute image processing software stored in a storage device or internal memory while utilizing the internal memory. The illustrated information processing device 22 has a display unit such as a liquid crystal display or organic EL for displaying the status of cell observation or cell culture.

[0049] If it is necessary to change the cell culture conditions based on the results of cell observation, the culture vessel 10 may be operated manually or controlled automatically. When operating or controlling the culture vessel 10, appropriate items may be selected from various factors such as the supply of culture medium, the supply of gas, temperature, humidity, atmospheric pressure, shaking method, and stirring method, and these may be the targets of operation or control. [Explanation of Symbols]

[0050] 10...Culture vessel, 11...Culture medium, 12...Gas, 13...Porous material on the inlet side, 14...Camera mounting section, 15...Flow channel, 16...Pump, 17...Porous material on the outlet side, 20...Camera, 21...Cable, 22...Information processing device, 30...Cells, 31...Cell aggregate or scaffolding material, 32...Liquid.

Claims

1. A cell observation device that uses a camera to observe cells in a culture vessel, A camera for observing cells in a culture vessel, A channel for discharging culture medium from the culture vessel, The system comprises a porous material disposed between the culture vessel and the flow channel, The porous material allows the liquid in the culture medium to pass through without passing through the cell aggregate or scaffold material to which the cells are attached. The camera is a cell observation device for observing cells contained in a cell mass attached to the porous material or cells present on a scaffolding material.

2. The cell observation apparatus according to claim 1, wherein the camera is positioned in the channel opposite the porous material.

3. The cell observation apparatus according to claim 1, wherein the flow path is provided with an inlet and an outlet for the culture vessel, and is equipped with a pump for circulating the culture medium from the inlet to the outlet when observing cells in the culture vessel using the camera.

4. The cell observation apparatus according to claim 3, wherein the pump is capable of backflowing the culture medium from the outlet towards the inlet.

5. The cell observation apparatus according to claim 3, wherein the flow path is provided with a porous material at the outlet.

6. The culture vessel comprises a flexible synthetic resin container for containing the culture medium, The cell observation apparatus according to claim 1, wherein the channel can be joined to the synthetic resin container.

7. A cell culture apparatus comprising a cell observation apparatus according to any one of claims 1 to 6 and the culture vessel.

8. A cell culture method comprising using the cell observation device according to any one of claims 1 to 6, observing the cells in the culture vessel with the camera while discharging the culture medium from the culture vessel into the channel.

Images