Cell culture vessel and cell culture device

Abstract

A cell culture vessel is provided. [Solution] A cell incubator for culturing cells inside, the width of the bottom surface is equal to or greater than the height of the sides, the cell incubator is provided with a flow path for supplying a fluid to the inside, and the inside can be closed. A cell incubator for culturing cells inside, the cell incubator has at least one of the bottom surface and top surface transparent, the cell incubator is provided with a flow path for supplying a fluid to the inside, and the inside can be closed.

Description

[Technical Field]

[0001] The present invention relates to cell technology and to a cell culture vessel and a cell culture device. [Background technology]

[0002] Embryonic stem cells (ES cells) are stem cells established from early human or mouse embryos. ES cells have the pluripotency to differentiate into all cell types present in the body. Currently, human ES cells are available for cell transplantation therapy for many diseases, including Parkinson's disease, juvenile diabetes, and leukemia. However, ES cell transplantation has its drawbacks. In particular, ES cell transplantation can trigger immune rejection similar to the rejection that occurs following unsuccessful organ transplants. Furthermore, the use of ES cells, which are established by destroying human embryos, has been criticized and opposed on ethical grounds.

[0003] Against this background, Professor Shinya Yamanaka of Kyoto University succeeded in establishing induced pluripotent stem cells (iPS cells) by introducing four genes: OCT3 / 4, KLF4, c-MYC, and SOX2 into somatic cells. This led to Professor Yamanaka receiving the Nobel Prize in Physiology or Medicine in 2012 (see, for example, Patent Documents 1 and 2). iPS cells are ideal pluripotent cells that are free from rejection and ethical issues. Therefore, iPS cells are expected to be used in cell transplantation therapy. [Prior art documents] [Patent documents]

[0004] [Patent Document 1] Patent No. 4183742 [Patent Document 2] Japanese Patent Application Laid-Open No. 2014-114997 Summary of the Invention [Problem to be solved by the invention]

[0005] There is a demand for an apparatus that can efficiently culture not only iPS cells but also various other cells. Therefore, one of the objects of the present invention is to provide a cell culture vessel and a cell culture apparatus. [Means for solving the problem]

[0006] According to an aspect of the present invention, there is provided a cell incubator for culturing cells therein, the width of the bottom surface being equal to or greater than the height of the sides, the cell incubator having a flow path for supplying a fluid to the inside, and the inside being closable.

[0007] In the cell culture vessel, at least one of the bottom surface and the top surface may be transparent.

[0008] According to an aspect of the present invention, there is provided a cell incubator for culturing cells therein, wherein at least one of the bottom surface and the top surface is transparent, the cell incubator has a flow path for supplying a fluid to the inside, and the inside can be closed.

[0009] The cell culture vessel may include a first housing having a bottom surface and a second housing placed on the first housing and having a top surface opposite the bottom surface, and the first housing and the second housing may be combined to form the interior.

[0010] In the cell culture device, the flow path may be provided in at least one of the first housing and the second housing.

[0011] The cell culture vessel may further include a temperature control unit that controls the temperature inside the cell culture vessel.

[0012] In the cell culture device, an inner culture container can be placed inside, and the flow path may supply a fluid into the inner culture container.

[0013] The cell culture vessel may further include a medium component permeable member disposed inside.

[0014] According to another aspect of the present invention, there is provided a cell culture device comprising a cell culture vessel for culturing cells therein and a variable volume container connected to the cell culture vessel, wherein the width of the bottom of the cell culture vessel is equal to or greater than the height of the sides, the cell culture vessel has a flow path for supplying fluid to the interior, the variable volume container is connected to the cell culture vessel via the flow path, fluid can move within the cell culture vessel and the variable volume container, and the interiors of the cell culture vessel and the variable volume container can be closed.

[0015] According to another aspect of the present invention, there is provided a cell culture device comprising a cell culture vessel for culturing cells therein and a variable volume container connected to the cell culture vessel, wherein at least one of the bottom and top surfaces is transparent, the cell culture vessel has a flow path for supplying a fluid to the interior, the variable volume container is connected to the cell culture vessel via the flow path, fluid can move within the cell culture vessel and the variable volume container, and the interiors of the cell culture vessel and the variable volume container can be closed.

[0016] In the cell culture device, the variable volume container may contain a substance, and the substance may come into contact with the cells by the movement of the fluid.

[0017] The cell culture device may further include a flow path for supplying cells into the cell culture vessel.

[0018] The cell culture device may further include a fluid machine for supplying cells into the cell culture vessel.

[0019] The cell culture device may further include a flow path for supplying a culture medium into the cell culture vessel.

[0020] The cell culture device may further include a flow path for supplying a cell release agent into the cell culture vessel.

[0021] The cell culture device may further include a flow path for discharging at least a portion of the cells detached from the inner surface of the cell culture vessel with the cell detachment agent to the outside of the cell culture vessel.

[0022] In the above cell culture device, at least a portion of the cells detached from the inner surface of the cell culture vessel with the cell detachment agent may be returned to the cell culture vessel.

[0023] The cell culture device may further include a flow path for supplying a cell cryopreservation solution into the cell culture vessel.

[0024] The cell culture device may further include a temperature control unit that controls the temperature inside the cell culture vessel.

[0025] In the above cell culture device, an inner culture container can be placed inside the cell culture vessel, and a culture medium may be supplied into the inner culture container.

[0026] The cell culture device may further include a medium component permeable member disposed inside the cell culture vessel. [Effects of the Invention]

[0027] According to the present invention, it is possible to provide a cell culture vessel and a cell culture device. [Brief explanation of the drawings]

[0028] [Figure 1] FIG. 1 is a schematic front view of a cell culture system according to an embodiment. [Figure 2] 1 is a schematic perspective view of a cell culture system according to an embodiment. [Figure 3] FIG. 1 is a schematic diagram of a mononuclear cell collector according to an embodiment. [Figure 4] FIG. 1 is a schematic cross-sectional view of a cell culture vessel according to an embodiment. [Figure 5] FIG. 1 is a schematic cross-sectional view of a cell culture vessel according to an embodiment. [Figure 6] FIG. 1 is a schematic cross-sectional view of a cell culture vessel according to an embodiment. [Figure 7] FIG. 1 is a schematic cross-sectional view of a cell culture vessel according to an embodiment. [Figure 8] FIG. 1 is a schematic front view of a cell culture system according to an embodiment. [Figure 9]1 is a schematic perspective view of a cell culture system according to an embodiment. [Figure 10] 1 is a micrograph of a cell aggregate according to Example 1. [Figure 11] 1 is a histogram showing the results of flow cytometry of iPS cells according to Example 1. [Figure 12] 1 shows the analysis results of fluorescence-activated cell sorting according to Example 2. [Figure 13] 1A is a micrograph of treated blood before it is placed in the mononuclear cell collector of Example 2, and FIG. 1B is a micrograph of a solution containing mononuclear cells collected from the mononuclear cell collector. [Figure 14] 10 is a graph showing the number of platelets in treated blood before it is placed in the mononuclear cell collector of Example 2, and the number of platelets in a solution containing mononuclear cells collected from the mononuclear cell collector. [Figure 15] 1A is a photograph of a culture medium containing treated blood containing platelets before being placed in the mononuclear cell collection device of Example 2, and FIG. 1B is a photograph of a culture medium containing a solution containing mononuclear cells from which platelets have been removed. [Figure 16] 10 is a micrograph of cells produced by the method for producing iPS cells according to Example 3. [Figure 17] 10 is a histogram showing the results of flow cytometry analysis of cells produced by the method for producing iPS cells according to Example 3. [Figure 18] 10 is a micrograph of cells produced by the method for producing iPS cells according to Example 4. [Figure 19] 10 is a histogram showing the results of flow cytometry analysis of cells produced by the method for producing iPS cells according to Example 4. [Figure 20] 10 is a micrograph of cells produced by the method for producing iPS cells according to Example 5. [Figure 21] 10 is a histogram showing the results of flow cytometry analysis of cells produced by the method for producing iPS cells according to Example 5. [Figure 22] 10 is a micrograph of cells produced by the method for producing iPS cells according to Example 6. [Figure 23] 10 is a histogram showing the results of flow cytometry analysis of cells produced by the method for producing iPS cells in Example 6. DETAILED DESCRIPTION OF THE INVENTION

[0029] An embodiment of the present invention will be described below. In the following description of the drawings, the same or similar parts are denoted by the same or similar reference numerals. However, the drawings are schematic. Therefore, specific dimensions and the like should be determined in light of the following description. Furthermore, it goes without saying that the dimensional relationships and ratios of parts may differ between the drawings.

[0030] As shown in FIGS. 1 and 2 , the cell culture device according to the embodiment includes a cell incubator 22 for culturing cells therein and a variable volume container 27 connected to the cell incubator 22. For example, the width of the bottom surface of the cell incubator 22 is equal to or greater than the height of the side surfaces. Here, the bottom surface refers to a surface that is approximately perpendicular to the direction of gravity. The cell incubator 22 includes a flow path 26 for supplying a fluid therein. The variable volume container 27 is connected to the cell incubator 22 via the flow path 26, allowing fluid to move through the cell incubator 22 and the variable volume container 27. The flow path 26 is connected to, for example, a side wall of the cell incubator 22. The flow path 26 may not be provided with any valves other than those for fluid machinery. The interiors of the cell incubator 22 and the variable volume container 27 can be closed. In the present disclosure, the term "fluid" includes both gases and liquids.

[0031] The cell culture device according to the embodiment includes a blood container 50 for containing blood, and an erythrocyte treatment agent container 53 for containing an erythrocyte sedimenting agent or an erythrocyte removing agent.

[0032] The blood container 50 contains blood. The blood container 50 may have a structure that allows the interior to be sealed from the outside air. The closed space including the interior of the blood container 50 may be configured to prevent exchange of gases, viruses, microorganisms, impurities, etc. with the outside. The blood container 50 may be embedded in a gas-impermeable material. At least a portion of the blood container 50 may be formed by carving into a member. At least a portion of the blood container 50 may be formed by carving into a member and overlapping recesses. The blood container 50 may have a variable volume. In this case, for example, the blood container 50 includes a syringe that contains a fluid and a plunger that is inserted into the syringe and movable within the syringe. The volume that can contain the fluid in the syringe can be changed by moving the plunger. Alternatively, the blood container 50 may be a flexible bellows or bag.

[0033] The red blood cell treatment agent container 53 contains an erythrocyte sedimentation agent or an erythrocyte removal agent. The red blood cell treatment agent container 53 may have a structure that allows the interior to be sealed off from the outside air. The closed space including the interior of the red blood cell treatment agent container 53 may be configured to prevent exchange of gases, viruses, microorganisms, impurities, etc. with the outside. The red blood cell treatment agent container 53 may be embedded in a gas-impermeable material. At least a portion of the red blood cell treatment agent container 53 may be formed by carving into a member. At least a portion of the red blood cell treatment agent container 53 may be formed by carving into a member and overlapping recesses. The red blood cell treatment agent container 53 may have a variable volume. In this case, for example, the red blood cell treatment agent container 53 includes a syringe that contains a fluid and a plunger that is inserted into the syringe and movable within the syringe, and the volume that can contain the fluid in the syringe can be changed by moving the plunger. Alternatively, the red blood cell treatment agent container 53 may be a flexible bellows or bag.

[0034] The cell culture device according to the embodiment further includes a mixer 57 that mixes, for example, blood with an erythrocyte sedimenting agent or an erythrocyte removing agent. The mixer 57 includes, for example, a meandering flow path through which a mixture of blood and an erythrocyte sedimenting agent or an erythrocyte removing agent flows. The meandering flow path may be spirally bent. The meandering flow path may meander. The cross-sectional area of ​​the meandering flow path may repeatedly increase and decrease. The mixer 57 may have a structure that allows the interior to be closed off from the outside air. The closed space including the interior of the mixer 57 may be configured to prevent exchange of gases, viruses, microorganisms, impurities, etc. with the outside. The mixer 57 may be embedded or encapsulated in a gas-impermeable material. At least a portion of the mixer 57 may be formed by being engraved into a member. At least a portion of the mixer 57 may be formed by being engraved into a member and overlapping recesses.

[0035] A flow path 51 is connected to the blood container 50 for sending at least blood from the blood container 50 to the mixer 57. The flow path 51 does not need to be provided with any valves other than those of the fluid machine. The red blood cell treatment agent container 53 is connected to a flow path 54 for sending at least an erythrocyte sedimenting agent or an erythrocyte removing agent from the erythrocyte treatment agent container 53 to the mixer 57. The flow path 54 does not need to be provided with any valves other than those of the fluid machine. The flow paths 51 and 54 merge into a flow path 56. The flow path 56 does not need to be provided with any valves other than those of the fluid machine. The flow path 56 is connected to the mixer 57. The mixer 57 is connected to a flow path 58 for sending the mixture of the blood and the erythrocyte sedimenting agent or the erythrocyte removing agent mixed in the mixer 57 into the red blood cell remover 11. The flow path 58 does not need to be provided with any valves other than those of the fluid machine.

[0036] The flow path 51 may be provided with a fluid machine 52, such as a pump, for moving the fluid within the flow path 51. A positive displacement pump can be used as the fluid machine 52. Examples of positive displacement pumps include reciprocating pumps, such as piston pumps, plunger pumps, and diaphragm pumps, and rotary pumps, such as gear pumps, vane pumps, and screw pumps. Examples of diaphragm pumps include tubing pumps and piezoelectric (piezo) pumps. Tubing pumps are sometimes called peristaltic pumps. A microfluidic chip module combining various types of pumps may also be used. The same applies to other fluid machines in this disclosure. Using a sealed pump, such as a peristaltic pump, tubing pump, or diaphragm pump, allows the pump to transport the fluid within the flow path without directly contacting the fluid within the flow path. The flow path 54 may be provided with a fluid machine 55, such as a pump, for moving the fluid within the flow path 54.

[0037] The channels 51, 54, 56, and 58 may have a structure that allows the interiors to be closed off from the outside air. The closed space including the interiors of the channels 51, 54, 56, and 58 may be configured to prevent exchange of gases, viruses, microorganisms, impurities, and the like with the outside. The channels 51, 54, 56, and 58 may be embedded or encapsulated in a gas-impermeable material. At least a portion of the channels 51, 54, 56, and 58 may be formed by being engraved into a member. At least a portion of the channels 51, 54, 56, and 58 may be formed by being engraved into a member and overlapping recesses.

[0038] When sending a mixture of blood and an erythrocyte sedimenting agent or an erythrocyte removing agent to the erythrocyte remover 11, the fluid machine 52 moves the blood in the blood container 50 into the mixer 57 via the flow paths 51 and 56. In addition, the fluid machine 55 moves the erythrocyte sedimenting agent or the erythrocyte removing agent in the erythrocyte treatment agent container 53 into the mixer 57 via the flow paths 54 and 56. It is also possible to provide a fluid machine in the flow paths 56 instead of providing a fluid machine in the flow paths 51 and 54, and the fluid machine provided in the flow path 56 moves the blood in the blood container 50 and the erythrocyte sedimenting agent or the erythrocyte removing agent in the erythrocyte treatment agent container 53 into the mixer 57. The blood and the erythrocyte sedimenting agent or the erythrocyte removing agent are mixed in the mixer 57. The mixture of blood and the erythrocyte sedimenting agent or erythrocyte removing agent mixed in the mixer 57 is sent to the erythrocyte remover 11 via a flow path 58 .

[0039] The red blood cell remover 11 may have a structure that allows the interior to be closed off from the outside air. The closed space including the interior of the red blood cell remover 11 may be configured to prevent exchange of gas, viruses, microorganisms, impurities, etc. with the outside. The red blood cell remover 11 may be embedded in a gas-impermeable substance. At least a portion of the red blood cell remover 11 may be formed by being engraved into a member. At least a portion of the red blood cell remover 11 may be formed by being engraved into a member with overlapping recesses. The volume of the red blood cell remover 11 may be changeable.

[0040] When blood is mixed with an erythrocyte sedimenting agent, erythrocytes are precipitated in the erythrocyte remover 11, and the erythrocytes are at least partially removed from the blood. When blood is mixed with an erythrocyte removing agent, erythrocytes are hemolyzed in the erythrocyte remover 11, and the erythrocytes can be at least partially removed from the blood.

[0041] The cell culture device according to the embodiment may further include a mononuclear cell collector 15 that receives treated blood from which red blood cells have been at least partially removed from the red blood cell remover 11 and collects mononuclear cells from the treated blood. The mononuclear cell collector 15 may have a structure that allows the interior to be sealed off from the outside air. The closed space including the interior of the mononuclear cell collector 15 may be configured to prevent exchange of gas, viruses, microorganisms, impurities, etc. with the outside. The mononuclear cell collector 15 may be embedded or encapsulated in a gas-impermeable material. At least a portion of the mononuclear cell collector 15 may be formed by being engraved into a member. At least a portion of the mononuclear cell collector 15 may be formed by being engraved into a member with overlapping recesses. The volume of the mononuclear cell collector 15 may be adjustable.

[0042] 3, for example, a first opening 115 is provided at the bottom of the mononuclear cell collector 15, and a second opening 116 is provided at the side of the mononuclear cell collector 15. The first opening 115 may be located below the second opening 116 in the direction of gravity.

[0043] A flow path 19 is connected to the first opening 115 of the mononuclear cell collector 15. The flow path 19 may not be provided with any valves other than the fluid machine. The flow path 19 may have a structure that allows the interior to be closed off from the outside air. The closed space including the interior of the flow path 19 may be configured to prevent exchange of gases, viruses, microorganisms, impurities, etc. with the outside. The flow path 19 may be embedded or encapsulated in a gas-impermeable material. At least a portion of the flow path 19 may be formed by being engraved into a member. At least a portion of the flow path 19 may be formed by being engraved into a member and overlapping recesses.

[0044] A flow path 117 is connected to the second opening 116 of the mononuclear cell collector 15. The flow path 117 may not be provided with any valves other than the fluid machine. The flow path 117 may have a structure that allows the interior to be closed off from the outside air. The closed space including the interior of the flow path 117 may be configured to prevent exchange of gases, viruses, microorganisms, impurities, etc. with the outside. The flow path 117 may be embedded or encapsulated in a gas-impermeable material. At least a portion of the flow path 117 may be formed by being engraved into a member. At least a portion of the flow path 117 may be formed by being engraved into a member and overlapping recesses. As shown in FIGS. 1 and 2, the flow path 117 is provided with a fluid machine 21, such as a pump, for moving a fluid within the flow path 117.

[0045] 3, the bottom of the mononuclear cell collector 15 may be funnel-shaped. In this case, for example, a first opening 115 is provided at the tip of the funnel-shaped bottom of the mononuclear cell collector 15, and a second opening 116 is provided on the side of the funnel-shaped bottom. The second opening 116 may be provided with a filter that does not allow mononuclear cells to pass through.

[0046] The mononuclear cell collector 15 can accommodate a diluent such as a buffer solution inside. As shown in FIGS. 1 and 2, the diluent may be introduced into the mononuclear cell collector 15 from a diluent container 61 that accommodates the diluent through a flow path 60. The flow path 60 does not need to be provided with any valves other than the fluid machine. The flow path 60 may be provided with a fluid machine 62 such as a pump for moving the fluid in the flow path 60. The volume of the diluent container 61 may be changeable. Furthermore, for example, the insides of the flow paths 19 and 117 are filled with the diluent.

[0047] At least one of the diluent container 61 and the flow path 60 may have a structure that allows the interior to be closed off from the outside air. The closed space including the interior of the diluent container 61 and the flow path 60 may be configured to prevent exchange of gas, viruses, microorganisms, impurities, etc. with the outside. The diluent container 61 and the flow path 60 may be embedded or encapsulated in a gas-impermeable material. At least a portion of the diluent container 61 and the flow path 60 may be formed by being engraved into a member. At least a portion of the diluent container 61 and the flow path 60 may be formed by being engraved into a member and overlapping recesses.

[0048] A flow path 17 is provided between the red blood cell remover 11 and the mononuclear cell collector 15 for sending treated blood from which red blood cells have been at least partially removed from the red blood cell remover 11 to the mononuclear cell collector 15. The flow path 17 may not be provided with any valves other than fluid machines. The flow path 17 may have a structure that allows the interior to be closed off from the outside air. The closed space including the interior of the flow path 17 may be configured to prevent exchange of gases, viruses, microorganisms, impurities, etc. with the outside. The flow path 17 may be embedded or encapsulated in a gas-impermeable material. At least a portion of the flow path 17 may be formed by carving into a member. At least a portion of the flow path 17 may be formed by carving into a member and overlapping recesses.

[0049] The flow path 17 is provided with a fluid machine 18 such as a pump for moving the fluid in the flow path 17 .

[0050] When the mononuclear cell collector 15 is filled with gas and diluent in advance, the fluid machine 18 sucks the treated blood from the red blood cell remover 11 through the flow path 17, and supplies the sucked treated blood from which the red blood cells have been at least partially removed into the mononuclear cell collector 15.

[0051] When red blood cells are sedimented in the red blood cell remover 11, the supernatant in the red blood cell remover 11 is sent to the mononuclear cell collector 15 as treated blood from which red blood cells have been at least partially removed.

[0052] The treated blood, from which red blood cells have been at least partially removed, sent to the mononuclear cell collector 15 is diluted with a diluent as shown in FIG. 3(a). In the diluted treated blood solution, platelets float, and mononuclear cells settle toward the bottom of the mononuclear cell collector 15. The diluent may contain an red blood cell removing agent. In this case, red blood cells remaining in the treated blood solution are hemolyzed. Alternatively, a red blood cell treatment agent container other than the red blood cell treatment agent container 53 may be connected to the mononuclear cell collector 15 via a flow path, and an red blood cell sedimentation agent or red blood cell removing agent may be supplied from the red blood cell treatment agent container to the mononuclear cell collector 15.

[0053] As shown in FIG. 3(b), the sedimented mononuclear cells accumulate at the tip of the funnel-shaped bottom of the mononuclear cell collector 15. After the mononuclear cells sediment in the diluted treated blood solution, as shown in FIG. 3(c), a fluid machine 21 provided in a flow path 117 connected to the second opening 116 of the mononuclear cell collector 15 aspirates the diluted treated blood solution, which is the supernatant. The suction force for aspirating the supernatant is set so as to make it difficult to aspirate the sedimented mononuclear cells. The supernatant contains platelets and hemolyzed red blood cells. Therefore, by aspirating and removing the supernatant from the mononuclear cell collector 15, the mononuclear cells can be separated from the platelets and red blood cells. The aspirated supernatant may be sent to the red blood cell remover 11 via a flow path 216 connected to the fluid machine 21 shown in FIGS. 1 and 2. The flow path 216 does not need to be provided with any valves other than the fluid machine. Alternatively, the aspirated supernatant may be sent to the second volume variable container 30, which will be described later, or to another container. Thereafter, the diluent may be supplied from the diluent container 61 to the mononuclear cell collector 15, and the supernatant may be aspirated repeatedly. Excess fluid in the red blood cell remover 11 may be sent to the second volume variable container 30, which will be described later, via the flow path 93. The flow path 93 does not need to be provided with any valves other than those for the fluid machinery.

[0054] The flow path 19 is provided with a mononuclear cell suction device 20 that suctions mononuclear cells accumulated at the bottom of the mononuclear cell collector 15. A fluid machine such as a pump can be used as the mononuclear cell suction device 20. The size of the first opening 115 shown in FIG. 3 is set, for example, so that when the mononuclear cell suction device 20 is not suctioning mononuclear cells, the mononuclear cells clog the first opening 115, but when the mononuclear cell suction device 20 is suctioning mononuclear cells, the mononuclear cells can pass through the first opening 115. When the mononuclear cell suction device 20 suctions mononuclear cells, the mononuclear cells move from inside the mononuclear cell collector 15 to the flow path 19.

[0055] The mononuclear cells in the mononuclear cell collector 15 may be moved to the flow path 19 by pressurizing the inside of the mononuclear cell collector 15. In this case, the flow path 19 may or may not be provided with a mononuclear cell suction device 20.

[0056] The cell incubator 22 for culturing cells shown in Figures 1 and 2 may have a structure that allows the interior to be closed off from the outside air, as shown in Figure 4. The closed space including the interior of the cell incubator 22 may be configured to prevent exchange of gases, viruses, microorganisms, impurities, etc. with the outside. The cell incubator 22 may be embedded or encapsulated in a gas-impermeable material. At least a portion of the cell incubator 22 may be formed by being engraved into a member. At least a portion of the cell incubator 22 may be formed by being engraved into a member and overlapping recesses.

[0057] Cells may be cultured in the cell culture vessel 22 either as adherent culture or in suspension. When cells are cultured as adherent culture, the interior of the cell culture vessel 22 may be coated with a cell adhesion coating agent such as Matrigel, collagen, polylysine, fibronectin, vitronectin, gelatin, or laminin. The following describes adherent culture as an example. The interior of the cell culture vessel 22 may be partitioned by a medium component permeable member that is impermeable to cells but allows medium components and waste products to pass through. The side walls of the cell culture vessel 22 may be coated with a cell-nonadhesive material such as poly-HEMA (poly 2-hydroxyethyl methacrylate) to prevent cells from adhering to the side walls, making the side walls nonadhesive to cells. The cell culture vessel 22 may be provided with a transparent window through which the interior can be observed. Examples of materials that can be used for the window include glass and resin. For example, a transparent window 125 may be provided on at least one of the bottom and top surfaces of the cell culture vessel 22. This allows the inside of the cell incubator 22 to be observed from the bottom side using a microscope or the like.

[0058] The cell incubator 22 may be provided with a temperature control unit for heating and cooling the window. The temperature control unit may be a transparent heater, such as a transparent conductive film, disposed on the window and heating the window. Alternatively, the cell incubator 22 may be provided with a temperature control unit for heating and cooling the housing. The temperature of the medium in the cell incubator 22 can be controlled by controlling the temperature of the housing with the temperature control unit. The cell incubator 22 may further be provided with a thermometer for measuring the temperature of the medium in the cell incubator 22. The thermometer may measure the temperature of the medium based on the temperature of the cell incubator 22 without contacting the medium, or may directly measure the temperature of the medium by contacting the medium. In this case, the temperature control unit may be feedback-controlled so that the temperature of the medium is maintained at a predetermined temperature. The temperature of the medium is controlled, for example, from 0°C to 45°C or from 20°C to 45°C.

[0059] The cell incubator 22 may be integrally molded. The cell incubator 22 may be manufactured by a 3D printer. Examples of 3D printer methods include material extrusion deposition, material jetting, binder jetting, and stereolithography. Alternatively, as shown in FIG. 5 , the cell incubator 22 may include a first housing 222 having a bottom surface and a second housing 223 disposed on the first housing 222 and having an upper surface opposite the bottom surface, and the first housing 222 and the second housing 223 may be combined to form the interior. A flow path connected to the cell incubator 22 may be provided in at least one of the first housing 222 and the second housing 223. A petri dish or the like may be placed inside the cell incubator 22 as an internal culture vessel. In this case, the flow path is configured to supply a fluid into the internal culture vessel.

[0060] As shown in FIGS. 1 and 2 , a flow path 19 is connected to the cell incubator 22. Cells are sent into the cell incubator 22 via the flow path 19. The flow path 19 is connected to, for example, the side wall of the cell incubator 22. A flow path 23 is connected to the flow path 19. The flow path 23 may not be provided with any valves other than the fluid machine. The flow path 23 may have a structure that allows the interior to be closed off from the outside air. The closed space including the interior of the flow path 23 may be configured to prevent exchange of gases, viruses, microorganisms, impurities, etc. with the outside. The flow path 23 may be embedded or encapsulated in a gas-impermeable material. At least a portion of the flow path 23 may be formed by being engraved into a member. At least a portion of the flow path 23 may be formed by engraving recesses into a member and overlapping them. The flow path 23 is provided with a fluid machine 24, such as a pump, for moving a fluid within the flow path 23.

[0061] A first culture medium container 25, which is a fluid container that contains a somatic cell culture medium such as a differentiated cell culture medium, or a stem cell culture medium suitable for iPS cells, ES cells, stem cells, etc., is connected to the flow channel 23. The culture medium may be a gel, a liquid, or a flowable solid. Examples of flowable solids include agar and temperature-sensitive gels.

[0062] When the medium is in a gel form, it may contain a polymer compound. The polymer compound may be, for example, at least one selected from the group consisting of gellan gum, deacylated gellan gum, hyaluronic acid, rhamsan gum, diutan gum, xanthan gum, carrageenan, fucoidan, pectin, pectic acid, pectinic acid, heparan sulfate, heparin, heparitin sulfate, keratosulfate, chondroitin sulfate, deltamannan sulfate, rhamnan sulfate, and salts thereof. The medium may also contain methylcellulose. By including methylcellulose, aggregation of cells is further suppressed.

[0063] Alternatively, the medium may contain at least one temperature-sensitive gel selected from poly(glycerol monomethacrylate) (PGMA), poly(2-hydroxypropyl methacrylate) (PHPMA), Poly(N-isopropylacrylamide) (PNIPAM), amine terminated, carboxylic acid terminated, maleimide terminated, N-hydroxysuccinimide (NHS) ester terminated, triethoxysilane terminated, Poly(N-isopropylacrylamide-co-acrylamide), Poly(N-isopropylacrylamide-co-acrylic acid), Poly(N-isopropylacrylamide-co-butylacrylate), Poly(N-isopropylacrylamide-co-methacrylic acid), Poly(N-isopropylacrylamide-co-methacrylic acid-co-octadecyl acrylate), and N-isopropylacrylamide.

[0064] In the present disclosure, gel-like medium or gel medium includes polymer medium.

[0065] When the cells sent from the flow channel 19 into the cell incubator 22 are mononuclear cells, which are somatic cells, a blood cell culture medium can be used as the somatic cell culture medium. The first culture medium container 25 may have a structure that allows the interior to be sealed off from the outside air. The closed space including the interior of the first culture medium container 25 may be configured to prevent exchange of gases, viruses, microorganisms, impurities, etc. with the outside. The first culture medium container 25 may be embedded in or encapsulated in a gas-impermeable material. At least a portion of the first culture medium container 25 may be formed by carving into a member. At least a portion of the first culture medium container 25 may be formed by carving into a member and overlapping recesses. The volume of the first culture medium container 25 may be changeable. In this case, for example, the first culture medium container 25 includes a syringe that contains the somatic cell culture medium and a plunger that is inserted into the syringe and movable within the syringe, and the volume of the somatic cell culture medium that can be contained in the syringe can be changed by moving the plunger. Alternatively, the first culture medium container 25 may be a flexible bellows or bag.

[0066] When mononuclear cells are sent from the mononuclear cell collector 15 to the flow path 19, the fluid machine 24 sends somatic cell culture medium from the first culture medium container 25 to the flow path 19 via the flow path 23. The first culture medium container 25 may reduce the volume capable of accommodating the somatic cell culture medium. The first culture medium container 25 may actively contract its volume, or may passively contract its volume by suction from inside the flow path 23. The somatic cell culture medium sent to the flow path 19 via the flow path 23 and the mononuclear cells in the flow path 19 are mixed and sent into the cell incubator 22. Pre-prepared mononuclear cells may also be supplied into the cell incubator 22. The cells sent to the cell incubator 22 are not limited to mononuclear cells and may be any type of cells, such as somatic cells.

[0067] At least one of the first culture medium container 25 and the flow path 23 may be provided with a temperature control device that adjusts the temperature of the culture medium in the first culture medium container 25. The fluid machine 24 may send the somatic cell culture medium from the first culture medium container 25 into the cell culture device 22 even after the cells have been sent into the cell culture device 22.

[0068] The cell incubator 22 is connected to a first variable volume container 27 via, for example, a flow path 26. The flow path 26 may have a structure that allows the interior to be closed off from the outside air. The closed space including the interior of the flow path 26 may be configured to prevent exchange of gases, viruses, microorganisms, impurities, etc. with the outside. The flow path 26 may be embedded in or encapsulated in a gas-impermeable material. At least a portion of the flow path 26 may be formed by being engraved into a member. At least a portion of the flow path 26 may be formed by being engraved into a member and overlapping recesses. The flow path 26 may be provided with a fluid machine 28 such as a pump for moving a fluid within the flow path 26.

[0069] The first volume variable container 27 may have a structure that allows the interior to be sealed off from the outside air. The closed space including the interior of the first volume variable container 27 may be configured to prevent exchange of gases, viruses, microorganisms, impurities, etc. with the outside. The first volume variable container 27 may be embedded in or encapsulated in a gas-impermeable material. At least a portion of the first volume variable container 27 may be formed by being engraved into a member. At least a portion of the first volume variable container 27 may be formed by being engraved into a member and overlapping recesses. The volume of the first volume variable container 27 may be changeable. In this case, for example, the first volume variable container 27 includes a syringe that contains a fluid and a plunger that is inserted into the syringe and movable within the syringe, and the volume that can contain the fluid in the syringe can be changed by moving the plunger. Alternatively, the first volume variable container 27 may be a flexible bellows or bag.

[0070] The second volume variable container 30 is connected to the cell incubator 22 via, for example, a flow path 29. The flow path 29 is connected to, for example, the side wall of the cell incubator 22. The flow path 29 may have a structure that allows the interior to be closed off from the outside air. The closed space including the interior of the flow path 29 may be configured to prevent exchange of gases, viruses, microorganisms, impurities, etc. with the outside. The flow path 29 may be embedded in or encapsulated in a gas-impermeable material. At least a portion of the flow path 29 may be formed by being engraved into a member. At least a portion of the flow path 29 may be formed by being engraved into a member and overlapping recesses. The flow path 29 may be provided with a fluid machine such as a pump for moving a fluid within the flow path 29. The flow path 29 may not be provided with any valves other than the fluid machine.

[0071] The second volume variable container 30 may have a structure that allows the interior to be sealed off from the outside air. The closed space including the interior of the second volume variable container 30 may be configured to prevent exchange of gases, viruses, microorganisms, impurities, etc. with the outside. The second volume variable container 30 may be embedded or encapsulated in a gas-impermeable material. At least a portion of the second volume variable container 30 may be formed by being engraved into a member. At least a portion of the second volume variable container 30 may be formed by being engraved into a member and overlapping recesses. The volume of the second volume variable container 30 may be changeable. In this case, for example, the second volume variable container 30 may include a syringe that contains a fluid and a plunger that is inserted into the syringe and movable within the syringe, and the volume that can contain the fluid in the syringe can be changed by moving the plunger. Alternatively, the second volume variable container 30 may be a flexible bellows or bag.

[0072] When somatic cells and somatic cell culture medium are sent into the cell incubator 22 from the flow path 19, gas such as air inside the cell incubator 22 moves into, for example, the second volume variable container 30, and the second volume variable container 30 expands its volume to receive the gas that has moved from inside the cell incubator 22. The second volume variable container 30 may actively expand its volume, or may passively expand its volume when subjected to pressure.

[0073] The first variable volume container 27 contains, for example, a substance such as an inducer that induces cells in a first state to become cells in a second state. The inducer may be RNA, a protein, or a chemical compound. The RNA may be modified or unmodified RNA. The first variable volume container 27 may contain, for example, a lipofection reagent. The inducer may be contained in a plasmid vector, or a viral vector or virus such as a retroviral vector, a lentiviral vector, or a Sendai virus vector, or a mixture thereof. In the present disclosure, induction refers to reprogramming, reprogramming, transformation, transdifferentiation or lineage reprogramming, differentiation induction, cell fate reprogramming, etc. Reprogramming factors include, for example, OCT3 / 4, SOX2, KLF4, and c-MYC.

[0074] When introducing an induction factor such as a reprogramming factor into somatic cells to produce iPS cells, a fluid machine 28 moves a somatic cell culture medium containing somatic cells from the cell incubator 22 into the first variable volume container 27 via the flow path 26. The first variable volume container 27 expands its volume to receive the somatic cell culture medium containing the somatic cells. The first variable volume container 27 may actively expand its volume or may passively expand its volume by receiving pressure. The second variable volume container 30, which contains gas, contracts its volume, and the gas is sent into the cell incubator 22. The second variable volume container 30 may actively contract its volume or may passively contract its volume by suction force from inside the cell incubator 22.

[0075] As the somatic cells move from inside the cell incubator 22 into the first variable volume container 27, they come into contact with the induction factors in the first variable volume container 27, and the induction factors are introduced into the somatic cells. The first variable volume container 27 may repeatedly expand and contract in volume to agitate the somatic cell culture medium containing the somatic cells and the induction factors.

[0076] A coating agent container 82, which is a fluid container that contains a coating agent for cell adhesion, such as matrigel, collagen, polylysine, fibronectin, vitronectin, gelatin, and laminin, is connected to the cell culture vessel 22 via, for example, a flow channel 81. The flow channel 81 is connected to, for example, the side wall of the cell culture vessel 22.

[0077] The flow path 81 may have a structure that allows the interior to be closed off from the outside air. The closed space including the interior of the flow path 81 may be configured to prevent exchange of gases, viruses, microorganisms, impurities, etc. with the outside. The flow path 81 may be embedded in or encapsulated in a gas-impermeable material. At least a portion of the flow path 81 may be formed by being engraved into a member. At least a portion of the flow path 81 may be formed by being engraved into a member and overlapping recesses. The flow path 81 may be provided with a fluid machine 83 such as a pump for moving a fluid within the flow path 81. The flow path 81 may not be provided with any valves other than the fluid machine.

[0078] The coating agent container 82 may have a structure that allows the interior to be sealed off from the outside air. The closed space including the interior of the coating agent container 82 may be configured to prevent exchange of gases, viruses, microorganisms, impurities, and the like with the outside. The coating agent container 82 may be embedded in a gas-impermeable material. At least a portion of the coating agent container 82 may be formed by being engraved into a member. At least a portion of the coating agent container 82 may be formed by being engraved into a member and overlapping recesses. The volume of the coating agent container 82 may be adjustable. In this case, for example, the coating agent container 82 may include a syringe that contains a fluid and a plunger that is inserted into the syringe and movable within the syringe, and the volume of the fluid that can be contained in the syringe can be adjusted by moving the plunger. Alternatively, the coating agent container 82 may be a flexible bellows or bag.

[0079] While the factors are being introduced into the cells in the first variable volume container 27, the fluid machine 83 moves the cell adhesive coating agent in the coating agent container 82 into the cell culture vessel 22 via the flow path 81. As a result, the bottom surface of the cell culture vessel 22 is covered with the cell adhesive coating agent.

[0080] When the cell adhesive coating agent is sent into the cell incubator 22 from the flow path 81, excess fluid in the cell incubator 22 moves, for example, into the second volume variable container 30, and the second volume variable container 30 expands its volume to receive the fluid that has moved from the cell incubator 22. The second volume variable container 30 may actively expand its volume, or may passively expand its volume when subjected to pressure.

[0081] A flow path 129 may be provided between the cell incubator 22 and the second variable volume container 30. The flow path 129 is connected to, for example, a side wall of the cell incubator 22. The flow path 129 may have a structure that allows the interior to be closed off from the outside air. The closed space including the interior of the flow path 129 may be configured to prevent exchange of gases, viruses, microorganisms, impurities, etc. with the outside. The flow path 129 may be embedded or encapsulated in a gas-impermeable material. At least a portion of the flow path 129 may be formed by being engraved into a member. At least a portion of the flow path 129 may be formed by being engraved into a member and overlapping recesses. The flow path 129 may be provided with a fluid machine 130 such as a pump for moving a fluid within the flow path 129. The flow path 129 may not be provided with any valves other than the fluid machine.

[0082] After the bottom surface of the cell culture vessel 22 has been covered with the cell adhesive coating agent for a predetermined time, the fluid machine 130 moves the cell adhesive coating agent in the cell culture vessel 22 into the second volume-variable container 30 via the flow path 129.

[0083] After the cell adhesive coating agent has been transferred into the second volume variable container 30, the fluid machine 28 transfers the somatic cell culture medium containing the somatic cells introduced with the inducer from the first volume variable container 27 into the cell incubator 22 via the flow path 26. The first volume variable container 27 contracts in volume. The second volume variable container 30 expands in volume to receive gas and / or liquid from inside the cell incubator 22.

[0084] If a cell adhesive coating agent is applied to the bottom surface of the cell culture vessel 22 in advance, or if a cell adhesive coating agent is placed in the cell culture vessel 22 in advance, the coating agent container 82 may be omitted.

[0085] As another example, the fluid machine 28 may transfer the induction factor in the first volume variable container 27 into the cell culture vessel 22 containing the cells via the flow path 26, without transferring the cells in the cell culture vessel 22 to the first volume variable container 27. At this time, the volume of the first volume variable container 27 may be contracted, and the volume of the second volume variable container 30 may be expanded. By transferring the induction factor from the first volume variable container 27 into the cell culture vessel 22, the induction factor comes into contact with the somatic cells in the cell culture vessel 22 and is introduced into the somatic cells. Note that the fluid machine 28 may transfer the induction factor in the first volume variable container 27 into the cell culture vessel 22 via the flow path 26 in multiple batches. This allows the induction factor to be introduced into the somatic cells in multiple batches.

[0086] A second culture medium container 32, which is a fluid container that contains a culture medium such as a stem cell culture medium or a somatic cell culture medium, is connected to the cell culture vessel 22, for example, via a flow path 31. The flow path 31 is connected to, for example, the side wall of the cell culture vessel 22. In the following, an example will be described in which the second culture medium container 32 contains a stem cell culture medium. The stem cell culture medium may be a gel, a liquid, or a flowable solid. The stem cell culture medium may contain agar or a temperature-sensitive gel. As the stem cell culture medium, an induction culture medium, an expansion culture medium, or a maintenance culture medium can be used.

[0087] The flow path 31 may have a structure that allows the interior to be closed off from the outside air. The closed space including the interior of the flow path 31 may be configured to prevent exchange of gases, viruses, microorganisms, impurities, etc. with the outside. The flow path 31 may be embedded in or encapsulated in a gas-impermeable material. At least a portion of the flow path 31 may be formed by being engraved into a member. At least a portion of the flow path 31 may be formed by being engraved into a member and overlapping recesses. The flow path 31 may be provided with a fluid machine 33 such as a pump for moving a fluid within the flow path 31. The flow path 31 may not be provided with any valves other than the fluid machine.

[0088] The second culture medium container 32 may have a structure that allows the interior to be sealed off from the outside air. The closed space including the interior of the second culture medium container 32 may be configured to prevent exchange of gases, viruses, microorganisms, impurities, etc. with the outside. The second culture medium container 32 may be embedded in a gas-impermeable material. At least a portion of the second culture medium container 32 may be formed by carving into a member. At least a portion of the second culture medium container 32 may be formed by carving into a member and overlapping recesses. The volume of the second culture medium container 32 may be adjustable. In this case, for example, the second culture medium container 32 includes a syringe that contains a fluid and a plunger that is inserted into the syringe and movable within the syringe, and the volume that can contain the fluid in the syringe can be adjusted by moving the plunger. Alternatively, the second culture medium container 32 may be a flexible bellows or bag.

[0089] At least one of the second culture medium container 32 and the flow path 31 may be provided with a temperature adjusting device that adjusts the temperature of the culture medium in the second culture medium container 32.

[0090] After a predetermined period of time has elapsed since the induction factor was introduced into the somatic cells, the fluid machine 33 transfers the stem cell medium from the second culture medium container 32 into the cell incubator 22 via the flow path 31. As shown in FIG. 6, the stem cell medium may be placed in a cell-free compartment 124 above the compartment 123 containing cells, which is separated by a medium component permeable member 122 in the cell incubator 22, in the direction of gravity. Alternatively, as shown in FIG. 7, the stem cell medium may be placed in a cell-free compartment 123 below the compartment 123 containing cells, which is separated by a medium component permeable member 122 in the cell incubator 22. In this case, cells are present in the compartment 124 above the direction of gravity. The second culture medium container 32 shown in FIGS. 1 and 2 , which has had the stem cell medium aspirated from inside, contracts in volume. The second culture medium container 32 may contract in volume actively or passively.

[0091] When stem cell culture medium is sent into the cell culture vessel 22 from the flow path 31, gas such as air and culture medium inside the cell culture vessel 22 move into the second volume variable container 30 via the flow path 29, for example, and the second volume variable container 30 expands its volume to receive the gas and culture medium that have moved from inside the cell culture vessel 22. The second volume variable container 30 may actively expand its volume, or may passively expand its volume when subjected to pressure.

[0092] The flow channel 29 may be in contact with a compartment in which cells are present and connected to a compartment in which cells are not present, among the compartments separated by the medium component permeable member in the cell culture vessel 22. Alternatively, the flow channel 29 may be in contact with a compartment in which cells are present, among the compartments separated by the medium component permeable member in the cell culture vessel 22. In this case, excess cells in the cell culture vessel 22 may be sent to the second volume variable container 30 via the flow channel 29.

[0093] Among the compartments separated by the medium component permeable member in the cell culture vessel 22, the medium in the compartment where cells are present and the medium in the compartment where cells are not present exchange medium components and waste products, for example, by osmotic pressure. Examples of the medium component permeable member that can be used include semipermeable membranes, meshes, and hollow fiber membranes. Semipermeable membranes include dialysis membranes. The medium component permeable member may be fixed inside the cell culture vessel 22 via a packing or the like. Among the compartments separated by the medium component permeable member in the cell culture vessel 22, at least one of the flow channels 19, 26, 90, and 129 may be connected to the compartment where cells are present, and at least one of the flow channels 29, 31, 81, 84, and 87 may be connected to the compartment where cells are not present. Alternatively, one or more hollow fibers may be disposed inside the cell culture vessel 22, and cells may be disposed inside the hollow fibers. In this case, for example, at least one of the flow paths 19, 26, 90, and 129 may communicate with the interior of the hollow fibers, and at least one of the flow paths 29, 31, 81, 84, and 87 may communicate with the exterior of the hollow fibers.

[0094] When the medium component permeable member is a semipermeable membrane, the molecular weight cutoff of the semipermeable membrane is, for example, 0.1 KDa or more, 10 KDa or more, or 50 KDa or more. Examples of semipermeable membranes include cellulose ester, ethyl cellulose, cellulose esters, regenerated cellulose, polysulfone, polyacrylonitrile, polymethyl methacrylate, ethylene-vinyl alcohol copolymer, polyester polymer alloy, polycarbonate, polyamide, cellulose acetate, cellulose diacetate, cellulose triacetate, cupric ammonium rayon, saponified cellulose, hemophane membrane, phosphatidylcholine membrane, and vitamin E-coated membrane.

[0095] When the medium component permeable member is a mesh, the mesh has pores smaller than the cells to be cultured in the cell culture vessel 22. The material of the mesh is, for example, resin or metal, but is not particularly limited. The surface of the medium component permeable member may be non-cell adhesive.

[0096] When the medium component permeable member is a hollow fiber membrane, the hollow fiber membrane has pores smaller than the cells cultured in the cell culture vessel 22. For example, cells may be cultured inside the hollow fiber membrane.

[0097] While cells are being cultured in the cell incubator 22, the fluid machine 33 may move the stem cell medium in the second culture medium container 32 into the cell incubator 22 via the flow path 31 at a predetermined timing. The medium may also be circulated between the second culture medium container 32 and the cell incubator 22. The second volume adjustable container 30 may expand its volume to receive the excess used stem cell medium in the cell incubator 22 due to the inflow of fresh stem cell medium. The fluid machine 33 may control the amount of medium fed or start and stop the medium feed in response to, for example, the state of the medium, the state of the cell clumps in the medium, the number of cells, the number of cell clumps, the turbidity of the medium, and changes in pH.

[0098] A detachment liquid container 85, which is a fluid container containing a cell detachment agent such as trypsin, Triple Select, Accutase, or EDTA, is connected to the cell incubator 22 via a flow path 84. The flow path 84 is connected to, for example, the side wall of the cell incubator 22.

[0099] The flow path 84 may have a structure that allows the interior to be closed off from the outside air. The closed space including the interior of the flow path 84 may be configured to prevent exchange of gases, viruses, microorganisms, impurities, etc. with the outside. The flow path 84 may be embedded in or encapsulated in a gas-impermeable material. At least a portion of the flow path 84 may be formed by being engraved into a member. At least a portion of the flow path 84 may be formed by being engraved into a member and overlapping recesses. The flow path 84 may be provided with a fluid machine 86 such as a pump for moving a fluid within the flow path 84. The flow path 84 is connected to the flow path 84. The flow path 84 may not be provided with any valves other than the fluid machine.

[0100] The remover liquid container 85 may have a structure that allows the interior to be sealed off from the outside air. The closed space including the interior of the remover liquid container 85 may be configured to prevent exchange of gases, viruses, microorganisms, impurities, etc. with the outside. The remover liquid container 85 may be embedded in a gas-impermeable material. At least a portion of the remover liquid container 85 may be formed by carving into a member. At least a portion of the remover liquid container 85 may be formed by carving into a member and overlapping recesses. The volume of the remover liquid container 85 may be adjustable. In this case, for example, the remover liquid container 85 may include a syringe that contains a fluid and a plunger that is inserted into the syringe and movable within the syringe, and the volume that can contain the fluid in the syringe can be adjusted by moving the plunger. Alternatively, the remover liquid container 85 may be a flexible bellows or bag.

[0101] At least one of the detachment liquid container 85 and the flow path 84 may be provided with a temperature regulator that regulates the temperature of the cell detachment agent in the detachment liquid container 85 .

[0102] The fluid machine 86 moves the cell release agent in the release liquid container 85 through the flow path 84 into the cell culture vessel 22. As a result, the cells adhering to the bottom surface of the cell culture vessel 22 are exposed to the cell release agent.

[0103] When the cell release agent is sent into the cell incubator 22 from the flow path 84, excess fluid in the cell incubator 22 moves, for example, into the second volume variable container 30, and the second volume variable container 30 expands its volume to accept the fluid that has moved from the cell incubator 22. The second volume variable container 30 may actively expand its volume, or may passively expand its volume when subjected to pressure.

[0104] After the cells in the cell culture vessel 22 are exposed to the cell release agent at a predetermined temperature for a predetermined time, the fluid machine 130 transfers the cell release agent from the cell culture vessel 22 into the second variable volume container 30 via the flow path 129. After a predetermined time has elapsed at a predetermined temperature, the cells are detached from the bottom surface of the cell culture vessel 22. Some or all of the detached cells in the cell culture vessel 22 may be sent to the second variable volume container 30 via the flow path 129. At least a portion of the cells sent out of the cell culture vessel 22 may be returned to the cell culture vessel 22. Then, stem cell medium is supplied from the second culture medium container 32 into the cell culture vessel 22. After the cells adhere to the bottom surface of the cell culture vessel 22 and a predetermined time has elapsed, the Sendai virus vector may be eliminated at a high temperature, such as 38°C. The introduction of factors into the cells may be repeated multiple times, such as two or three times. In this way, the cell culture vessel and cell culture device according to this embodiment can function as a cell induction container and a cell induction device. Thereafter, the cells in the cell culture vessel 22 are detached from the bottom surface of the cell culture vessel 22 .

[0105] A cryopreservation liquid container 88, which is a fluid container that contains a cell cryopreservation liquid, is connected to the cell incubator 22 via, for example, a flow path 87. The flow path 87 is connected to, for example, the side wall of the cell incubator 22.

[0106] The flow path 87 may have a structure that allows the interior to be closed off from the outside air. The closed space including the interior of the flow path 87 may be configured to prevent exchange of gases, viruses, microorganisms, impurities, etc. with the outside. The flow path 87 may be embedded in or encapsulated in a gas-impermeable material. At least a portion of the flow path 87 may be formed by being engraved into a member. At least a portion of the flow path 87 may be formed by being engraved into a member and overlapping recesses. The flow path 87 may be provided with a fluid machine 89 such as a pump for moving a fluid within the flow path 87. The flow path 87 may not be provided with any valves other than the fluid machine.

[0107] The cryopreservation liquid container 88 may have a structure that allows the interior to be sealed off from the outside air. The closed space, including the interior of the cryopreservation liquid container 88, may be configured to prevent exchange of gases, viruses, microorganisms, impurities, etc. with the outside. The cryopreservation liquid container 88 may be embedded in a gas-impermeable material. At least a portion of the cryopreservation liquid container 88 may be formed by being engraved into a member. At least a portion of the cryopreservation liquid container 88 may be formed by being engraved into a member and overlapping recesses. The cryopreservation liquid container 88 may have a variable volume. In this case, for example, the cryopreservation liquid container 88 includes a syringe that contains a fluid and a plunger that is inserted into the syringe and movable within the syringe, and the volume that can contain the fluid in the syringe can be changed by moving the plunger. Alternatively, the cryopreservation liquid container 88 may be a flexible bellows or bag.

[0108] For example, after iPS cells are produced from somatic cells introduced with an induction factor in the cell culture vessel 22, the fluid machine 89 moves the cell cryopreservation solution in the cryopreservation solution container 88 into the cell culture vessel 22 via the flow path 87. As a result, the cells in the cell culture vessel 22 are immersed in the cell cryopreservation solution.

[0109] When the cell cryopreservation solution is sent into the cell incubator 22 from the flow path 87, excess fluid in the cell incubator 22 moves, for example, into the second volume variable container 30, and the second volume variable container 30 expands its volume to receive the fluid that has moved from the cell incubator 22. The second volume variable container 30 may actively expand its volume, or may passively expand its volume when subjected to pressure.

[0110] A cell cryopreservation container 91, which is a fluid container that contains a cell cryopreservation liquid, is connected to the cell incubator 22, for example, via a flow channel 90. The flow channel 90 is connected to, for example, the side wall of the cell incubator 22.

[0111] The flow path 90 may have a structure that allows the interior to be closed off from the outside air. The closed space including the interior of the flow path 90 may be configured to prevent exchange of gases, viruses, microorganisms, impurities, etc. with the outside. The flow path 90 may be embedded in or encapsulated in a gas-impermeable material. At least a portion of the flow path 90 may be formed by being engraved into a member. At least a portion of the flow path 90 may be formed by being engraved into a member and overlapping recesses. The flow path 90 may be provided with a fluid machine 92 such as a pump for moving a fluid within the flow path 90. The flow path 90 may not be provided with any valves other than the fluid machine.

[0112] The cell cryopreservation container 91 may have a structure that allows the interior to be sealed off from the outside air. The closed space, including the interior of the cell cryopreservation container 91, may be configured to prevent exchange of gases, viruses, microorganisms, impurities, etc. with the outside. The cell cryopreservation container 91 may be embedded in a gas-impermeable material. At least a portion of the cell cryopreservation container 91 may be formed by carving into a member. At least a portion of the cell cryopreservation container 91 may be formed by carving into a member and overlapping recesses. The cell cryopreservation container 91 may have a variable volume. In this case, for example, the cell cryopreservation container 91 includes a syringe that contains a fluid and a plunger that is inserted into the syringe and movable within the syringe, and the volume that can contain the fluid in the syringe can be changed by moving the plunger. Alternatively, the cell cryopreservation container 91 may be a flexible bellows or bag.

[0113] A fluid machine 92 sends the cell cryopreservation solution containing the cells in the cell incubator 22 to a cell cryopreservation container 91. The cell cryopreservation container 91 can be removed from the flow path 90 and sealed. The cell cryopreservation container 91 is placed in, for example, a freezer.

[0114] According to this embodiment, cells, microorganisms, viruses, dust, and the like present outside the cell incubator 22 are prevented from entering the sealed cell incubator 22, thereby maintaining the cleanliness of the cell incubator 22. Therefore, the cell incubator 22 does not need to be placed in a clean room. Carbon dioxide gas, nitrogen gas, oxygen gas, and the like may or may not be supplied to the closed system in which the cells exist. When supplying gas to the closed system, the gas may be supplied, for example, via a gas exchange filter to a flow path or the like, or the gas may be supplied into the cell incubator 22.

[0115] According to the cell culture device of the embodiment, for example, cells are cultured in a completely closed system, which makes it possible to reduce the risk of cross-contamination due to cells leaking from the culture device. Furthermore, even if cells are infected with a virus such as HIV hepatitis virus, it is possible to reduce the risk of infection to the operator due to cell leakage. Furthermore, it is possible to reduce the risk of the medium in the cell culture device being contaminated by bacteria, viruses, mold, etc. in the air outside the cell culture device. Furthermore, according to the cell culture device of the embodiment, it is also possible to culture cells without using a CO2 incubator.

[0116] As described above, the present invention has been described by way of embodiments. However, the descriptions and drawings that form part of this disclosure should not be understood as limiting the present invention. Various alternative embodiments, working examples, and operational techniques will become apparent to those skilled in the art from this disclosure. For example, the cells introduced into the cell culture vessel 22 shown in FIGS. 1 and 2 are not limited to blood cells such as monocytes. The cells introduced into the cell culture vessel 22 may be stem cells, fibroblasts, neurons, retinal epithelial cells, hepatocytes, beta cells, kidney cells, mesenchymal stem cells, blood cells, megakaryocytes, T cells, chondrocytes, cardiomyocytes, muscle cells, vascular cells, epithelial cells, pluripotent stem cells, ES cells, iPS cells, or other somatic cells. The cells introduced into the cell culture vessel 22 are optional.

[0117] Furthermore, although the embodiment has been described as an example of producing iPS cells from mononuclear cells in the cell culture vessel 22, differentiated cells such as fibroblasts, neurons, retinal epithelial cells, hepatocytes, β cells, kidney cells, mesenchymal stem cells, blood cells, megakaryocytes, T cells, chondrocytes, cardiomyocytes, muscle cells, vascular cells, epithelial cells, pluripotent stem cells, ES cells, iPS cells, or other somatic cells may also be produced from stem cells in the cell culture vessel 22. The stem cells may be iPS cells, embryonic stem cells (ES cells), somatic stem cells, or other artificially induced stem cells. In this case, for example, the first variable volume container 27 contains a differentiation inducer therein. Note that cells may be cultured in the cell culture vessel 22 without inducing the cells.

[0118] Furthermore, as described above, cells prepared in advance may be supplied into the cell incubator 22. In this case, as shown in Figures 8 and 9, the cells prepared in advance may be contained in a cell container 215, and the cells in the cell container 215 may be sent to the flow path 19. Thus, it should be understood that the present invention encompasses various embodiments and the like. [Example]

[0119] Example 1 This example demonstrates that cells can be cultured in a completely closed environment without medium or gas exchange. Growth factors were added to a medium (StemSpan H3000, registered trademark, STEMCELL Technologies Inc.), and deacylated gellan gum was further added to the medium to prepare a gel medium.

[0120] Place the prepared gel medium in a 15 mL tube and add 2 × 10 5 100 blood cells were seeded. The 15 mL tube was then placed in a CO2 incubator and the blood cells (mononuclear cells) were cultured for 7 days. A Sendai virus vector carrying OCT3 / 4, SOX2, KLF4, and cMYC was then added to the gel medium at a multiplicity of infection (MOI) of 10.0, and the blood cells were infected with the Sendai virus.

[0121] After adding the Sendai virus to the gel medium, 15 mL of gelled stem cell medium (DMEM / F12 containing 20% ​​KnockOut SR (registered trademark, ThermoFisher Scientific)) was added to the gel medium, and 15 mL of the medium containing the Sendai virus-infected cells was placed in a sealable cell culture vessel, and the gel medium was poured into the cell culture vessel. The inside of the cell culture vessel was then sealed to completely prevent gas exchange between the inside and outside of the cell culture vessel.

[0122] The cells transfected with the reprogramming factors were then cultured in suspension in a cell culture vessel. Thereafter, 2 mL of gel medium in the medium holding tank 40 was replaced with 2 mL of fresh gel medium every two days.

[0123] After 15 days, the cells were observed under a microscope, and it was confirmed that ES cell-like colonies had formed, as shown in Figure 10. Furthermore, the cells were fixed with 4% paraformaldehyde, and the expression level of the cell surface antigen TRA-1-60 in the fixed cells was measured using a flow cytometer. As shown in Figure 11, over 90% were TRA-1-60 positive, confirming almost complete reprogramming. Therefore, it was demonstrated that iPS cells can be induced from somatic cells other than stem cells in a completely closed environment without medium or gas exchange.

[0124] Example 2 The blood was treated with an erythrocyte sedimenting agent to obtain treated blood from which red blood cells were at least partially removed. The treated blood was treated with antibodies to surface cell markers and analyzed by fluorescence-activated cell sorting (FACS). The results are shown in Figure 12. The treated blood contained CD3-positive cells, CD14-positive cells, CD31-positive cells, CD33-positive cells, CD34-positive cells, CD19-positive cells, CD41-positive cells, CD42-positive cells, and CD56-positive cells.

[0125] The treated blood from which red blood cells had been at least partially removed was placed in a mononuclear cell collector as shown in Figure 3, diluted with buffer, and the supernatant was removed. Mononuclear cells were then recovered from the mononuclear cell collector. As shown in Figure 13(a), the treated blood before being placed in the mononuclear cell collector contained a large number of platelets. On the other hand, as shown in Figure 13(b), the solution containing mononuclear cells recovered from the mononuclear cell collector had almost all of the platelets removed. Figure 14 shows a graph showing the number of platelets in the treated blood before being placed in the mononuclear cell collector and the number of platelets in the solution containing mononuclear cells recovered from the mononuclear cell collector per unit area.

[0126] When treated blood containing platelets before being placed in the mononuclear cell collector was placed in culture medium, it aggregated, as shown in Figure 15(a). In contrast, when a solution containing mononuclear cells collected from the mononuclear cell collector and from which platelets had been removed was placed in culture medium, it did not aggregate, as shown in Figure 15(b).

[0127] Example 3 Deacylated gellan gum was added to the blood medium to prepare a gel medium. The prepared gel medium was placed in a laminin-coated 6-well dish, and 2 × 10 5 Blood cells (monocytes) were seeded onto the 6-well dish. The 6-well dish was then placed in a CO2 incubator at 37°C and cultured for 7 days. A Sendai virus vector (CytoTune-iPS2.0, ThermoFisher Scientific) carrying OCT3 / 4, SOX2, KLF4, and cMYC was then added to the blood growth medium at a multiplicity of infection (MOI) of 5, and the blood cells were infected with the Sendai virus.

[0128] Two days after adding Sendai virus to the blood growth medium, the cells were left in the 6-well dish and the medium was replaced with 500 μL of stem cell medium (DMEM / F12 containing 20% ​​KnockOut SR (registered trademark, ThermoFisher SCIENTIFIC)) or StemFit.

[0129] Fifteen days after adding Sendai virus to the blood growth medium, the cells were observed under a microscope, confirming the formation of ES cell-like colonies, as shown in Figure 16. Furthermore, the cells were fixed with 4% paraformaldehyde, and the expression level of the cell surface antigen TRA-1-60 in the fixed cells was measured using a flow cytometer. As shown in Figure 17, the induced cells were nearly 100% TRA-1-60 positive, confirming almost complete reprogramming. Therefore, it was demonstrated that it is possible to reprogram cells by introducing reprogramming factors into cells in a cell culture vessel and culturing the cells transfected with the reprogramming factors in the same cell culture vessel.

[0130] Example 4 Deacylated gellan gum was added to the blood medium to prepare a gel medium. The prepared gel medium was placed in a laminin-coated flask, and 5 × 10 5 Blood cells (mononuclear cells) were seeded onto the plate. The plate was then placed in a CO2 incubator at 37°C and cultured for 7 days. A Sendai virus vector (CytoTune-iPS2.0, ThermoFisher Scientific) carrying OCT3 / 4, SOX2, KLF4, and cMYC was then added to the blood growth medium at a multiplicity of infection (MOI) of 5, and the blood cells were infected with the Sendai virus.

[0131] Two days after adding Sendai virus to the blood growth medium, the flask was completely filled with stem cell medium (DMEM / F12 containing 20% ​​KnockOut SR (registered trademark, ThermoFisher SCIENTIFIC)) or StemFit to ensure that no air remained in the flask, and the flask was capped to prevent gas exchange with the outside, sealing the inside of the flask to prevent the penetration of cells, microorganisms, impurities, etc.

[0132] Fifteen days after adding Sendai virus to the blood growth medium, the cells were observed under a microscope, confirming the formation of ES cell-like colonies, as shown in Figure 18. Furthermore, the cells were fixed with 4% paraformaldehyde, and the expression level of the cell surface antigen TRA-1-60 in the fixed cells was measured using a flow cytometer. As shown in Figure 19, the cells after induction were nearly 100% TRA-1-60 positive, confirming almost complete reprogramming. Therefore, it was demonstrated that it is possible to reprogram cells by introducing reprogramming factors into cells in a cell culture vessel and culturing the cells introduced with the reprogramming factors in the same closed cell culture vessel.

[0133] Example 5 Place 2 × 10 cells in liquid, non-gel-like blood growth medium into a laminin-coated 6-well dish. 5 Blood cells (monocytes) were seeded onto the 6-well dish. The 6-well dish was then placed in a CO2 incubator at 37°C and cultured for 7 days. A Sendai virus vector (CytoTune-iPS2.0, ThermoFisher Scientific) carrying OCT3 / 4, SOX2, KLF4, and cMYC was then added to the blood growth medium at a multiplicity of infection (MOI) of 5, and the blood cells were infected with the Sendai virus.

[0134] Two days after adding Sendai virus to the blood growth medium, the cells were left in the 6-well dish and the medium was replaced with 500 μL of stem cell medium (DMEM / F12 containing 20% ​​KnockOut SR (registered trademark, ThermoFisher SCIENTIFIC)) or StemFit.

[0135] Fifteen days after adding Sendai virus to the blood growth medium, the cells were observed under a microscope, confirming the formation of ES cell-like colonies, as shown in Figure 20. Furthermore, the cells were fixed with 4% paraformaldehyde, and the expression level of the cell surface antigen TRA-1-60 in the fixed cells was measured using a flow cytometer. As shown in Figure 21, after induction, nearly 100% of the cells were TRA-1-60 positive, confirming almost complete reprogramming. Therefore, it was demonstrated that it is possible to reprogram cells by introducing reprogramming factors into cells in a cell culture vessel and culturing the cells with the reprogramming factors in the same cell culture vessel.

[0136] Example 6 Place 5 x 10 cells in liquid, non-gel-like blood growth medium in a laminin-coated flask. 5 Blood cells (mononuclear cells) were seeded into the flasks. The flasks were then placed in a CO2 incubator at 37°C and cultured for 7 days. A Sendai virus vector (CytoTune-iPS2.0, ThermoFisher Scientific) carrying OCT3 / 4, SOX2, KLF4, and cMYC was then added to the blood growth medium at a multiplicity of infection (MOI) of 5, and the blood cells were infected with the Sendai virus.

[0137] Two days after adding Sendai virus to the blood growth medium, the flask was completely filled with stem cell medium (DMEM / F12 containing 20% ​​KnockOut SR (registered trademark, ThermoFisher SCIENTIFIC)) or StemFit to ensure that no air remained in the flask, and the flask was capped to prevent gas exchange with the outside, sealing the inside of the flask to prevent the penetration of cells, microorganisms, impurities, etc.

[0138] Fifteen days after adding Sendai virus to the blood growth medium, the cells were observed under a microscope, confirming the formation of ES cell-like colonies, as shown in Figure 22. Furthermore, the cells were fixed with 4% paraformaldehyde, and the expression level of the cell surface antigen TRA-1-60 in the fixed cells was measured using a flow cytometer. As shown in Figure 23, the induced cells were nearly 100% TRA-1-60 positive, confirming almost complete reprogramming. Therefore, it was demonstrated that it is possible to reprogram cells by introducing reprogramming factors into cells in a cell culture vessel and culturing the cells introduced with the reprogramming factors in the same closed cell culture vessel. [Explanation of symbols]

[0139] 11...Red blood cell remover, 15...Mononuclear cell collector, 17...Flow path, 18...Fluid machinery, 19...Flow path, 20...Mononuclear cell suction device, 21...Fluid machinery, 22...Cell culture vessel, 23...Flow path, 24...Fluid machinery, 25...Culture medium container, 26...Flow path, 27...Variable volume container, 28...Fluid machinery, 29...Flow path, 30...Variable volume container, 31...Flow path, 32...Culture medium container, 33...Fluid machinery, 40...Culture medium holding tank, 50...Blood container, 51...Flow path, 52...Fluid machinery, 53...Red blood cell treatment agent container, 54...Flow path, 55...Fluid machinery, 56...Flow path, 57... Mixer, 58...flow path, 60...flow path, 61...dilution liquid container, 62...fluid machinery, 81...flow path, 82...coating agent container, 83...fluid machinery, 84...flow path, 85...stripping liquid container, 86...fluid machinery, 87...flow path, 88...cryopreservation liquid container, 89...fluid machinery, 90...flow path, 91...cell cryopreservation container, 92...fluid machinery, 93...flow path, 115...opening, 116...opening, 117...flow path, 122...medium component permeable member, 123...compartment, 124...compartment, 129...flow path, 130...fluid machinery, 215...cell container, 216...flow path

Claims

1. A cell incubator for culturing cells therein (excluding cell incubators with variable volume); a first variable volume container connected to the cell culture vessel and containing a reprogramming factor; a second variable volume container connected to the cell incubator; Equipped with The width of the bottom surface of the cell culture vessel is equal to or greater than the height of the side surface, (i) when the fluid in the second variable volume container moves into the cell culture vessel, the volume of the second variable volume container contracts, and the fluid in the cell culture vessel moves into the first variable volume container; The volume of the first variable volume container expands, or (ii) when the fluid in the first volume variable container moves into the cell culture vessel, the volume of the first volume variable container contracts, and the fluid in the cell culture vessel moves into the second volume variable container, causing the volume of the second volume variable container to expand; the cell culture vessel is embedded with a gas-impermeable material, and the interiors of the cell culture vessel, the first variable volume container, and the second variable volume container are closable; The fluid movement brings the reprogramming factors into contact with cells in a first state, which are somatic cells other than stem cells, to induce cells in a second state, which are stem cells. Cell culture equipment.

2. A cell incubator for culturing cells therein (excluding cell incubators with variable volume); a first variable volume container connected to the cell culture vessel and containing a reprogramming factor; a second variable volume container connected to the cell incubator; Equipped with At least one of the bottom and top surfaces is transparent; (i) when the fluid in the second volume adjustable container moves into the cell culture vessel, the volume of the second volume adjustable container contracts, and the fluid in the cell culture vessel moves into the first volume adjustable container, causing the volume of the first volume adjustable container to expand; or (ii) when the fluid in the first volume variable container moves into the cell culture vessel, the volume of the first volume variable container contracts, and the fluid in the cell culture vessel moves into the second volume variable container, causing the volume of the second volume variable container to expand; the cell culture vessel is embedded with a gas-impermeable material, and the interiors of the cell culture vessel, the first variable volume container, and the second variable volume container are closable; The fluid movement brings the reprogramming factors into contact with cells in a first state, which are somatic cells other than stem cells, to induce cells in a second state, which are stem cells. Cell culture equipment.

3. The cell culture device according to claim 1 or 2, further comprising a flow path for supplying the cells into the cell culture vessel.

4. The cell culture device according to claim 1 , further comprising a fluid machine for supplying the cells into the cell culture vessel.

5. The cell culture device according to claim 1 , further comprising a flow path for supplying a culture medium into the cell culture vessel.

6. The cell culture device according to claim 1 , further comprising a flow path for supplying a cell release agent into the cell culture vessel.

7. The cell culture device according to claim 6 , further comprising a flow path for discharging at least a portion of the cells detached from the inner surface of the cell culture vessel by the cell release agent to the outside of the cell culture vessel.

8. The cell culture device according to claim 6 or 7, wherein at least a portion of the cells detached from the inner surface of the cell culture vessel by the cell release agent are returned to the cell culture vessel.

9. The cell culture device according to claim 1 , further comprising a flow path for supplying a cell cryopreservation solution into the cell culture vessel.

10. The cell culture device according to claim 1 , further comprising a temperature adjusting unit that adjusts the temperature inside the cell culture vessel.

11. The cell culture device according to claim 1 , wherein an inner culture container can be placed inside the cell culture vessel.

12. The cell culture device according to claim 1 , further comprising a medium component permeable member disposed inside the cell culture vessel.

13. The cell culture vessel is a first housing having the bottom surface; a second housing disposed on the first housing and having a top surface opposite the bottom surface; Equipped with The first housing and the second housing are combined to form the interior. The cell culture device according to any one of claims 1 to 12.

14. The cell culture device according to claim 13 , further comprising a flow path connected to the first variable volume container, the flow path being provided in at least one of the first housing and the second housing.

15. The cell culture device according to claim 13 , further comprising a flow path connected to the second variable volume container, the flow path being provided in at least one of the first housing and the second housing.

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