Cell culture device

Abstract

A cell culture device (200) is provided with: a cell culture vessel (22) for culturing cells; a first variable volume container (27) connected to the cell culture device (22); and a second variable-volume container (30) connected to the cell culture device (22), the volume of the first variable-volume container (27) being reduced and the volume of the second variable-volume container (30) being expanded when a fluid in the first variable-volume container (27) moves into the cell culture device (22).

Description

[0001] This application is a divisional application of an application with an international application date of June 10, 2020, an international application number of PCT / JP2020 / 022843, an application number entering China of 202080038395.8, and an invention name of "Red blood cell removal device, monocyte recovery device, cell culture device, cell culture system". Technical Field

[0002] The present invention relates to a cell culture device. Background Art

[0003] Embryonic stem cells (ES cells) are stem cells established from early embryos of humans and mice. ES cells have the pluripotency to differentiate into all cells existing in an organism. Currently, human ES cells can be used for cell transplantation therapy for various diseases such as Parkinson's disease, juvenile diabetes and leukemia. However, there are obstacles to the transplantation of ES cells. In particular, the transplantation of ES cells may cause the same immune rejection reaction as the rejection reaction that occurs after unsuccessful organ transplantation. In addition, there are many criticisms or objections to the use of ES cells established by destroying human embryos from an ethical point of view.

[0004] Under such circumstances, Professor Shinya Yamanaka of Kyoto University successfully established induced pluripotent stem cells (iPS cells) by introducing four genes: OCT3 / 4, KLF4, c-MYC, and SOX2 into somatic cells. As a result, Professor Yamanaka won the 2012 Nobel Prize in Physiology or Medicine (for example, see Patent Documents 1 and 2). iPS cells are ideal pluripotent cells without rejection reactions and ethical issues. Therefore, the application of iPS cells in cell transplantation therapy is expected.

[0005] Prior art literature Patent Literature Patent Document 1: Japanese Patent No. 4183742 Patent Document 2: Japanese Patent Application Publication No. 2014-114997 Summary of the invention

[0006] Technical issues iPS cells are sometimes induced from blood cells. Without being limited to the use of inducing iPS cells, a technology capable of effectively treating blood cells is desired. In addition, without being limited to iPS cells, a device capable of efficiently culturing various cells is desired. Therefore, one of the objects of the present invention is to provide a red blood cell removal device, a monocyte recovery device, a cell culture device, a cell culture system, a cell culture method, and a monocyte recovery method.

[0007] Technical Solution According to an aspect of the present invention, there is provided an erythrocyte removal device including a blood container for containing blood, an erythrocyte remover for receiving blood from the blood container and removing at least part of erythrocytes from the blood, and a flow path for at least transferring blood from the blood container to the erythrocyte remover.

[0008] In the above-mentioned red blood cell removal device, the interior of the flow path for conveying blood from the blood container to the red blood cell remover may be sealed from the outside air.

[0009] The above-mentioned red blood cell removal device may also include a monocyte recoverer and a flow path, wherein the monocyte recoverer receives processed blood obtained by at least partially removing red blood cells from the red blood cell remover and recovers monocytes from the processed blood, and the flow path is used to at least transport the processed blood obtained by at least partially removing red blood cells from the red blood cell remover to the monocyte recoverer.

[0010] In the above-mentioned red blood cell removal device, the red blood cell remover may be capable of removing internal gas.

[0011] The red blood cell removal device may further include a flow path through which the treated blood from which the red blood cells are at least partially removed flows, and the interior of the flow path through which the treated blood from which the red blood cells are at least partially removed flows may be sealed from the outside air.

[0012] In the above-mentioned red blood cell removal device, the monocyte recovery device may be capable of removing internal gas.

[0013] In the above-mentioned red blood cell removal device, the interior of the blood container and the interior of the red blood cell remover may be sealed from the outside air.

[0014] In the above-mentioned red blood cell removal device, the interior of the mononuclear cell recovery device may be sealed from the outside air.

[0015] In the above-mentioned red blood cell removal device, the closed space including the interior of the blood container and the interior of the red blood cell remover may not exchange gas with the outside.

[0016] In the above-mentioned red blood cell removal device, the blood container and the red blood cell remover may be embedded.

[0017] In the above-mentioned red blood cell removal device, at least a portion of the blood container and / or at least a portion of the red blood cell remover may be formed by carving into the member.

[0018] In the above-mentioned red blood cell removal device, a monocyte recovering device may be embedded.

[0019] In the above-mentioned red blood cell removal device, at least a part of the monocyte recovering device may be formed by carving into the member.

[0020] In the above-mentioned erythrocyte removal device, blood may be mixed with at least one of an erythrocyte sedimentation agent and an erythrocyte removing agent in the erythrocyte remover.

[0021] The erythrocyte removal device may further include an erythrocyte treating agent container containing at least one of an erythrocyte sedimentation agent and an erythrocyte removing agent, and the erythrocyte remover may receive at least one of the erythrocyte sedimentation agent and the erythrocyte removing agent from the erythrocyte treating agent container.

[0022] The erythrocyte removal device may further include a mixer for mixing the blood with at least one of the erythrocyte sedimentation agent and the erythrocyte removing agent, and the erythrocyte removal device may receive the blood mixed with at least one of the erythrocyte sedimentation agent and the erythrocyte removing agent from the mixer.

[0023] In the above-mentioned erythrocyte removal device, the mixer may include a bent flow path through which a mixed solution of blood and at least one of the erythrocyte sedimentation agent and the erythrocyte removing agent flows.

[0024] The above-mentioned red blood cell removal device may further include a flow path for transferring blood from at least the blood container to the red blood cell remover.

[0025] The above-mentioned erythrocyte removal device may include a vacuum container connected to a flow path for at least transferring blood from the blood container to the erythrocyte removal device, and capable of evacuating the interior thereof.

[0026] The erythrocyte removal device may further include an erythrocyte treating agent container containing at least one of the erythrocyte sedimentation agent and the erythrocyte removing agent, and a flow path for conveying at least one of the erythrocyte sedimentation agent and the erythrocyte removing agent from the erythrocyte treating agent container to the erythrocyte remover.

[0027] The above-mentioned red blood cell removal device may further include a fluid machine for transferring blood from at least the blood container to the red blood cell remover.

[0028] In the above-mentioned red blood cell removal device, the blood container may be capable of changing the volume of the blood container.

[0029] In the above-mentioned erythrocyte removal device, the erythrocyte remover may be capable of changing the volume of the erythrocyte remover.

[0030] In the above-mentioned red blood cell removal device, the mononuclear cell recovering device may be capable of changing the volume of the mononuclear cell recovering device.

[0031] In the above-mentioned red blood cell removing device, the red blood cell treating agent container may be capable of changing the volume of the red blood cell treating agent container.

[0032] In the above-mentioned erythrocyte removal device, erythrocytes may be precipitated in the erythrocyte remover, and a supernatant in the erythrocyte remover may be sent to the monocyte recovery device in the form of treated blood from which erythrocytes are at least partially removed.

[0033] The above-mentioned red blood cell removal device may further include a flow path for conveying the treated blood obtained by at least partially removing red blood cells from the red blood cell remover to the monocyte recovery device.

[0034] The above-mentioned red blood cell removal device may further include a fluid machine for transferring the treated blood obtained by at least partially removing red blood cells from the red blood cell remover to the monocyte recovery device.

[0035] In the above-mentioned red blood cell removal device, the treated blood from which red blood cells are at least partially removed may be diluted in the monocyte recovery device.

[0036] In the above-mentioned red blood cell removal device, the monocytes are allowed to settle in the monocyte recovery device.

[0037] In the above-mentioned red blood cell removal device, platelets can be suspended in the diluent used to treat blood.

[0038] In the above-mentioned red blood cell removing device, red blood cells may be hemolyzed by the red blood cell removing agent in the diluent for treating blood.

[0039] The red blood cell removal device may further include a diluent container that contains a diluent for diluting the treated blood from which the red blood cells are at least partially removed.

[0040] In the above-mentioned red blood cell removal device, the diluting liquid may be a buffer solution.

[0041] In the above-mentioned red blood cell removal device, the diluent container may be capable of changing the volume of the diluent container.

[0042] In the above-mentioned red blood cell removal device, the supernatant in the monocyte recovery device may be removed after the monocytes are precipitated in the monocyte recovery device.

[0043] In the above-mentioned red blood cell removal device, platelets suspended in the supernatant can be removed by removing the supernatant.

[0044] In the above-mentioned red blood cell removal device, the hemolyzed red blood cell component suspended in the supernatant can be removed by removing the supernatant.

[0045] In the above-mentioned red blood cell removal device, a first opening may be provided at the bottom of the mononuclear cell recovery device, and a second opening may be provided at a position higher than the first opening in the gravity direction.

[0046] In the above-mentioned red blood cell removal device, the bottom of the mononuclear cell recovery device may be funnel-shaped, a first opening may be provided at a front end of the funnel-shaped bottom, and a second opening may be provided at a side surface of the funnel-shaped bottom.

[0047] In the above-mentioned red blood cell removal device, when treated blood from which red blood cells are at least partially removed is introduced into the monocyte recovery device, the monocytes can be accumulated at the bottom and the supernatant can be discharged from the second opening.

[0048] In the above-mentioned red blood cell removal device, platelets suspended in the supernatant can be removed by discharging the supernatant.

[0049] In the above-mentioned erythrocyte removal device, the hemolyzed erythrocyte component suspended in the supernatant can be removed by discharging the supernatant.

[0050] The red blood cell removal device may further include a monocyte aspirating device for aspirating monocytes from the first opening.

[0051] In the above-mentioned red blood cell removal device, the size of the first opening may be set so that the monocytes block the first opening when the monocytes are not sucked by the monocyte suction device.

[0052] The above-mentioned erythrocyte removal device may further include a flow path for conveying the fluid in the erythrocyte removal device to the blood container.

[0053] The above-mentioned red blood cell removal device may further include at least one of a fluid machine for transferring blood from the blood container to the red blood cell remover and a fluid machine for transferring fluid in the red blood cell remover to the blood container.

[0054] The above-mentioned erythrocyte removal device may further include a flow path for conveying the fluid in the monocyte recovery device to the erythrocyte removal device.

[0055] The above-mentioned red blood cell removal device may further include at least one of a fluid machine for conveying the processed blood obtained by at least partially removing red blood cells from the red blood cell remover to the monocyte recoverer and a fluid machine for conveying the fluid in the monocyte recoverer to the red blood cell remover.

[0056] According to an embodiment of the present invention, a monocyte recovery device is provided, which comprises a recovery container for accommodating a solution containing monocytes, wherein the bottom of the recovery container is funnel-shaped, a first opening is provided at the front end of the funnel-shaped bottom, and a second opening is provided on the side of the funnel-shaped bottom.

[0057] In the above-mentioned monocyte recovery device, when the solution is introduced into the recovery container, the monocytes are accumulated at the front end of the funnel-shaped bottom, and the solution is discharged from the second opening.

[0058] The monocyte recovery device may further include a monocyte suction device for suctioning monocytes accumulated at the front end of the funnel-shaped bottom.

[0059] In the above-mentioned monocyte recovery device, the size of the first opening may be set such that when the monocytes are not sucked by the monocyte suction device, the monocytes block the first opening.

[0060] According to an aspect of the present invention, there is provided a cell culture apparatus including a cell culture vessel for culturing cells and a variable volume container connected to the cell culture vessel, wherein a fluid can move in the cell culture vessel and the variable volume container.

[0061] The cell culture apparatus may include at least a first volume-variable container and a second volume-variable container as the volume-variable containers.

[0062] In the above cell culture apparatus, when the fluid in the cell culture vessel moves into the first variable volume container, the volume of the first variable volume container may expand and the volume of the second variable volume container may contract.

[0063] In the above cell culture apparatus, when the fluid in the first variable volume container moves into the cell culture chamber, the volume of the first variable volume container may contract and the volume of the second variable volume container may expand.

[0064] In the above cell culture apparatus, when the fluid in the second variable volume container moves into the cell culture chamber, the volume of the second variable volume container contracts and the volume of the first variable volume container expands.

[0065] In the above cell culture apparatus, the interior of the cell culture chamber, the interior of the first variable volume container, and the interior of the second variable volume container may be sealed from the outside air.

[0066] In the above-mentioned cell culture apparatus, the cell culture vessel, the first variable volume container, and the second variable volume container may be embedded.

[0067] In the above cell culture apparatus, at least a portion of the cell culture vessel, at least a portion of the first variable volume container, and at least a portion of the second variable volume container may be formed by carving into the member.

[0068] In the above cell culture apparatus, the first variable volume container may contain a substance, and the substance may be brought into contact with the cells by movement of a fluid.

[0069] In the above cell culture device, the substance may be an induction factor, and the induction factor may be introduced into the cells by movement of a fluid.

[0070] The cell culture apparatus may further include a fluid machine for moving the fluid in the cell culture chamber to the first variable volume container.

[0071] The cell culture apparatus may further include a fluid machine for moving the fluid in the cell culture chamber to the second variable volume container.

[0072] The cell culture apparatus may further include a flow channel for supplying cells into the cell culture chamber.

[0073] The cell culture apparatus may further include a flow channel for supplying a culture solution connected to the flow channel for supplying cells into the cell culture chamber.

[0074] In the above cell culture apparatus, the cells and the culture solution may be mixed in a flow channel for supplying the cells into the cell culture vessel, and the culture solution containing the cells may be supplied into the cell culture vessel.

[0075] In the above cell culture apparatus, when the cells are introduced into the cell culture vessel from the flow channel for supplying the cells, the volume of at least one of the first variable volume container and the second variable volume container may expand.

[0076] The cell culture apparatus may further include a fluid machine for supplying cells into the cell culture vessel.

[0077] In the above cell culture device, the cells may be somatic cells or stem cells.

[0078] The cell culture apparatus may further include a fluid container for containing a fluid to be supplied into the cell culture chamber.

[0079] In the above cell culture device, the fluid may be a somatic cell culture medium or a stem cell culture medium.

[0080] In the above cell culture device, the stem cell culture medium may be an induction medium, an expansion medium or a maintenance medium.

[0081] In the above cell culture apparatus, when the fluid is supplied from the fluid container into the cell culture apparatus, the volume of at least one of the first variable volume container and the second variable volume container may expand.

[0082] The cell culture apparatus may further include a fluid machine for supplying a fluid into the cell culture chamber.

[0083] The cell culture apparatus may further include a temperature regulating unit for regulating the temperature in the cell culture chamber.

[0084] In the above-mentioned cell culture device, cells can be adhered and cultured in the cell culture vessel.

[0085] In the above-mentioned cell culture device, the inside of the cell culture vessel may be coated with a cell-adhesive coating agent.

[0086] In the above-mentioned cell culture apparatus, cells can be cultured in a suspended state in the cell culture vessel.

[0087] The cell culture apparatus may further include a hollow fiber membrane disposed in the cell culture chamber.

[0088] In the above cell culture device, cells can be cultured inside the hollow fiber membrane.

[0089] In the above cell culture apparatus, the cells in the cell culture vessel can be moved to the variable volume container.

[0090] The above-mentioned cell culture device may also include a flow path connected to the cell culture vessel and a fluid machine arranged in the flow path, the fluid machine sucks the cells in the cell culture vessel into the flow path, and returns the cells in the flow path to the cell culture vessel, thereby performing at least one of cell subculture and expansion culture.

[0091] In the above cell culture apparatus, the flow channel may have a structure for dividing the cell clusters.

[0092] According to an aspect of the present invention, there is provided a cell culture system including a monocyte collector for collecting monocytes from blood and a cell culture device for receiving monocytes from the monocyte collector.

[0093] In the cell culture system, the monocyte recovering device may receive treated blood from which red blood cells are at least partially removed, and recover monocytes from the treated blood.

[0094] The cell culture system may further include a red blood cell remover for supplying treated blood from which red blood cells are at least partially removed to the monocyte recovering device.

[0095] The cell culture system may further include a blood container for supplying blood from which red blood cells have been at least partially removed to the red blood cell remover.

[0096] The cell culture system may include a variable volume container connected to the cell culture apparatus, and when the fluid in the cell culture apparatus moves to the variable volume container, the volume of the variable volume container expands.

[0097] The above-mentioned cell culture system can have a first variable volume container connected to the cell culture device and a second variable volume container connected to the cell culture device. If the fluid in the cell culture device moves to the first variable volume container, the volume of the first variable volume container expands and the volume of the second variable volume container shrinks.

[0098] In the above cell culture system, the interior of the monocyte recoverer and the interior of the cell culture vessel may be sealed from the outside air.

[0099] In the above cell culture system, the interior of the red blood cell remover may be sealed from the outside air.

[0100] In the above cell culture system, the interior of the blood container may be sealed from the outside air.

[0101] In the above cell culture system, the interior of the first variable volume container and the interior of the second variable volume container may be sealable from the outside air.

[0102] In the above-mentioned cell culture system, a blood container, a red blood cell remover, a monocyte recoverer, and a cell culture vessel may be embedded.

[0103] In the above cell culture system, at least a portion of the blood container, at least a portion of the red blood cell remover, at least a portion of the monocyte recoverer, and at least a portion of the cell culture vessel may be formed by carving into the member.

[0104] In the above cell culture system, the first variable volume container and the second variable volume container may be embedded.

[0105] In the above cell culture system, at least a portion of the first variable volume container and at least a portion of the second variable volume container may be formed by carving into the member.

[0106] In the above cell culture system, the interior of the first variable volume container and the interior of the second variable volume container may not exchange gas with the outside.

[0107] According to an aspect of the present invention, there is provided a cell culture method, comprising introducing factors into cells in a cell culture vessel, and culturing the cells into which the factors have been introduced in the same cell culture vessel as the cell culture vessel.

[0108] In the above cell culture method, the cell culture chamber may be closed while the factor is introduced into the cells and the cells into which the factor is introduced are cultured.

[0109] In the above cell culture method, a variable volume container may be connected to the cell culture apparatus so that the fluid moves in the cell culture apparatus and the variable volume container.

[0110] In the above-mentioned cell culture method, the factors may be supplied from the variable-volume container.

[0111] In the above cell culture method, cells in the first state into which the factor has been introduced can be induced to cells in the second state in the same cell culture vessel.

[0112] In the above cell culture method, the first state may be a differentiated state, and the second state may be an undifferentiated state.

[0113] In the above cell culture method, the first state may be a dedifferentiated state, and the second state may be a differentiated state.

[0114] In the above cell culture method, the first state may be a dedifferentiated state, and the second state may be a dedifferentiated state different from the first state.

[0115] In the above-mentioned cell culture method, the cells in the first state may be somatic cells.

[0116] In the above cell culture method, the cells in the first state may be blood cells.

[0117] In the above cell culture method, the cells in the first state may be monocytes.

[0118] In the above cell culture method, the cells in the second state may be stem cells.

[0119] In the above cell culture method, the cells in the second state may be iPS cells.

[0120] In the above cell culture method, the cells in the first state may be stem cells.

[0121] In the above cell culture method, the cells in the first state may be iPS cells.

[0122] In the above cell culture method, the cells in the second state may be somatic cells.

[0123] In the above cell culture method, the cells in the first state may be somatic cells, and the cells in the second state may be somatic cells different from the cells in the first state.

[0124] In the above-mentioned cell culture method, the cells in the first state may be blood cells from which red blood cells are at least partially removed.

[0125] In the above-mentioned cell culture method, the cells in the first state may be blood cells from which platelets have been at least partially removed.

[0126] In the above-mentioned cell culture method, the factor may be a factor that induces cells in the first state to be cells in the second state.

[0127] In the above-mentioned cell culture method, the factor may be a factor that induces a specific cell state.

[0128] In the above cell culture method, the factor may be a reprogramming factor.

[0129] In the above-mentioned cell culture method, the factor may be a differentiation-inducing factor.

[0130] In the above-mentioned cell culture method, the cells into which the factor has been introduced can be recovered from the cell culture vessel, and the cells can be returned to the same cell culture vessel as the cell culture vessel to perform subculture or expansion culture on the cells.

[0131] According to the present invention, a method for recovering monocytes is provided, comprising: treating blood to produce treated blood from which red blood cells are at least partially removed; diluting the treated blood; allowing the monocytes contained in the diluted treated blood to settle; removing the supernatant of the diluted treated blood; and recovering the monocytes.

[0132] In the above-mentioned monocyte recovery method, treated blood can be prepared in an erythrocyte remover, the treated blood can be diluted, monocytes can be precipitated, and supernatant can be removed in the monocyte recovery device, and the erythrocyte remover and the monocyte recovery device can be closed.

[0133] In the above-mentioned method for recovering mononuclear cells, the blood may be treated with an erythrocyte sedimentation agent or an erythrocyte removing agent.

[0134] In the above-mentioned method for recovering monocytes, the treated blood may be diluted with a phosphate buffer.

[0135] In the above-mentioned method for recovering monocytes, the supernatant of the diluted treated blood may contain platelets.

[0136] In the above-mentioned method for recovering monocytes, red blood cells may be at least partially removed from the recovered monocytes.

[0137] In the above-mentioned method for recovering monocytes, platelets may be at least partially removed from the recovered monocytes.

[0138] Effects of the Invention According to the present invention, a red blood cell removal device, a monocyte recovery device, a cell culture device, a cell culture system, a cell culture method, and a monocyte recovery method can be provided. BRIEF DESCRIPTION OF THE DRAWINGS

[0139] Figure 1This is a schematic diagram of the cell culture system according to the first embodiment.

[0140] Figure 2 This is a schematic diagram of the monocyte collecting device according to the first embodiment.

[0141] Figure 3 This is a schematic diagram of the red blood cell removal device according to the second embodiment.

[0142] Figure 4 This is a schematic diagram of the red blood cell removal device according to the third embodiment.

[0143] Figure 5 This is a microscopic photograph of the cell cluster of Example 1.

[0144] Figure 6 This is a histogram showing the results of flow cytometry of iPS cells in Example 1.

[0145] Figure 7 This is the analysis result of fluorescence activated cell sorting in Example 2.

[0146] Figure 8 (a) is a microscope photograph of the treated blood before being placed in the monocyte recovery device of Example 2, Figure 8 (b) is a microscopic photograph of a solution containing monocytes recovered from the monocyte recoverer.

[0147] Fig. 9 This is a graph showing the number of platelets in the treated blood before being introduced into the monocyte recovering device of Example 2 and the number of platelets in the solution containing monocytes recovered from the monocyte recovering device.

[0148] Fig.10 (a) is a photograph of a culture medium to which treated blood containing platelets is added before being placed in the monocyte recovery device of Example 2. Fig.10 (b) is a photograph of a culture medium to which a solution containing monocytes from which platelets were removed was added.

[0149] Fig.11 This is a microscopic photograph of cells produced by the method for producing iPS cells in Example 3.

[0150] Fig.12 This is a histogram showing the results of analyzing cells produced by the method for producing iPS cells in Example 3 using flow cytometry.

[0151] Fig.13 This is a microscopic photograph of cells produced by the method for producing iPS cells in Example 4.

[0152] Fig.14This is a histogram showing the results of analyzing cells produced by the method for producing iPS cells in Example 4 using flow cytometry.

[0153] Fig.15 This is a microscopic photograph of cells produced by the method for producing iPS cells in Example 5.

[0154] Fig.16 This is a histogram showing the results of analyzing cells produced by the method for producing iPS cells in Example 5 using flow cytometry.

[0155] Fig.17 This is a microscopic photograph of cells produced by the method for producing iPS cells in Example 6.

[0156] Fig.18 This is a histogram showing the results of analyzing cells produced by the method for producing iPS cells in Example 6 using flow cytometry.

[0157] Explanation of symbols 10…blood container, 11…erythrocyte remover, 12…flow path, 13…flow path, 14…fluid machinery, 15…mononuclear cell recovery device, 16…flow path, 17…flow path, 18…fluid machinery, 19…flow path, 20…mononuclear cell aspiration device, 21…fluid machinery, 22…cell culture apparatus, 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, 34…flow path, 35… Variable volume container, 36 ... flow path, 37 ... fluid machinery, 38 ... flow path, 39 ... 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 ... diluent container, 70 ... vacuum container, 71 ... vacuum container, 100 ... red blood cell removal device, 101 ... red blood cell removal device, 115 ... opening, 116 ... opening, 117 ... flow path, 200 ... cell culture device DETAILED DESCRIPTION

[0158] The following is an explanation of the embodiments of the present invention. In the following description of the accompanying drawings, the same or similar parts are represented by the same or similar symbols. However, the accompanying drawings are schematic. Therefore, specific dimensions and the like should be judged by comparing with the following description. In addition, of course, the drawings also include parts with different dimensional relationships and ratios.

[0159] (First embodiment) like Figure 1 As shown, the red blood cell removal device 100 of the first embodiment includes a blood container 10 for storing blood, and a red blood cell remover 11 for receiving blood from the blood container 10 and removing at least part of red blood cells from the blood.

[0160] The blood container 10 contains blood inside. The blood container 10 may have a structure capable of sealing the inside from the outside air. The closed space including the inside of the blood container 10 may be configured not to exchange gas with the outside. The blood container 10 may be embedded and embedded in a gas impermeable substance. At least a part of the blood container 10 may be formed by carving into a member. At least a part of the blood container 10 may be formed by carving into a component and overlapping recesses. The blood container 10 may be capable of changing the volume of the blood container 10.

[0161] The erythrocyte remover 11, for example, contains an erythrocyte sedimentation agent or an erythrocyte removing agent inside. The erythrocyte remover 11 may have a structure capable of sealing the inside from the outside air. The closed space including the inside of the erythrocyte remover 11 may be configured so as not to exchange gas, viruses, microorganisms, impurities, etc. with the outside. The erythrocyte remover 11 may be embedded and embedded in a gas impermeable substance. At least a portion of the erythrocyte remover 11 may be formed by engraving into a component. At least a portion of the erythrocyte remover 11 may be formed by engraving into a component and overlapping recesses. The erythrocyte remover 11 may be capable of changing the volume of the erythrocyte remover 11.

[0162] A flow path 13 for conveying blood from the blood container 10 to the red blood cell remover 11 is provided between the blood container 10 and the red blood cell remover 11. The flow path 13 may have a structure capable of sealing the interior from the outside air. The closed space inside including the flow path 13 may be configured so as not to exchange gas, viruses, microorganisms, impurities, etc. with the outside. The flow path 13 may be embedded and embedded in a gas impermeable substance. At least a portion of the flow path 13 may be formed by engraving into a component. At least a portion of the flow path 13 may be formed by engraving into a component and overlapping recesses.

[0163] In addition, a flow path 12 for conveying a fluid such as a gas such as air from the red blood cell remover 11 to the blood container 10 is provided between the blood container 10 and the red blood cell remover 11. The flow path 12 may have a structure capable of sealing the interior from the outside air. The closed space inside including the flow path 12 may be configured so as not to exchange gas, viruses, microorganisms, impurities, etc. with the outside. The flow path 12 may be embedded and embedded in a gas impermeable substance. At least a portion of the flow path 12 may be formed by engraving into a component. At least a portion of the flow path 12 may be formed by engraving into a component and overlapping recesses.

[0164] Each of the blood container 10 and the flow paths 12 and 13 can be connected by a connector. The connector can be a sterile connector. The connector can be a needleless connector. The needleless connector can be a partition membrane type or a mechanical valve type.

[0165] The flow path 13 is provided with a fluid machine 14 such as a pump for moving the fluid in the flow path 13. It should be noted that the fluid machine may be provided in the flow path 12 or in both the flow path 12 and the flow path 13. It should be noted that in the present invention, fluid includes both gas and liquid.

[0166] As the fluid machine 14, a positive displacement pump can be used. Examples of positive displacement pumps include reciprocating pumps including piston pumps, plunger pumps and diaphragm pumps, or rotary pumps including gear pumps, vane pumps and screw pumps. Examples of diaphragm pumps include tubular pumps and piezo pumps. Tubular pumps are sometimes also referred to as peristaltic pumps. In addition, a microfluidic chip module that combines various pumps can also be used. The same is true for other fluid machines in the present invention. If a closed pump such as a peristaltic pump, a tubular pump and a diaphragm pump is used, the fluid can be transported without the pump being in direct contact with the fluid inside the flow path.

[0167] In the case where the erythrocyte remover 11 is previously filled with gas and erythrocyte sedimentation agent, when the fluid machine 14 draws blood from the blood container 10 via the flow path 13 and supplies the drawn blood to the erythrocyte remover 11, the gas in the erythrocyte remover 11 is pushed by the pressure and transported to the blood container 10 via the flow path 12. In this way, by transporting the blood in the blood container 10 to the erythrocyte remover 11 and transporting the gas in the erythrocyte remover 11 to the blood container 10, the pressures in the blood container 10 and the erythrocyte remover 11 can be equalized.

[0168] It should be noted that the fluid machine 14 may also suck the gas in the erythrocyte remover 11 via the flow path 13 and supply the sucked gas to the blood container 10. In this case, the blood in the blood container 10 is pushed by the gas pressure and transported to the erythrocyte remover 11 via the flow path 12. In this way, by removing the gas in the erythrocyte remover 11, the blood in the blood container 10 can also be transported to the erythrocyte remover 11.

[0169] The blood sent into the erythrocyte remover 11 contacts the erythrocyte sedimentation agent or erythrocyte removing agent in the erythrocyte remover 11. The fluid machine 14 can repeatedly suck the fluid from the erythrocyte remover 11 and send the fluid into the erythrocyte remover 11 to stir the blood. When the erythrocyte sedimentation agent is contained in the erythrocyte remover 11, the erythrocytes are precipitated in the erythrocyte remover 11, and the erythrocytes are at least partially removed from the blood. When the erythrocyte removing agent is contained in the erythrocyte remover 11, the erythrocytes are hemolyzed in the erythrocyte remover 11, and the erythrocytes are at least partially removed from the blood.

[0170] The red blood cell removal device 100 may further include a monocyte recovery device 15, which receives processed blood from the red blood cell removal device 11 after at least partially removing red blood cells, and recovers monocytes from the processed blood. The monocyte recovery device 15 may have a structure that can seal the interior relative to the external air. The internal closed space including the monocyte recovery device 15 may be configured not to exchange gas, viruses, microorganisms and impurities with the outside. The monocyte recovery device 15 may be embedded and embedded in a gas impermeable material. At least a portion of the monocyte recovery device 15 may be formed by engraving into a component. At least a portion of the monocyte recovery device 15 may be formed by engraving into a component and overlapping recesses. The monocyte recovery device 15 may be able to change the volume of the monocyte recovery device 15.

[0171] like Figure 2 As shown, 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 is located lower than the second opening 116 in the gravity direction.

[0172] The first opening 115 of the monocyte recovery device 15 is connected to a flow path 19. The flow path 19 may have a structure capable of sealing the interior from the outside air. The closed space inside the flow path 19 may be configured not to exchange gas, viruses, microorganisms, impurities, etc. with the outside. The flow path 19 may be embedded and embedded in a gas impermeable substance. At least a portion of the flow path 19 may be formed by engraving into a component. At least a portion of the flow path 19 may be formed by engraving into a component and overlapping recesses.

[0173] The second opening 116 of the monocyte recovery device 15 is connected to a flow path 117. The flow path 117 may have a structure capable of sealing the interior from the outside air. The closed space inside the flow path 117 may be configured so as not to exchange gas, viruses, microorganisms, impurities, etc. with the outside. The flow path 117 may be embedded and embedded in a gas impermeable material. At least a portion of the flow path 117 may be formed by carving into a component. At least a portion of the flow path 117 may be formed by carving into a component and overlapping recesses. Figure 1 As shown, a fluid machine 21 such as a pump for moving the fluid in the flow path 117 is provided in the flow path 117 .

[0174] like Figure 2 As shown, the bottom of the monocyte recovery device 15 may be funnel-shaped. In this case, for example, a first opening 115 is provided at the front end of the funnel-shaped bottom of the monocyte recovery device 15, and a second opening 116 is provided at the side of the funnel-shaped bottom. A filter that monocytes cannot pass through may be provided at the second opening 116.

[0175] The monocyte recovery device 15 can contain a diluent such as a buffer solution. Figure 1 The diluent container 61 is introduced into the mononuclear cell recovery device 15 via the flow path 60. The volume of the diluent container 61 can be changed. For example, the flow paths 19 and 117 are filled with diluent.

[0176] At least one of the dilution liquid container 61 and the flow path 60 may have a structure capable of sealing the interior from the outside air. The closed space including the dilution liquid container 61 and the flow path 60 may be configured so as not to exchange gas, viruses, microorganisms, impurities, etc. with the outside. The dilution liquid container 61 and the flow path 60 may be embedded and embedded in a gas impermeable substance. At least a portion of the dilution liquid container 61 and the flow path 60 may be formed by engraving into a component. At least a portion of the dilution liquid container 61 and the flow path 60 may be formed by engraving into a component and overlapping recesses.

[0177] A flow path 17 for conveying the processed blood obtained by at least partially removing red blood cells from the red blood cell remover 11 to the monocyte recoverer 15 is provided between the red blood cell remover 11 and the monocyte recoverer 15. The flow path 17 may have a structure capable of sealing the interior relative to the outside air. The closed space inside including the flow path 17 may be configured so as not to exchange gas, viruses, microorganisms, impurities, etc. with the outside. The flow path 17 may be embedded and embedded in a gas impermeable substance. At least a portion of the flow path 17 may be formed by engraving into a component. At least a portion of the flow path 17 may be formed by engraving into a component and overlapping recesses.

[0178] In addition, a flow path 16 for conveying fluids such as gases such as air from the monocyte recoverer 15 to the red blood cell remover 11 is provided between the red blood cell remover 11 and the monocyte recoverer 15. The flow path 16 may have a structure capable of sealing the interior relative to the outside air. The internal closed space including the flow path 16 may be configured not to exchange gases, viruses, microorganisms, impurities, etc. with the outside. The flow path 16 may be embedded and embedded in a gas impermeable substance. At least a portion of the flow path 16 may be formed by engraving into a component. At least a portion of the flow path 16 may be formed by engraving into a component and overlapping recesses.

[0179] The flow path 17 is provided with a fluid machine 18 such as a pump for moving the fluid in the flow path 17. It should be noted that the fluid machine may be provided in the flow path 16, or in both the flow path 16 and the flow path 17.

[0180] In the case where the monocyte remover 15 is filled with gas and diluent in advance, if the fluid machine 18 draws the processed blood from which the red blood cells are at least partially removed in the red blood cell remover 11 through the flow path 17 and supplies the drawn processed blood from which the red blood cells are at least partially removed to the monocyte remover 15, the gas in the monocyte remover 15 is pushed by the pressure and transported to the red blood cell remover 11 through the flow path 16. In this way, by transporting the processed blood from which the red blood cells are at least partially removed in the red blood cell remover 11 to the monocyte remover 15 and by transporting the gas in the monocyte remover 15 to the red blood cell remover 11, the pressures in the red blood cell remover 11 and the monocyte remover 15 can be averaged. The diluent can be repeatedly supplied from the diluent container 61.

[0181] It should be noted that the fluid machine 18 can suck the gas in the monocyte collector 15 via the flow path 17, and supply the sucked gas to the erythrocyte remover 11. In this case, the processed blood obtained by at least partially removing the erythrocytes in the erythrocyte remover 11 is pushed by the gas pressure and transported to the monocyte remover 15 via the flow path 16. In this way, by removing the gas in the monocyte remover 15, the processed blood obtained by at least partially removing the erythrocytes in the erythrocyte remover 11 can also be transported to the monocyte remover 15.

[0182] When the erythrocytes are precipitated in the erythrocyte remover 11 , the supernatant in the erythrocyte remover 11 is sent to the monocyte recovery device 15 in the form of treated blood from which the erythrocytes are at least partially removed.

[0183] The processed blood from which the red blood cells are at least partially removed and which is sent to the mononuclear cell recovery device 15 is as follows: Figure 2 As shown in (a), the treated blood solution is diluted with a diluent. In the diluted treated blood solution, platelets are suspended and monocytes settle to the bottom of the monocyte recovery device 15. It should be noted that the diluent may contain a red blood cell removing agent. In this case, the red blood cells remaining in the treated blood solution are hemolyzed.

[0184] like Figure 2 As shown in (b), the precipitated monocytes are accumulated at the front end of the funnel-shaped bottom of the monocyte recovery device 15. After precipitation in the diluted treated blood solution, as shown in Figure 2 As shown in (c), the flow path 117 connected to the second opening 116 of the monocyte recovery device 15 is provided. Figure 1 The fluid machine 21 shown sucks the diluted treated blood solution as the supernatant. The suction force for sucking the supernatant is set to be difficult to suck. Figure 2 The supernatant contains platelets and hemolyzed erythrocytes. Therefore, by removing the supernatant by suction from the monocyte recovery device 15, the monocytes can be separated from the platelets and erythrocytes. The supernatant thus sucked can be transferred to Figure 1 The erythrocyte remover 11 or the blood container 10 is shown. In addition, a gas having a volume equivalent to that of the supernatant sucked from the monocyte recovering device 15 can be transported from the erythrocyte remover 11 or the blood container 10 to the monocyte recovering device 15.

[0185] The flow path 19 is provided with a monocyte suction device 20 for suctioning monocytes accumulated at the bottom of the monocyte recovery device 15. As the monocyte suction device 20, a fluid machine such as a pump can be used. Figure 2 The size of the first opening 115 shown is set, for example, in the following manner: when the monocyte aspirating device 20 does not aspirate monocytes, the monocytes are blocked in the first opening 115, and when the monocyte aspirating device 20 aspirates monocytes, the monocytes can pass through the first opening 115. When the monocyte aspirating device 20 aspirates monocytes, the monocytes move from the monocyte collecting device 15 to the flow path 19.

[0186] It should be noted that the monocytes in the monocyte recovering device 15 may be moved to the flow path 19 by pressurizing the inside of the monocyte recovering device 15. In this case, the monocyte aspirating device 20 may or may not be provided in the flow path 19.

[0187] like Figure 1As shown, the cell culture device 200 of the first embodiment includes a cell culture vessel 22 for culturing cells. The cell culture vessel 22 may have a structure capable of sealing the interior from the outside air. The closed space including the interior of the cell culture vessel 22 may be configured so as not to exchange gas, viruses, microorganisms, impurities, etc. with the outside. The cell culture vessel 22 may be embedded and embedded in a gas impermeable substance. At least a portion of the cell culture vessel 22 may be formed by engraving into a component. At least a portion of the cell culture vessel 22 may be formed by engraving into a component and overlapping recesses.

[0188] In the cell culture vessel 22, cells can be subjected to adhesion culture or suspension culture. In the case of adhesion culture of cells, the inside of the cell culture vessel 22 can be coated with a cell adhesion coating agent such as matrix glue, collagen, polylysine, fibronectin, vitronectin and laminin. The following is an explanation using suspension culture as an example. The inside of the cell culture vessel 22 can be divided by a medium component permeable member that is impermeable to cells but permeable to medium components and waste. The inner wall of the cell culture vessel 22 can also be coated with a cell non-adhesive substance such as poly-HEMA (poly 2-hydroxyethyl methacrylate: poly (methacrylate-2-hydroxyethyl ester)) in a cell non-adhesive manner, so that the inner wall of the cell culture vessel 22 becomes cell non-adhesive. A window that can observe the inside can also be provided in the cell culture vessel 22. As the material of the window, for example, glass and resin can be used.

[0189] A temperature regulating unit for heating and cooling the window may be provided in the cell culture vessel 22. The temperature regulating unit may be a transparent heater such as a transparent conductive film that is arranged on the window and heats the window. Alternatively, the cell culture vessel 22 may be provided with a temperature regulating unit for heating and cooling the frame. By regulating the temperature of the frame using the temperature regulating unit, the temperature of the culture medium in the cell culture vessel 22 can be regulated. The cell culture vessel 22 may also be provided with a thermometer for measuring the temperature of the culture medium in the cell culture vessel 22. The thermometer may measure the temperature of the culture medium based on the temperature of the cell culture vessel 22 without contacting the culture medium, or may directly measure the temperature of the culture medium by contacting the culture medium. In this case, the temperature regulating unit may also be feedback-controlled so that the temperature of the culture medium becomes a predetermined temperature. The temperature of the culture medium is adjusted to, for example, 20°C to 45°C.

[0190] The cell culture vessel 22 is connected to a flow path 19. Cells are transported into the cell culture vessel 22 via the flow path 19. The flow path 23 is connected to the flow path 19. The flow path 23 may have a structure capable of sealing the interior from the outside air. The closed space including the interior of the flow path 23 may be constructed so as not to exchange gas, viruses, microorganisms, impurities, etc. with the outside. The flow path 23 may be embedded and embedded in a gas impermeable substance. At least a portion of the flow path 23 may be formed by engraving into a component. At least a portion of the flow path 23 may be formed by engraving into a component and overlapping recesses. A fluid machine 24 such as a pump for moving the fluid in the flow path 23 is provided in the flow path 23.

[0191] A first culture medium container 25, which is a fluid container for containing a somatic cell culture medium such as a differentiation cell culture medium, is connected to the flow path 23. The somatic cell culture medium may be a gel or a liquid.

[0192] When the culture medium is gelatinous, the culture medium may contain a polymer compound. The polymer compound may be, for example, at least one selected from gellan gum, deacylated gellan gum, hyaluronic acid, neutral gum (Rhamsan gum), diutangum (diutangum), xanthan gum, carrageenan, fucoidan, pectin, pectic acid, pectinic acid, heparan sulfate, heparan, heparan sulfate, keratan sulfate, chondroitin sulfate, dermatan sulfate, rhamnosyl sulfate and their salts. In addition, the culture medium may contain methylcellulose. By containing methylcellulose, the aggregation of cells is further suppressed.

[0193] Alternatively, the culture medium may comprise a member selected from the group consisting of poly(glycerolmonomethacrylate) (PGMA), poly(2-hydroxypropylmethacrylate) (PHPMA), poly(N-isopropylacrylamide) (PNIPAM), amine terminated, carboxylic acid terminated, maleimide terminated, N-hydroxysuccinimide ester terminated, triethoxysilane terminated, poly(N-isopropylacrylamide-co-acrylamide), poly(N-isopropylacrylamide-co-acrylic acid) and poly(N-isopropylacrylamide-co-acrylic acid). A small amount of temperature-sensitive gel selected from poly (N-isopropylacrylamide-co-butylacrylate), poly (N-isopropylacrylamide-co-methacrylic acid), poly (N-isopropylacrylamide-co-methacrylic acid), poly (N-isopropylacrylamide-co-methacrylic acid-co-octadecyl acrylate) and N-isopropylacrylamide.

[0194] It should be noted that, in the present invention, the gel-like culture medium or gel culture medium includes a polymer culture medium.

[0195] In the case where the cells transported from the flow path 19 to the cell culture device 22 are mononuclear cells as somatic cells, a blood cell culture medium, for example, can be used as the somatic cell culture medium. The first culture medium container 25 may have a structure capable of sealing the interior relative to the outside air. The closed space including the interior of the first culture medium container 25 may be configured so as not to exchange gas, viruses, microorganisms, impurities, etc. with the outside. The first culture medium container 25 may be embedded and embedded in a gas impermeable substance. At least a portion of the first culture medium container 25 may be formed by engraving into a component. At least a portion of the first culture medium container 25 may be formed by engraving into a component and overlapping recesses. The first culture medium container 25 may be capable of changing the volume of the first culture medium container 25. In this case, for example, the first culture medium container 25 includes a syringe for accommodating a somatic cell culture medium and a plunger inserted into the syringe and capable of moving in the syringe, and the volume of the syringe capable of accommodating a somatic cell culture medium can be changed by moving the plunger. Alternatively, the first culture medium container 25 may also be a bellows or bag having flexibility.

[0196] If monocytes are transported from the monocyte recovery device 15 to the flow path 19, the fluid machine 24 transports the 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 reduces the volume that can accommodate the somatic cell culture medium. It should be noted that the first culture medium container 25 can actively shrink the volume, or passively shrink the volume by using the suction force from the inside of the flow path 23. The somatic cell culture medium transported to the flow path 19 via the flow path 23 is mixed with the monocytes in the flow path 19 and transported to the cell culture device 22.

[0197] The first culture medium container 25 may be provided with a temperature adjustment device for adjusting the temperature of the culture medium in the first culture medium container 25 .

[0198] The cell culture device 22 is connected to a first variable volume container 27, for example, via a flow path 26. The flow path 26 may have a structure capable of sealing the interior from the outside air. The closed space inside the flow path 26 may be configured so as not to exchange gas, viruses, microorganisms, impurities, etc. with the outside. The flow path 26 may be embedded and embedded in a gas impermeable substance. At least a portion of the flow path 26 may be formed by engraving into a component. At least a portion of the flow path 26 may be formed by engraving into a component and overlapping recesses. A fluid machine 28 such as a pump for moving the fluid in the flow path 26 may be provided in the flow path 26.

[0199] The first variable volume container 27 may have a structure capable of sealing the interior relative to the outside air. The closed space including the interior of the first variable volume container 27 may be configured so as not to exchange gas, virus, microorganism, impurity, etc. with the outside. The first variable volume container 27 may be embedded and embedded in a gas impermeable substance. At least a portion of the first variable volume container 27 may be formed by carving into a component. At least a portion of the first variable volume container 27 may be formed by carving into a component and overlapping recesses. The first variable volume container 27 may be capable of changing the volume of the first variable volume container 27. In this case, for example, the first variable volume container 27 includes a syringe for containing a fluid and a plunger inserted into the syringe and movable in the syringe, and the volume of the syringe capable of containing the fluid can be changed by moving the plunger. Alternatively, the first variable volume container 27 may also be a bellows or bag having flexibility.

[0200] The cell culture device 22 is connected to a second variable volume container 30, for example, via a flow path 29. The flow path 29 may have a structure capable of sealing the interior from the outside air. The closed space including the interior of the flow path 29 may be configured so as not to exchange gas, viruses, microorganisms, impurities, etc. with the outside. The flow path 29 may be embedded in and embedded in a gas impermeable substance. At least a portion of the flow path 29 may be formed by engraving into a component. At least a portion of the flow path 29 may be formed by engraving into a component and overlapping recesses. A fluid machine such as a pump for moving the fluid in the flow path 29 may be provided in the flow path 29.

[0201] The second variable volume container 30 may have a structure capable of sealing the interior relative to the outside air. The closed space including the interior of the second variable volume container 30 may be configured not to exchange gas, virus, microorganism and impurities with the outside. The second variable volume container 30 may be embedded and embedded in a gas impermeable substance. At least a portion of the second variable volume container 30 may be formed by engraving into a component. At least a portion of the second variable volume container 30 may be formed by engraving into a component and overlapping recesses. The second variable volume container 30 may be capable of changing the volume of the second variable volume container 30. In this case, for example, the second variable volume container 30 is provided with a syringe containing a fluid and a plunger inserted into the syringe and movable in the syringe, and the volume in the syringe that can contain the fluid can be changed by moving the plunger. Alternatively, the second variable volume container 30 may also be a bellows or bag with flexibility.

[0202] If the mononuclear cell and somatic cell culture medium is introduced into the cell culture vessel 22 from the flow path 19, the gas such as air in the cell culture vessel 22 moves to the second variable volume container 30, and the second variable volume container 30 expands its volume to receive the gas moved from the cell culture vessel 22. It should be noted that the second variable volume container 30 can actively expand its volume or passively expand its volume by receiving pressure.

[0203] The first variable volume container 27, for example, contains induction factors and other substances such as factors that induce cells in the first state to cells in the second state. The induction factor can be RNA, protein, or compound. RNA can be modified RNA or non-modified RNA. The first variable volume container 27 can contain, for example, a lipid transfection reagent. The induction factor can 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. In the present invention, induction refers to reprogramming, initialization, conversion, transdifferentiation (Transdifferentiation or Lineage reprogramming: transdifferentiation or lineage reprogramming), differentiation induction, and cell fate change (Cell fatereprogramming), etc. Reprogramming factors include, for example, OCT3 / 4, SOX2, KLF4, and c-MYC. When induction factors such as reprogramming factors are introduced into monocytes to make iPS cells, the fluid machine 28 moves the somatic cell culture medium containing monocytes in the cell culture device 22 to the first variable volume container 27 via the flow path 26. In addition, the first variable volume container 27 expands its volume and receives the somatic cell culture medium containing monocytes. It should be noted that the first variable volume container 27 can actively expand its volume, or passively expand its volume by receiving pressure. The second variable volume container 30 containing gas shrinks its volume, and the contained gas is sent to the cell culture device 22. It should be noted that the second variable volume container 30 can actively shrink its volume, or passively shrink its volume by the suction force from the inside of the cell culture device 22.

[0204] The monocytes move from the cell culture vessel 22 into the first variable volume container 27, and come into contact with the induction factor in the first variable volume container 27, thereby introducing the induction factor into the monocytes. It should be noted that the first variable volume container 27 can repeatedly expand and contract its volume to stir the somatic cell culture medium containing the monocytes and the induction factor.

[0205] After a predetermined period of time, the fluid machine 28 moves the somatic cell culture medium containing the mononuclear cells introduced with the induction factor in the first variable volume container 27 into the cell culture apparatus 22 via the flow path 26. The first variable volume container 27 contracts in volume. In addition, the second variable volume container 30 expands in volume and receives gas from the cell culture apparatus 22.

[0206] Alternatively, when introducing induction factors such as reprogramming factors into monocytes to produce iPS cells, the fluid machinery 28 can move the induction factors in the first variable volume container 27 into the cell culture vessel 22 via the flow path 26. At this time, the first variable volume container 27 can shrink the volume, and the second variable volume container 30 can expand the volume. The induction factor moves from the first variable volume container 27 into the cell culture vessel 22, thereby contacting the monocytes in the cell culture vessel 22, and introducing the induction factor into the monocytes. It should be noted that the fluid machinery 28 can also move the induction factor in the first variable volume container 27 into the cell culture vessel 22 in multiple times via the flow path 26. Thus, the induction factor is introduced into the monocytes in multiple times.

[0207] The cell culture device 22 is connected to a second culture medium container 32 as a fluid container for containing a culture medium such as a stem cell culture medium or a somatic cell culture medium, for example, via a flow path 31. Hereinafter, an example in which the second culture medium container 32 contains a stem cell culture medium will be described. The stem cell culture medium may be a gel or a liquid. As the stem cell culture medium, an induction culture medium, an expansion culture medium, and a maintenance culture medium may be used.

[0208] The flow path 31 may have a structure capable of sealing the interior from the outside air. The closed space including the interior of the flow path 31 may be configured so as not to exchange gas, viruses, microorganisms, impurities, etc. with the outside. The flow path 31 may be embedded and embedded in a gas impermeable substance. At least a portion of the flow path 31 may be formed by carving into a component. At least a portion of the flow path 31 may be formed by carving into a component and overlapping recesses. A fluid machine 33 such as a pump for moving the fluid in the flow path 31 may be provided in the flow path 31.

[0209] The second culture medium container 32 may have a structure capable of sealing the interior from the outside air. The closed space including the interior of the second culture medium container 32 may be configured so as not to exchange gas, virus, microorganism, impurity, etc. with the outside. The second culture medium container 32 may be embedded and embedded in a gas impermeable substance. At least a portion of the second culture medium container 32 may be formed by carving into a component. At least a portion of the second culture medium container 32 may be formed by carving into a component and overlapping recesses. The second culture medium container 32 may be capable of changing the volume of the second culture medium container 32. In this case, for example, the second culture medium container 32 includes a syringe for containing a fluid and a plunger inserted into the syringe and movable in the syringe, and the volume in the syringe that can contain the fluid can be changed by moving the plunger. Alternatively, the second culture medium container 32 may also be a flexible bellows or bag.

[0210] The second culture medium container 32 may be provided with a temperature adjustment device for adjusting the temperature of the culture medium in the second culture medium container 32 .

[0211] The cell culture device 22 is connected to the third variable volume container 35, for example, via the flow path 34. The flow path 34 may have a structure capable of sealing the interior from the outside air. The closed space inside including the flow path 34 may be configured so as not to exchange gas, viruses, microorganisms, impurities, etc. with the outside. The flow path 34 may be embedded and embedded in a gas impermeable substance. At least a portion of the flow path 34 may be formed by engraving into a component. At least a portion of the flow path 34 may be formed by engraving into a component and overlapping recesses.

[0212] The third variable volume container 35 may have a structure capable of sealing the interior relative to the outside air. The closed space including the interior of the third variable volume container 35 may be configured not to exchange gas, virus, microorganisms and impurities with the outside. The third variable volume container 35 may be embedded and embedded in a gas impermeable material. At least a portion of the third variable volume container 35 may be formed by engraving into a component. At least a portion of the third variable volume container 35 may be formed by engraving into a component and overlapping recesses. The third variable volume container 35 may be capable of changing the volume of the third variable volume container 35. In this case, for example, the third variable volume container 35 is provided with a syringe containing a fluid and a plunger inserted into the syringe and movable in the syringe, and the volume of the fluid contained in the syringe can be changed by the movement of the plunger. Alternatively, the third variable volume container 35 may also be a bellows or bag having flexibility.

[0213] After a predetermined period of time has passed since the induction factor was introduced into the monocytes, the fluid machine 33 moves the stem cell culture medium in the second culture medium container 32 into the cell culture vessel 22 via the flow path 31. The stem cell culture medium can contact the partition in which cells exist among the partitions divided by the culture medium component permeable member in the cell culture vessel 22, and be added to the partition in which cells do not exist. The second culture medium container 32 from which the stem cell culture medium is sucked shrinks its volume. It should be noted that the second culture medium container 32 can shrink its volume actively or passively. The third variable volume container 35 expands its volume and receives the remaining fluid in the cell culture vessel 22 due to the inflow of the stem cell culture medium through the flow path 34. The flow path 34 can contact the partition in which cells exist among the partitions divided by the culture medium component permeable member in the cell culture vessel 22, and be connected to the partition in which cells do not exist. It should be noted that the third variable volume container 35 can expand its volume actively or passively under pressure.

[0214] Alternatively, the flow path 34 may be in contact with a partition in which cells exist among the partitions partitioned by the medium component permeable member in the cell culture vessel 22. In this case, the remaining cells in the cell culture vessel 22 may be sent to the third variable volume container 35 via the flow path 34.

[0215] The culture medium in the partitions divided by the culture medium component permeation member in the cell culture apparatus 22, where the cells are present, and the culture medium in the partitions where the cells are not present, exchange culture medium components and wastes, for example, by osmotic pressure. As the culture medium component permeation member, for example, a semipermeable membrane, a mesh, and a hollow fiber membrane can be used. The semipermeable membrane includes a dialysis membrane.

[0216] When the culture 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. The semipermeable membrane is formed of, for example, 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, cuprammonium rayon, saponified cellulose, hemoform membrane, phosphatidylcholine membrane, vitamin E coating, etc.

[0217] When the culture component permeable member is a mesh, the mesh has pores smaller than the cells cultured in the cell culture chamber 22. The material of the mesh is, for example, resin and metal, but is not particularly limited. The surface of the culture component permeable member may be cell non-adhesive.

[0218] When the culture component permeating member is a hollow fiber membrane, the hollow fiber membrane has pores smaller than those of the cells cultured in the cell culture vessel 22. For example, the cells can be cultured inside the hollow fiber membrane.

[0219] During the cell culture in the cell culture apparatus 22, at a predetermined timing, the fluid machine 33 moves the stem cell culture medium in the second culture medium container 32 into the cell culture apparatus 22 via the flow path 31. The third variable volume container 35 expands its volume to receive the remaining used stem cell culture medium that flows in through fresh stem cell culture medium. The fluid machine 33 can control the amount of culture medium delivered, or start and end the delivery of the culture medium, for example, based on the state of the culture medium, the state of the cell clusters in the culture medium, the number of cells, the number of cell clusters, the turbidity of the culture medium, and the pH change.

[0220] The partitions in which cells can exist in the partitions divided by the medium component permeable member in the cell culture device 22 are connected to a fluid machine 37 such as a pump via a flow path 36. The flow path 36 can have a structure that can seal the inside relative to the outside air. The closed space inside including the flow path 36 can be configured not to exchange gas, viruses, microorganisms, impurities, etc. with the outside. The flow path 36 can be embedded and embedded in a gas impermeable material. At least a portion of the flow path 36 can be formed by engraving into a member. At least a portion of the flow path 36 can be formed by engraving into a component and overlapping the recesses.

[0221] For example, in order to control cell aggregation, the fluid machine 37 circulates the culture medium between the partitions in the cell culture vessel 22 where cells exist and the flow path 36. The fluid machine 37 can circulate the culture medium all the time or at any time. Alternatively, the fluid machine 37 can stir the culture medium by reciprocating between the partitions in the cell culture vessel 22 where cells exist and the flow path 36. The fluid machine 37 can stir the culture medium all the time or at any time. For example, the fluid machine 37 can control the amount of culture medium delivered, or start and end the delivery of the culture medium, according to the state of the culture medium, the state of cell clusters in the culture medium, the number of cells, the number of cell clusters, the turbidity of the culture medium, and the pH change.

[0222] By aspirating the cells in the cell culture vessel 22 into the flow channel 36 and returning the cells to the cell culture vessel 22, the cells can be subcultured and expanded. The flow channel 36 may have a structure for dividing the cell clusters. For example, the flow channel 36 may have a meandering structure or a structure with increasing and decreasing diameters, so that the cell clusters flowing in the flow channel 36 can be divided.

[0223] The partitions in which cells do not exist in the partitions divided by the medium component permeable member in the cell culture device 22 may be connected to a fluid machine 39 such as a pump via a flow path 38. The flow path 38 may have a structure capable of sealing the interior from the outside air. The internal closed space including the flow path 38 may be configured so as not to exchange gas, viruses, microorganisms, impurities, etc. with the outside. The flow path 38 may be embedded and embedded in a gas impermeable material. At least a portion of the flow path 38 may be formed by engraving into a member. At least a portion of the flow path 38 may be formed by engraving into a component and overlapping recesses.

[0224] For example, in order to increase the chance of the culture medium contacting the culture medium component permeable member, the fluid machine 39 circulates between the partitions in the culture medium cell culture vessel 22 divided by the culture medium component permeable member where no cells exist and the flow path 38. The fluid machine 39 can circulate the culture medium all the time or at any time. Alternatively, the fluid machine 39 can reciprocate the culture medium between the partitions in the cell culture vessel 22 divided by the culture medium component permeable member where no cells exist and the flow path 38 to stir the culture medium. The fluid machine 39 can stir the culture medium all the time or at any time. For example, the fluid machine 39 can control the amount of culture medium delivered, or start and end the delivery of the culture medium, according to the state of the culture medium, the state of the cell clusters in the culture medium, the number of cells, the number of cell clusters, the turbidity of the culture medium, and the pH changes.

[0225] For example, iPS cells are prepared from monocytes into which induction factors have been introduced in the cell culture vessel 22, and after expansion culture, the iPS cells are collected from the cell culture vessel 22. The iPS cells can form cell clusters (colonies) in the cell culture vessel 22.

[0226] According to the view of the inventors, cells can be cultured in a completely closed, sealed space, so it is not necessary to actively supply carbon dioxide gas, nitrogen gas, oxygen, etc. to the cell culture vessel 22. Therefore, the cell culture vessel 22 may not be placed in a CO2 incubator. In addition, cells, microorganisms, viruses, dust, etc. that exist outside the cell culture vessel 22 will not enter the sealed cell culture vessel 22, so the cleanliness of the cell culture vessel 22 is maintained. Therefore, the cell culture vessel 22 may not be placed in a clean room. However, it does not necessarily prevent the supply of carbon dioxide gas, nitrogen gas, oxygen, etc. to the closed system where the cells exist.

[0227] According to the cell culture device 200 of the embodiment, cells are cultured in a completely closed system, so the risk of cross contamination caused by the leakage of cells from the culture device can be reduced. In addition, for example, even if the cells are infected with viruses such as HIV and hepatitis virus, the risk of infection to operators caused by the leakage of cells can be reduced. In addition, the risk of the culture medium in the cell culture device being contaminated by bacteria, viruses, molds, etc. in the air outside the cell culture device can be reduced. In addition, according to the cell culture device of the embodiment, cells can also be cultured without using a CO2 incubator.

[0228] (Second embodiment) like Figure 3 As shown, the red blood cell removal device 101 of the second embodiment includes a blood container 50 for containing blood, and a red blood cell treating agent container 53 for containing a red blood cell sedimentation agent or a red blood cell removing agent.

[0229] The blood container 50 contains blood inside. The blood container 50 may have a structure capable of sealing the inside from the outside air. The closed space including the inside of the blood container 50 may be configured so as not to exchange gas, virus, microorganism, impurity, etc. with the outside. The blood container 50 may be embedded and embedded in a gas impermeable substance. At least a part of the blood container 50 may be formed by carving into a component. At least a part of the blood container 50 may be formed by carving into a component and overlapping recesses. The blood container 50 may be capable of changing the volume of the blood container 50. In this case, for example, the blood container 50 includes a syringe containing a fluid and a plunger inserted into the syringe and capable of moving in the syringe, and the volume of the fluid that can be contained in the syringe can be changed by moving the plunger. Alternatively, the blood container 50 may also be a bellows or bag having flexibility.

[0230] The red blood cell treatment agent container 53 contains a red blood cell sedimentation agent or a red blood cell removal agent inside. The red blood cell treatment agent container 53 may have a structure capable of sealing the inside from the outside air. The closed space including the inside of the red blood cell treatment agent container 53 may be configured so as not to exchange gas, virus, microorganisms, impurities, etc. with the outside. The red blood cell treatment agent container 53 may be embedded and embedded in a gas impermeable substance. At least a portion of the red blood cell treatment agent container 53 may be formed by engraving into a component. At least a portion of the red blood cell treatment agent container 53 may be formed by engraving into a component and overlapping recesses. The red blood cell treatment agent container 53 may be capable of changing the volume of the red blood cell treatment agent container 53. In this case, for example, the red blood cell treatment agent container 53 includes a syringe containing a fluid and a plunger inserted into the syringe and capable of moving in the syringe, and the volume of the syringe capable of containing the fluid can be changed by moving the plunger. Alternatively, the red blood cell treatment agent container 53 may also be a bellows or bag having flexibility.

[0231] The red blood cell removal device 101 of the second embodiment further includes a mixer 57 for mixing blood with an red blood cell sedimentation agent or a red blood cell removal agent, for example. The mixer 57 includes, for example, a curved flow path for the mixed solution of blood and an red blood cell sedimentation agent or a red blood cell removal agent to flow. The curved flow path can be curved in a spiral shape. In the curved flow path, the flow path can meander. In the curved flow path, the cross-sectional area can be repeatedly increased and decreased. The mixer 57 can have a structure capable of sealing the interior from the outside air. The closed space including the interior of the mixer 57 can be configured so that there is no exchange of gas, virus, microorganism, impurity, etc. with the outside. The mixer 57 can be embedded and embedded in a gas impermeable substance. At least a portion of the mixer 57 can be formed by engraving into a component. At least a portion of the mixer 57 can be formed by engraving into a component and overlapping the recesses.

[0232] The blood container 50 is connected to a flow path 51 for transferring at least blood from the blood container 50 to the mixer 57. The red blood cell processing agent container 53 is connected to a flow path 54 for transferring at least a red blood cell sedimentation agent or a red blood cell removing agent from the red blood cell processing agent container 53 to the mixer 57. The flow paths 51 and 54 merge with a flow path 56. The flow path 56 is connected to the mixer 57. The mixer 57 is connected to a flow path 58 for transferring a mixed solution of blood and a red blood cell sedimentation agent or a red blood cell removing agent mixed in the mixer 57 to the red blood cell removing device 11.

[0233] A fluid machine 52 such as a pump for moving the fluid in the flow path 51 may be provided in the flow path 51. A fluid machine 55 such as a pump for moving the fluid in the flow path 54 may be provided in the flow path 54.

[0234] The flow paths 51, 54, 56, 58 may have a structure capable of sealing the interior from the outside air. The closed space including the interior of the flow paths 51, 54, 56, 58 may be configured so as not to exchange gas, viruses, microorganisms, impurities, etc. with the outside. The flow paths 51, 54, 56, 58 may be embedded and embedded in a gas impermeable substance. At least a portion of the flow paths 51, 54, 56, 58 may be formed by engraving into a member. At least a portion of the flow paths 51, 54, 56, 58 may be formed by engraving into a member and overlapping recesses.

[0235] When the mixed solution of blood and erythrocyte sedimentation agent or erythrocyte removing agent is sent to the erythrocyte remover 11, the fluid machine 52 moves the blood in the blood container 50 to the mixer 57 via the flow paths 51 and 56. In addition, the fluid machine 55 moves the erythrocyte sedimentation agent or erythrocyte removing agent in the erythrocyte treating agent container 53 to the mixer 57 via the flow paths 54 and 56. It should be noted that the fluid machine may be provided in the flow path 56 instead of 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 sedimentation agent or erythrocyte removing agent in the erythrocyte treating agent container 53 to the mixer 57. In the mixer 57, the blood and the erythrocyte sedimentation agent or erythrocyte removing agent are mixed. The mixed solution of the blood and the erythrocyte sedimentation agent or erythrocyte removing agent mixed in the mixer 57 is sent to the erythrocyte remover 11 via the flow path 58. In the red blood cell removal device 11, the red blood cell sedimentation or hemolysis is the same as in the first embodiment. In addition, other components of the red blood cell removal device 101 of the second embodiment may be the same as those of the red blood cell removal device 100 of the first embodiment.

[0236] (Third embodiment) like Figure 4 As shown, the red blood cell removal device 101 of the third embodiment includes a vacuum container 70 which is provided in the flow path 51 for conveying at least blood from the blood container 50 to the mixer 57 and can evacuate the interior thereof.

[0237] The vacuum container 70 may have a structure capable of sealing the interior from the outside air. The closed space including the interior of the vacuum container 70 may be configured not to exchange gas, viruses, microorganisms, impurities, etc. with the outside. The vacuum container 70 may be embedded and embedded in a gas impermeable substance. At least a portion of the vacuum container 70 may be formed by carving into a component. At least a portion of the vacuum container 70 may be formed by carving into a component and overlapping recesses. The vacuum container 70 may be capable of changing the volume of the vacuum container 70. The vacuum container 70 may also be a bellows or bag having flexibility.

[0238] The erythrocyte removal device 101 of the third embodiment includes a vacuum container 71 which is provided in the flow path 54 for conveying at least the erythrocyte sedimentation agent or the erythrocyte removing agent from the erythrocyte treating agent container 53 to the mixer 57 and can evacuate the interior thereof.

[0239] The vacuum container 71 may have a structure capable of sealing the interior from the outside air. The closed space including the interior of the vacuum container 71 may be configured so as not to exchange gas, viruses, microorganisms, impurities, etc. with the outside. The vacuum container 71 may be embedded and embedded in a gas impermeable substance. At least a portion of the vacuum container 71 may be formed by carving into a component. At least a portion of the vacuum container 71 may be formed by carving into a component and overlapping recesses. The vacuum container 71 may be capable of changing the volume of the vacuum container 71. The vacuum container 71 may also be a flexible bellows or bag.

[0240] When the blood container 50 is connected to the flow path 51 in a state where the vacuum container 70 is previously evacuated, the blood in the blood container 50 moves into the vacuum container 70, and further moves into the mixer 57 via the flow paths 51 and 56. When the red blood cell processing agent container 53 is connected to the flow path 54 in a state where the vacuum container 71 is previously evacuated, the red blood cell sedimentation agent or red blood cell removing agent in the red blood cell processing agent container 53 moves into the vacuum container 71, and further moves into the mixer 57 via the flow paths 54 and 56.

[0241] The other components of the red blood cell removal device 101 of the third embodiment may be the same as those of the second embodiment.

[0242] (Fourth embodiment) Can also be omitted Figure 4 The vacuum containers 70 and 71 shown in the figure are used to evacuate the red blood cell remover 11 in advance. When the blood container 50 is connected to the flow path 51 and the red blood cell treating agent container 53 is connected to the flow path 54 in the state where the red blood cell remover 11 is evacuated in advance, the blood in the blood container 50 moves to the mixer 57 via the flow paths 51 and 56, and the red blood cell sedimentation agent or red blood cell removing agent in the red blood cell treating agent container 53 moves to the mixer 57 via the flow paths 54 and 56. In addition, the blood and the red blood cell sedimentation agent or red blood cell removing agent mixed in the mixer 57 move to the red blood cell remover 11 via the flow path 58.

[0243] Alternatively, when the flow path 51 and the flow path 54 are closed by valves or the like, the interior of the erythrocyte remover 11 is made vacuum, and the valves of the flow paths 51 and 54 are opened, the blood in the blood container 50 moves into the mixer 57 via the flow paths 51 and 56, and the erythrocyte sedimentation agent or erythrocyte removing agent in the erythrocyte treating agent container 53 moves into the mixer 57 via the flow paths 54 and 56. Furthermore, the blood and the erythrocyte sedimentation agent or erythrocyte removing agent mixed in the mixer 57 move into the erythrocyte remover 11 via the flow path 58.

[0244] (Other Implementations) As described above, the present invention is described through the embodiments, but it should not be understood that the description and drawings constituting part of this disclosure limit the present invention. According to the present invention, a person skilled in the art should understand various alternative embodiments, embodiments, and application techniques. For example, Figure 1 The cells of the cell culture vessel 22 shown are not limited to monocytes. The cells delivered to the cell culture vessel 22 may be stem cells, fibroblasts or other somatic cells. The cells delivered to the cell culture vessel 22 are arbitrary.

[0245] In addition, in the first embodiment, an example of producing iPS cells from monocytes in the cell culture vessel 22 is described, but differentiated cells such as neural cells can also be produced from stem cells in the cell culture vessel 22. Stem cells can be iPS cells, embryonic stem cells (ES cells), adult stem cells, or other artificially induced stem cells. In this case, for example, the first variable volume container 27 contains differentiation inducing factors. In this way, it should be understood that the present invention includes various embodiments, etc.

[0246] Example (Example 1) This example shows that cells can be cultured in a completely closed environment without medium replacement or gas replacement. Growth factors were added to a culture medium (StemSpan H3000, registered trademark, STEMCELL Technologies Inc.), and deacylated gellan gum was further added to the culture medium to prepare a gel culture medium.

[0247] Place the prepared gel culture medium into a 15 mL tube and inoculate 2 × 10 5 Blood cells. Then, the 15 mL tube was placed in a CO2 incubator and the blood cells (monocytes) were cultured for 7 days. Then, Sendai virus vectors carrying OCT3 / 4, SOX2, KLF4, and cMYC were added to the gel culture medium at a multiplicity of infection (MOI) of 10.0 to infect the blood cells with Sendai virus.

[0248] After adding Sendai virus to the gel culture medium, 15 mL of gelled stem cell culture medium (DMEM / F12 containing 20% ​​KnockOut SR (registered trademark, ThermoFisher SCIENTIFIC)) was added to the gel culture medium, 15 mL of the culture medium containing cells infected with Sendai virus was placed in a sealable cell culture chamber, and the gel culture chamber was injected into the cell culture chamber. Then, the interior of the cell culture chamber was sealed so that no gas exchange occurred between the interior and the exterior of the cell culture chamber.

[0249] Suspension culture of cells into which the initialization factor has been introduced is started in the cell culture apparatus. Then, 2 mL of the gel culture medium in the culture medium holding tank 40 is replaced with 2 mL of fresh gel culture medium every 2 days.

[0250] After 15 days, the cells were observed under a microscope. Figure 5 In addition, the cells were fixed with 4% paraformaldehyde, and the expression level of cell surface antigen TRA-1-60 in the fixed cells was measured using flow cytometry. The results are as follows: Figure 6 As shown in the figure, more than 90% of the cells were TRA-1-60 positive, confirming that they were almost completely reprogrammed. This showed that iPS cells can be induced from somatic cells other than stem cells in a completely closed environment without changing the culture medium or gas.

[0251] (Example 2) The blood is treated with an erythrocyte sedimentation agent to obtain treated blood from which erythrocytes are at least partially removed. The treated blood is treated with antibodies against surface cell markers and analyzed by fluorescence activated cell sorting (FACS), and the results are shown in Figure 7 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.

[0252] The processed blood, from which red blood cells have been at least partially removed, is placed in Figure 2 The mononuclear cells are then collected from the mononuclear cell collection device. Figure 8 As shown in (a) of FIG. 1 , the treated blood before being placed in the mononuclear cell recovery device contains a large number of platelets. Figure 8 As shown in (b), the platelets in the solution containing monocytes recovered from the monocyte recovery device are almost completely removed. The graph showing the number of platelets in the treated blood before being placed in the monocyte recovery device and the number of platelets in the solution containing monocytes recovered from the monocyte recovery device per the same area is shown in Fig. 9 .

[0253] If the processed blood containing platelets before being placed in the mononuclear cell collector is placed in the culture medium, Fig.10 In contrast, if the solution containing monocytes from which platelets have been removed and recovered from the monocyte recovery device is placed in a culture medium, as shown in (a), Fig.10 As shown in (b), no agglutination occurred.

[0254] (Example 3) Deacylated gellan gum was added to the blood culture medium to prepare a gel culture medium. The prepared gel culture medium was placed in a 6-well dish coated with laminin and inoculated with 2×10 5 Blood cells (monocytes) were cultured in a 6-well dish in a CO2 incubator at 37°C for 7 days. Sendai virus vectors (CytoTune-iPS2.0, ThermoFisher SCIENTIFIC) carrying OCT3 / 4, SOX2, KLF4, and cMYC were then added to the blood proliferation medium at a multiplicity of infection (MOI) of 5 to infect blood cells with Sendai virus.

[0255] After the cells were placed in a 6-well dish, Sendai virus was added to the blood proliferation medium and the medium was replaced with 500 μL of stem cell medium (DMEM / F12 containing 20% ​​KnockOut SR (registered trademark, ThermoFisher SCIENTIFIC)) or Stem Fit two days later.

[0256] After 15 days of adding Sendai virus to the blood proliferation medium, the cells were observed under a microscope. Fig.11 As shown in FIG. 1 , it was confirmed that ES cell-like colonies were formed. In addition, 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. The results are as follows: Fig.12 As shown, it was confirmed that almost 100% of the induced cells were TRA-1-60 positive and almost completely reprogrammed. Therefore, it was shown that the cells can be reprogrammed by introducing reprogramming factors into the cells in a cell culture vessel and culturing the cells introduced with the reprogramming factors in the same cell culture vessel.

[0257] (Example 4) Deacylated gellan gum was added to the blood culture medium to prepare a gel culture medium. The prepared gel culture medium was placed in a laminin-coated flask and inoculated with 5×10 5 Blood cells (monocytes) were cultured in a 37°C CO2 incubator for 7 days. Sendai virus vectors (CytoTune-iPS2.0, ThermoFisherSCIENTIFIC) carrying OCT3 / 4, SOX2, KLF4, and cMYC were then added to the blood proliferation medium at a multiplicity of infection (MOI) of 5 to infect the blood cells with Sendai virus.

[0258] Two days after adding Sendai virus to the blood proliferation medium, the flask was completely filled with stem cell culture medium (DMEM / F12 containing 20% ​​KnockOut SR (registered trademark, ThermoFisher SCIENTIFIC)) or StemFit so that no air remained in the flask, and the flask was capped so that no gas exchange with the outside occurred, and the interior of the flask was sealed in a way that cells, microorganisms, impurities, etc. could not pass through.

[0259] After 15 days of adding Sendai virus to the blood proliferation medium, the cells were observed under a microscope. Fig.13 As shown in FIG. 1 , it was confirmed that ES cell-like colonies were formed. In addition, 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. The results are as follows: Fig.14 As shown, the induced cells were almost 100% TRA-1-60 positive and almost completely reprogrammed. Therefore, it was shown that the cells can be reprogrammed by introducing reprogramming factors into the cells in a cell culture vessel and culturing the cells introduced with the reprogramming factors in the same closed cell culture vessel.

[0260] (Example 5) Non-gel liquid blood proliferation medium was placed in a 6-well dish coated with laminin and 2×10 5 Blood cells (monocytes) were cultured in a 6-well dish in a CO2 incubator at 37°C for 7 days. Sendai virus vectors (CytoTune-iPS2.0, ThermoFisher SCIENTIFIC) carrying OCT3 / 4, SOX2, KLF4, and cMYC were then added to the blood proliferation medium at a multiplicity of infection (MOI) of 5 to infect blood cells with Sendai virus.

[0261] Two days after adding Sendai virus to the blood growth medium with the cells placed in a 6-well dish, the medium was replaced with 500 μL of stem cell medium (DMEM / F12 containing 20% ​​KnockOut SR (registered trademark, ThermoFisher SCIENTIFIC)) or Stem Fit.

[0262] Fifteen days after the blood proliferation medium was supplemented with Sendai virus, the cells were observed under a microscope. Fig.15 As shown in FIG. 1 , it was confirmed that ES cell-like colonies were formed. In addition, 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. The results are as follows: Fig.16As shown, it was confirmed that almost 100% of the induced cells were TRA-1-60 positive and almost completely reprogrammed. Therefore, it was shown that the cells can be reprogrammed by introducing reprogramming factors into the cells in a cell culture vessel and culturing the cells introduced with the reprogramming factors in the same cell culture vessel.

[0263] (Example 6) Non-gel liquid blood proliferation medium was placed in a laminin-coated flask and 5×10 5 Blood cells (monocytes) were added to the flask in a CO2 incubator at 37°C and the blood cells were cultured for 7 days. Then, Sendai virus vectors (CytoTune-iPS2.0, ThermoFisher SCIENTIFIC) carrying OCT3 / 4, SOX2, KLF4, and cMYC were added to the blood proliferation medium at a multiplicity of infection (MOI) of 5 to infect the blood cells with Sendai virus.

[0264] Two days after adding Sendai virus to the blood proliferation medium, the flask was completely filled with stem cell culture medium (DMEM / F12 containing 20% ​​KnockOut SR (registered trademark, ThermoFisher SCIENTIFIC)) or StemFit so that no air remained in the flask, and the flask was capped so that no gas exchange with the outside occurred, and the interior of the flask was sealed in a way that cells, microorganisms, impurities, etc. could not pass through.

[0265] After 15 days of adding Sendai virus to the blood proliferation medium, the cells were observed under a microscope. Fig.17 As shown in FIG. 1 , it was confirmed that ES cell-like colonies were formed. In addition, 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. The results are as follows: Fig.18 As shown, it was confirmed that almost 100% of the induced cells were TRA-1-60 positive and almost completely reprogrammed. Therefore, it was shown that the cells can be reprogrammed by introducing reprogramming factors into the cells in a cell culture vessel and culturing the cells introduced with the reprogramming factors in the same closed cell culture vessel.

Claims

1. A cell culture device, characterized in that: have: A cell culture apparatus, which is used to culture cells; a first variable-volume container connected to the cell culture apparatus; and a second variable volume container connected to the cell culture apparatus, When the fluid in the first variable volume container moves into the cell culture apparatus, the volume of the first variable volume container contracts and the volume of the second variable volume container expands.

2. The cell culture device according to claim 1, characterized in that: The interior of the cell culture chamber, the interior of the first variable volume container, and the interior of the second variable volume container can be sealed from the outside air.

3. The cell culture device according to claim 1 or 2, characterized in that: The first variable-volume container contains a substance.

4. The cell culture device according to any one of claims 1 to 3, characterized in that: The device further comprises a flow channel for supplying the cells into the cell culture device.

5. The cell culture device according to any one of claims 1 to 4, characterized in that: A fluid machine for supplying the cells into the cell culture apparatus is also provided.

6. The cell culture device according to any one of claims 1 to 5, characterized in that: A fluid container for containing the fluid supplied to the cell culture apparatus is further provided.

7. The cell culture device according to any one of claims 1 to 6, characterized in that: The fluid is a somatic cell culture medium or a stem cell culture medium.

8. The cell culture device according to any one of claims 1 to 7, characterized in that: The device further includes a temperature regulating unit for regulating the temperature in the cell culture chamber.

9. The cell culture device according to any one of claims 1 to 8, characterized in that: The cells are cultured in an adherent manner in the cell culture apparatus.

10. The cell culture device according to any one of claims 1 to 8, characterized in that: The cells are cultured in suspension in the cell culture vessel.

11. The cell culture device according to any one of claims 1 to 10, characterized in that: The invention further comprises a hollow fiber membrane disposed in the cell culture vessel.

12. The cell culture device according to any one of claims 1 to 11, characterized in that: The cells in the cell culture vessel can be moved to the first variable-volume container.

13. The cell culture device according to any one of claims 1 to 3, characterized in that: It also includes a flow path connected to the cell culture apparatus and a fluid machine disposed in the flow path, The fluid machine sucks the cells in the cell culture device into the flow channel and returns the cells in the flow channel to the cell culture device, thereby performing at least one of subculture and expansion culture of the cells.

14. The cell culture device according to claim 13, characterized in that: The flow channel has a structure for dividing the cell clusters.

Images