Method for culturing cells and method for recovering mononuclear cells

Abstract

Provided is a method for culturing cells, in which a factor is introduced into cells in a cell culture device, and the cells into which the factor has been introduced are cultured in the same cell culture device. Also provided is a method for recovering mononuclear cells, which includes: processing blood to produce processed blood from which red blood cells have been at least partially removed; diluting the processed blood; allowing mononuclear cells contained in the diluted processed blood to settle; removing the supernatant of the diluted processed blood; and recovering the mononuclear cells.

Description

Technical Field

[0001] This invention relates to cell technology, specifically to a red blood cell removal device, a mononuclear cell recovery device, a cell culture device, a cell culture system, a cell culture method, and a method for recovering mononuclear cells. Background Technology

[0002] Embryonic stem cells (ES cells) are stem cells derived from early human and mouse embryos. ES cells possess pluripotency, capable of differentiating into all cell types found in an organism. Currently, human ES cells are used in cell transplantation therapies for various diseases, including Parkinson's disease, juvenile diabetes, and leukemia. However, ES cell transplantation also faces obstacles. In particular, ES cell transplantation may induce the same immune rejection response as that following unsuccessful organ transplantation. Furthermore, the application of ES cells derived from the destruction of human embryos has drawn considerable criticism and opposition from an ethical standpoint.

[0003] Against this backdrop, Professor Shinya Yamanaka of Kyoto University successfully created induced pluripotent stem cells (iPS cells) by introducing four genes—OCT3 / 4, KLF4, c-MYC, and SOX2—into somatic cells. For this achievement, Professor Yamanaka was awarded the 2012 Nobel Prize in Physiology or Medicine (see, for example, patent documents 1 and 2). iPS cells are ideal pluripotent cells, free from rejection reactions and ethical concerns. Therefore, the application of iPS cells in cell transplantation therapy is anticipated.

[0004] Existing technical documents

[0005] Patent documents

[0006] Patent Document 1: Japanese Patent No. 4183742

[0007] Patent Document 2: Japanese Patent Application Publication No. 2014-114997 Summary of the Invention

[0008] Technical issues

[0009] iPS cells are sometimes induced from blood cells. Beyond the use of induced iPS cells, techniques for efficiently processing blood cells are desired. Furthermore, beyond iPS cells, devices for efficiently culturing various cell types are desired. Therefore, one object of the present invention is to provide a red blood cell removal device, a mononuclear cell recoverer, a cell culture device, a cell culture system, a cell culture method, and a method for recovering mononuclear cells.

[0010] Technical solution

[0011] According to the present invention, a red blood cell removal device is provided, comprising a blood container for containing blood, a red blood cell remover for receiving blood from the blood container and removing red blood cells from the blood in at least a portion thereof, and a flow path for conveying blood from the blood container to the red blood cell remover.

[0012] In the aforementioned red blood cell removal device, the interior of the flow path used to deliver blood from the blood container to the red blood cell remover can be sealed relative to the outside air.

[0013] The aforementioned red blood cell removal device may further include a mononuclear cell recoverer and a flow path, wherein the mononuclear cell recoverer receives processed blood obtained by removing at least partially red blood cells from the red blood cell remover and recovers mononuclear cells from the processed blood, and the flow path is used to transport the processed blood obtained by removing at least partially red blood cells from the red blood cell remover to the mononuclear cell recoverer.

[0014] In the aforementioned red blood cell removal device, the red blood cell remover may be capable of removing internal gases.

[0015] The aforementioned red blood cell removal device may further include a flow path for processing blood flow obtained by at least partially removing red blood cells, and the interior of the flow path for processing blood flow obtained by at least partially removing red blood cells can be sealed relative to the outside air.

[0016] In the aforementioned red blood cell removal device, a mononuclear cell recoverer may be used to remove internal gases.

[0017] In the aforementioned red blood cell removal device, the interior of the blood container and the interior of the red blood cell remover can be sealed relative to the outside air.

[0018] In the aforementioned red blood cell removal device, the interior of the mononuclear cell recycler can be sealed relative to the outside air.

[0019] In the aforementioned red blood cell removal device, the enclosed space containing the blood container and the red blood cell remover can be kept separate from the outside gas exchange.

[0020] In the aforementioned red blood cell removal device, the blood container and the red blood cell remover can be embedded.

[0021] In the aforementioned 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 can be formed by being engraved into the components.

[0022] In the aforementioned red blood cell removal device, a mononuclear cell recycler can be embedded.

[0023] In the aforementioned red blood cell removal device, at least a portion of the mononuclear cell recoverer can be formed by being engraved into the component.

[0024] In the aforementioned red blood cell removal device, blood may be mixed with at least one of a red blood cell settling agent and a red blood cell removal agent within the red blood cell remover.

[0025] The aforementioned red blood cell removal device may further include a red blood cell treatment agent container that contains at least one of a red blood cell settling agent and a red blood cell removal agent, and the red blood cell remover receives at least one of the red blood cell settling agent and the red blood cell removal agent from the red blood cell treatment agent container.

[0026] The aforementioned red blood cell removal device may further include a mixer for mixing blood with at least one of a red blood cell settling agent and a red blood cell removal agent, wherein the red blood cell remover receives blood mixed with at least one of the red blood cell settling agent and the red blood cell removal agent from the mixer.

[0027] In the aforementioned red blood cell removal device, the mixer may have a tortuous flow path for the flow of a mixture of blood and at least one of the red blood cell settling agent and red blood cell removal agent.

[0028] The aforementioned red blood cell removal device may also include a flow path for at least transporting blood from a blood container to the red blood cell remover.

[0029] The aforementioned red blood cell removal device may include a vacuum container connected to a flow path for at least transporting blood from a blood container to the red blood cell remover, enabling the interior to be vacuumed.

[0030] The aforementioned red blood cell removal device may further include a red blood cell treatment agent container for containing at least one of a red blood cell settling agent and a red blood cell removal agent, and a flow path for conveying at least one of the red blood cell settling agent and the red blood cell removal agent from the red blood cell treatment agent container to the red blood cell remover.

[0031] The aforementioned red blood cell removal device may also include fluid machinery for at least transporting blood from a blood container to the red blood cell remover.

[0032] In the aforementioned red blood cell removal device, the blood container may be able to change its volume.

[0033] In the above-mentioned red blood cell removal device, the volume of the red blood cell remover can be changed.

[0034] In the aforementioned red blood cell removal device, the mononuclear cell recycler may be able to change its volume.

[0035] In the aforementioned red blood cell removal device, the volume of the red blood cell treatment agent container can be changed.

[0036] In the aforementioned red blood cell removal device, red blood cells may settle within the red blood cell remover, and the supernatant within the red blood cell remover may be transported to a mononuclear cell recoverer in the form of processed blood obtained by removing at least some of the red blood cells.

[0037] The aforementioned red blood cell removal device may further include a flow path for transporting processed blood, from the red blood cell remover to the mononuclear cell collector, at least partially with red blood cells removed.

[0038] The aforementioned red blood cell removal device may further include fluid machinery for conveying processed blood, from the red blood cell remover to the mononuclear cell recoverer, at least partially with red blood cells removed.

[0039] In the aforementioned red blood cell removal device, the processed blood, from which at least some red blood cells have been removed, may be diluted within a mononuclear cell recoverer.

[0040] In the aforementioned red blood cell removal device, mononuclear cells can settle within the mononuclear cell reclaimer.

[0041] In the aforementioned red blood cell removal device, platelets can be suspended in a diluent used to process the blood.

[0042] In the aforementioned red blood cell removal device, the red blood cells may be hemolyzed by a red blood cell removal agent in a blood diluent.

[0043] The aforementioned red blood cell removal device may further include a dilution container containing a dilution solution for diluting the processed blood obtained after at least partial removal of red blood cells.

[0044] In the above-mentioned red blood cell removal device, the diluent can be a buffer solution.

[0045] In the aforementioned red blood cell removal device, the volume of the dilution liquid container can be changed.

[0046] In the above-mentioned red blood cell removal device, the supernatant in the mononuclear cell collector can be removed after the mononuclear cells settle in the mononuclear cell collector.

[0047] In the aforementioned red blood cell removal device, platelets suspended in the supernatant can be removed by removing the supernatant.

[0048] In the aforementioned red blood cell removal device, hemolyzed red blood cells suspended in the supernatant can be removed by removing the supernatant.

[0049] In the aforementioned red blood cell removal device, a first opening may be provided at the bottom of the mononuclear cell collector, and a second opening may be provided at a position higher than the first opening in the direction of gravity.

[0050] In the aforementioned red blood cell removal device, the bottom of the mononuclear cell collector may be funnel-shaped, with a first opening at the front end of the funnel-shaped bottom and a second opening on the side of the funnel-shaped bottom.

[0051] In the aforementioned red blood cell removal device, if processed blood, from which red blood cells have been at least partially removed, is introduced into the mononuclear cell recoverer, mononuclear cells can accumulate at the bottom, and the supernatant can be discharged from the second opening.

[0052] In the aforementioned red blood cell removal device, platelets suspended in the supernatant can be removed by discharging the supernatant.

[0053] In the aforementioned red blood cell removal device, hemolyzed red blood cell components suspended in the supernatant can be removed by discharging the supernatant.

[0054] The aforementioned red blood cell removal device may also include a mononuclear cell aspiration device for aspirating mononuclear cells from the first opening.

[0055] In the aforementioned red blood cell removal device, the size of the first opening can be set such that, when the mononuclear cells are not aspirated by the mononuclear cell aspiration device, the mononuclear cells block the first opening.

[0056] The aforementioned red blood cell removal device may also include a flow path for conveying fluid within the red blood cell remover to a blood container.

[0057] The aforementioned red blood cell removal device may further include at least one of a fluid mechanism for conveying blood from a blood container to a red blood cell remover, and a fluid mechanism for conveying fluid within the red blood cell remover to a blood container.

[0058] The aforementioned red blood cell removal device may also include a flow path for conveying fluid from the mononuclear cell recoverer to the red blood cell remover.

[0059] The aforementioned red blood cell removal device may further include at least one of the following: fluid machinery for conveying processed blood, from the red blood cell remover to the mononuclear cell recoverer, having at least partially removed the red blood cells; and fluid machinery for conveying fluid from the mononuclear cell recoverer to the red blood cell remover.

[0060] According to the present invention, a mononuclear cell recoverer is provided, which includes a recovery container for containing a solution containing mononuclear cells, 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.

[0061] In the above-mentioned mononuclear cell recoverer, if a solution is introduced into the recovery container, the mononuclear cells accumulate at the front end of the bottom of the funnel shape, and the solution is discharged from the second opening.

[0062] The aforementioned mononuclear cell collector may also include a mononuclear cell aspiration device for aspirating mononuclear cells that have accumulated at the front end of the funnel-shaped bottom.

[0063] In the aforementioned mononuclear cell recycler, the size of the first opening can be set such that, when the mononuclear cells are not aspirated by the mononuclear cell suction device, the mononuclear cells block the first opening.

[0064] According to the present invention, a cell culture apparatus is provided, comprising a cell culture vessel for culturing cells and a variable-volume container connected to the cell culture vessel, wherein fluid is capable of moving within the cell culture vessel and the variable-volume container.

[0065] The cell culture apparatus described above may have at least a first variable volume container and a second variable volume container as variable volume containers.

[0066] In the cell culture apparatus described above, if the fluid in the cell culture vessel moves into the first variable volume container, the volume of the first variable volume container expands, and the volume of the second variable volume container contracts.

[0067] In the cell culture apparatus described above, if the fluid in the first variable volume container moves into the cell culture vessel, the volume of the first variable volume container shrinks and the volume of the second variable volume container expands.

[0068] In the cell culture apparatus described above, if the fluid in the second variable volume container moves into the cell culture vessel, the volume of the second variable volume container shrinks, and the volume of the first variable volume container expands.

[0069] In the aforementioned cell culture apparatus, the interior of the cell culture vessel, the interior of the first variable-volume container, and the interior of the second variable-volume container can be sealed relative to the outside air.

[0070] In the aforementioned cell culture apparatus, the cell culture vessel, the first variable-volume container, and the second variable-volume container can be embedded.

[0071] In the aforementioned 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 can be formed by being etched into the components.

[0072] In the cell culture apparatus described above, a first variable-volume container may be used to hold the substance, and the substance may come into contact with the cells through the movement of fluid.

[0073] In the cell culture apparatus described above, the substance can be the inducing factor, which is introduced into the cells through the movement of fluid.

[0074] The aforementioned cell culture apparatus may also include fluid machinery for moving fluid within the cell culture vessel to a first variable-volume container.

[0075] The aforementioned cell culture apparatus may also include fluid machinery for moving fluid within the cell culture vessel to a second variable-volume container.

[0076] The aforementioned cell culture apparatus may also include a flow path for supplying cells into the cell culture vessel.

[0077] The cell culture apparatus described above may also include a flow path for supplying culture medium, which is connected to a flow path for supplying cells into the cell culture vessel.

[0078] In the cell culture apparatus described above, cells and culture medium may be mixed in a flow path for supplying cells into the cell culture vessel, and culture medium containing cells may be supplied into the cell culture vessel.

[0079] In the cell culture apparatus described above, at least one of the first variable-volume container and the second variable-volume container may expand in volume when cells are introduced into the cell culture vessel from the flow path used for supplying cells.

[0080] The aforementioned cell culture apparatus may also include fluid machinery for supplying cells into the cell culture vessel.

[0081] In the cell culture apparatus described above, the cells can be somatic cells or stem cells.

[0082] The aforementioned cell culture apparatus may also include a fluid container for containing the fluid supplied to the cell culture vessel.

[0083] In the above-mentioned cell culture device, the fluid can be somatic cell culture medium or stem cell culture medium.

[0084] In the cell culture apparatus described above, the stem cell culture medium can be an induction medium, an expansion medium, or a maintenance medium.

[0085] In the cell culture apparatus described above, at least one of the first variable-volume container and the second variable-volume container may expand in volume when fluid is supplied from the fluid container into the cell culture vessel.

[0086] The aforementioned cell culture apparatus may also include fluid machinery for supplying fluid into the cell culture vessel.

[0087] The aforementioned cell culture apparatus may also include a temperature control unit for regulating the temperature within the cell culture vessel.

[0088] In the cell culture apparatus described above, cells can be adhered and cultured within the cell culture vessel.

[0089] In the above-mentioned cell culture device, a cell adhesion coating agent can be used to coat the inside of the cell culture vessel.

[0090] In the cell culture apparatus described above, cells can be cultured in suspension within the cell culture vessel.

[0091] The aforementioned cell culture device may also include a hollow fiber membrane disposed within the cell culture vessel.

[0092] In the cell culture apparatus described above, cells can be cultured on the inside of a hollow fiber membrane.

[0093] In the cell culture apparatus described above, cells within the cell culture vessel can be moved to a variable-volume container.

[0094] The aforementioned cell culture apparatus may further include a flow path connected to a cell culture vessel and a fluid mechanism disposed in the flow path. The fluid mechanism draws cells from 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 passage culture and amplification culture.

[0095] In the cell culture apparatus described above, the flow path may have a structure that divides the cell clusters.

[0096] According to the present invention, a cell culture system is provided, comprising a mononuclear cell recycler for recovering mononuclear cells from blood and a cell culture device for receiving mononuclear cells from the mononuclear cell recycler.

[0097] In the cell culture system described above, a mononuclear cell recoverer may be used to receive processed blood from which red blood cells have been at least partially removed, and to recover mononuclear cells from the processed blood.

[0098] The cell culture system described above may also include a red blood cell remover for supplying processed blood, which has had at least partially red blood cells removed, to a mononuclear cell recycler.

[0099] The cell culture system described above may also include a blood container for supplying blood, before the red blood cells have been at least partially removed, to the red blood cell remover.

[0100] The cell culture system described above can have a variable-volume container connected to a cell culture vessel. If fluid in the cell culture vessel moves to the variable-volume container, the volume of the variable-volume container expands.

[0101] The cell culture system described above may have a first variable volume container connected to a cell culture vessel and a second variable volume container connected to a cell culture vessel. If the fluid in the cell culture vessel 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 contracts.

[0102] In the cell culture system described above, the interior of the mononuclear cell recycler and the interior of the cell culture vessel can be sealed relative to the outside air.

[0103] In the cell culture system described above, the interior of the red blood cell remover can be sealed relative to the outside air.

[0104] In the cell culture system described above, the interior of the blood container can be sealed relative to the outside air.

[0105] In the cell culture system described above, the interiors of the first variable-volume container and the second variable-volume container can be sealed relative to the outside air.

[0106] In the cell culture system described above, blood containers, red blood cell removers, mononuclear cell recoverers, and cell culture vessels can be embedded.

[0107] In the aforementioned 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 mononuclear cell recycler, and at least a portion of the cell culture vessel can be formed by being engraved into the components.

[0108] In the cell culture system described above, the first variable-volume container and the second variable-volume container can be embedded.

[0109] In the cell culture system described above, at least a portion of the first variable volume container and at least a portion of the second variable volume container can be formed by being engraved into the components.

[0110] In the cell culture system described above, the interiors of the first variable-volume container and the second variable-volume container may not exchange gases with the outside.

[0111] According to the present invention, a cell culture method is provided, which involves introducing factors into cells in a cell culture vessel and culturing the cells with introduced factors in the same cell culture vessel as the cell culture vessel.

[0112] In the above-mentioned cell culture method, the cell culture vessel may be closed during the process of introducing factors into the cells and culturing the cells with introduced factors.

[0113] In the above cell culture method, a variable volume container can be connected to the cell culture vessel, allowing fluid to move within the cell culture vessel and the variable volume container.

[0114] In the cell culture methods described above, the supply factor can be supplied from a variable-volume container.

[0115] In the cell culture method described above, cells in state 1 that have been introduced with the factor can be induced to become cells in state 2 within the same cell culture vessel.

[0116] In the above cell culture method, the first state can be the differentiated state, and the second state can be the undifferentiated state.

[0117] In the above cell culture method, the first state can be the dedifferentiation state, and the second state can be the differentiation state.

[0118] In the above cell culture method, the first state can be a dedifferentiated state, and the second state can be a dedifferentiated state different from the first state.

[0119] In the cell culture methods described above, the cells in the first state can be somatic cells.

[0120] In the cell culture methods described above, the cells in the first state can be blood cells.

[0121] In the cell culture method described above, the cells in state 1 can be monocytes.

[0122] In the cell culture methods described above, the cells in the second state can be stem cells.

[0123] In the cell culture method described above, the cells in the second state can be iPS cells.

[0124] In the cell culture methods described above, the cells in the first state can be stem cells.

[0125] In the cell culture method described above, the cells in state 1 can be iPS cells.

[0126] In the cell culture method described above, the cells in the second state can be somatic cells.

[0127] In the above cell culture method, the cells in the first state can be somatic cells, and the cells in the second state can be somatic cells that are different from those in the first state.

[0128] In the cell culture method described above, the cells in state 1 can be blood cells with at least some red blood cells removed.

[0129] In the cell culture method described above, the cells in state 1 can be blood cells from which platelets have been at least partially removed.

[0130] In the cell culture method described above, the factor can be a factor that induces cells in state 1 to cells in state 2.

[0131] In the cell culture methods described above, the factors can be those that induce specific cell states.

[0132] In the cell culture methods described above, the factor can be an initialization factor.

[0133] In the cell culture methods described above, the factors can be differentiation-inducing factors.

[0134] In the cell culture method described above, cells that have been introduced with the factor can be recovered from the cell culture vessel and returned to the same cell culture vessel for passage culture or expansion culture.

[0135] According to the present invention, a method for recovering mononuclear cells is provided, comprising: processing blood to prepare processed blood by removing at least some red blood cells; diluting the processed blood; causing the mononuclear cells contained in the diluted processed blood to settle; removing the supernatant of the diluted processed blood; and recovering the mononuclear cells.

[0136] In the above-mentioned method for recovering mononuclear cells, blood can be prepared in a red blood cell remover, and the blood can be diluted, mononuclear cells can be settled, and supernatant can be removed in a mononuclear cell recoverer. The red blood cell remover and mononuclear cell recoverer are then sealed.

[0137] In the above-mentioned method for recovering mononuclear cells, the blood can be treated using erythrocyte sedimentation agents or erythrocyte removal agents.

[0138] In the above method for recovering mononuclear cells, the processed blood can be diluted with phosphate buffer.

[0139] In the above-mentioned method for recovering mononuclear cells, the supernatant of the diluted processed blood may contain platelets.

[0140] In the above-described method for recovering mononuclear cells, red blood cells can be at least partially removed from the recovered mononuclear cells.

[0141] In the above-mentioned method for recovering monocytes, platelets can be at least partially removed from the recovered monocytes.

[0142] Invention Effects

[0143] According to the present invention, a red blood cell removal device, a mononuclear cell recoverer, a cell culture device, a cell culture system, a cell culture method, and a mononuclear cell recovery method can be provided. Attached Figure Description

[0144] Figure 1 This is a schematic diagram of the cell culture system according to the first embodiment.

[0145] Figure 2 This is a schematic diagram of the mononuclear cell recycler of the first embodiment.

[0146] Figure 3 This is a schematic diagram of the red blood cell removal device according to the second embodiment.

[0147] Figure 4 This is a schematic diagram of the red blood cell removal device according to the third embodiment.

[0148] Figure 5 This is a microscope image of the cell cluster from Example 1.

[0149] Figure 6 This is a histogram showing the results of flow cytometry of iPS cells in Example 1.

[0150] Figure 7 This is the analysis result of fluorescence-activated cell sorting in Example 2.

[0151] Figure 8 (a) is a microscopic photograph of processed blood before it was placed into the mononuclear cell recoverer of Example 2. Figure 8 (b) is a microscopic photograph of a solution containing mononuclear cells recovered from a mononuclear cell recycler.

[0152] Figure 9 This is a graph showing the number of platelets in the processed blood before it was placed into the mononuclear cell recoverer of Example 2 and the number of platelets in the solution containing mononuclear cells recovered from the mononuclear cell recoverer.

[0153] Figure 10 (a) is a photograph of culture medium containing processed blood containing platelets added before it is placed into the mononuclear cell recoverer of Example 2. Figure 10 (b) is a photograph of a culture medium containing a solution of monocytes from which platelets have been removed.

[0154] Figure 11 These are microscope images of cells produced using the iPS cell preparation method described in Example 3.

[0155] Figure 12 This is a histogram showing the results of analyzing cells prepared by the iPS cell preparation method of Example 3 using flow cytometry.

[0156] Figure 13 These are microscope images of cells produced using the iPS cell preparation method described in Example 4.

[0157] Figure 14This is a histogram showing the results of analyzing cells prepared by the iPS cell preparation method of Example 4 using flow cytometry.

[0158] Figure 15 These are microscope images of cells produced using the iPS cell production method described in Example 5.

[0159] Figure 16 This is a histogram showing the results of analyzing cells prepared by the iPS cell preparation method of Example 5 using flow cytometry.

[0160] Figure 17 These are microscope images of cells produced using the iPS cell production method described in Example 6.

[0161] Figure 18 This is a histogram showing the results of analyzing cells prepared by the iPS cell preparation method of Example 6 using flow cytometry.

[0162] Symbol Explanation

[0163] 10. Blood container, 11. Red blood cell remover, 12. Flow path, 13. Flow path, 14. Fluid dynamics, 15. Mononuclear cell collector, 16. Flow path, 17. Flow path, 18. Fluid dynamics, 19. Flow path, 20. Mononuclear cell aspiration device, 21. Fluid dynamics, 22. Cell culture vessel, 23. Flow path, 24. Fluid dynamics, 25. Culture medium container, 26. Flow path, 27. Variable volume container, 28. Fluid dynamics, 29. Flow path, 30. Variable volume container, 31. Flow path, 32. Culture medium container, 33. Fluid dynamics, 34. Flow path, 35. 36. Variable volume container; 37. Flow path; 38. Fluid dynamics; 39. Fluid dynamics; 40. Culture medium holding tank; 50. Blood container; 51. Flow path; 52. Fluid dynamics; 53. Red blood cell treatment container; 54. Flow path; 55. Fluid dynamics; 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 Implementation

[0164] Hereinafter, embodiments of the present invention will be described. In the following drawings, the same or similar parts are indicated by the same or similar symbols. However, the drawings are schematic. Therefore, specific dimensions, etc., should be determined in conjunction with the following description. Furthermore, the drawings naturally include portions with different dimensional relationships and ratios.

[0165] (First Embodiment)

[0166] like Figure 1 As shown, the red blood cell removal apparatus 100 of the first embodiment includes a blood container 10 for containing blood and a red blood cell remover 11 for receiving blood from the blood container 10 and removing red blood cells from the blood in at least a portion thereof.

[0167] Blood container 10 contains blood internally. Blood container 10 may have a structure capable of sealing the interior relative to external air. The enclosed space including the interior of blood container 10 may be configured to prevent gas exchange with the outside. Blood container 10 may be embedded and encased in a gas-impermeable material. At least a portion of blood container 10 may be formed by engraving into a component. At least a portion of blood container 10 may be formed by engraving into a component and overlapping recesses. Blood container 10 may have its volume varied.

[0168] The red blood cell remover 11 may contain, for example, a red blood cell settling agent or a red blood cell remover. The red blood cell remover 11 may have a structure capable of sealing the interior relative to the outside air. The enclosed space including the interior of the red blood cell remover 11 may be configured to prevent exchange of gases, viruses, microorganisms, and impurities with the outside. The red blood cell remover 11 may be embedded and encased in a gas-impermeable material. At least a portion of the red blood cell remover 11 may be formed by etching into a component. At least a portion of the red blood cell remover 11 may be formed by etching into a component and overlapping recesses. The volume of the red blood cell remover 11 may be variable.

[0169] 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 its interior relative to the outside air. The enclosed space including the interior of the flow path 13 may be configured to prevent the exchange of gases, viruses, microorganisms, and impurities with the outside. The flow path 13 may be embedded and encased in a gas-impermeable material. At least a portion of the flow path 13 may be formed by etching it into a component. At least a portion of the flow path 13 may be formed by etching it into a component and overlapping the recesses.

[0170] Additionally, a flow path 12 is provided between the blood container 10 and the red blood cell remover 11 for conveying fluids such as air or gas from the red blood cell remover 11 to the blood container 10. The flow path 12 may have a structure capable of sealing its interior relative to external air. The enclosed space including the interior of the flow path 12 can be configured to prevent exchange of gases, viruses, microorganisms, and impurities with the outside. The flow path 12 may be embedded and encased in a gas-impermeable material. At least a portion of the flow path 12 can be formed by etching it into a component. At least a portion of the flow path 12 can be formed by etching it into a component and overlapping the recesses.

[0171] Each of the blood container 10 and flow paths 12 and 13 can be connected via a connector. The connector can be a sterile connector. The connector can be a needleless connector. The needleless connector can be a diaphragm type or a mechanical valve type.

[0172] A fluid machine 14, such as a pump, is provided in flow path 13 to move the fluid within it. It should be noted that the fluid machine can be provided in flow path 12, or in both flow path 12 and flow path 13. It should also be noted that in this invention, the fluid includes both gas and liquid.

[0173] As fluid machinery 14, positive displacement pumps 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 piezoelectric pumps. Tubular pumps are sometimes also called peristaltic pumps. Alternatively, microfluidic chip modules combining various pumps can also be used. The same applies to other fluid machinery in this invention. If closed-loop pumps such as peristaltic pumps, tubular pumps, and diaphragm pumps are used, fluid can be delivered without the pump directly contacting the fluid inside the flow path.

[0174] When the red blood cell remover 11 is pre-filled with gas and red blood cell settling agent, if the fluid machinery 14 draws blood from the blood container 10 via the flow path 13 and supplies the drawn blood to the red blood cell remover 11, the gas in the red blood cell remover 11 is pressure-driven 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 red blood cell remover 11 and the gas in the red blood cell remover 11 to the blood container 10, the pressure in the blood container 10 and the red blood cell remover 11 can be averaged.

[0175] It should be noted that the fluid mechanism 14 can also draw gas from the red blood cell remover 11 via the flow path 13 and supply the drawn gas to the blood container 10. In this case, the blood in the blood container 10 is propelled by the gas pressure and transported to the red blood cell remover 11 via the flow path 12. Thus, by removing the gas from the red blood cell remover 11, the blood in the blood container 10 can also be transported to the red blood cell remover 11.

[0176] Blood introduced into the red blood cell remover 11 comes into contact with a red blood cell settling agent or a red blood cell remover within the red blood cell remover 11. The fluid machinery 14 can repeatedly agitate the blood by drawing fluid from and releasing fluid into the red blood cell remover 11. When the red blood cell remover 11 contains a red blood cell settling agent, red blood cells settle within the red blood cell remover 11, at least partially removing red blood cells from the blood. When the red blood cell remover 11 contains a red blood cell remover, red blood cells hemolyze within the red blood cell remover 11, at least partially removing red blood cells from the blood.

[0177] The red blood cell removal device 100 may further include a mononuclear cell collector 15, which receives processed blood from the red blood cell remover 11 after at least partial removal of red blood cells, and recovers mononuclear cells from the processed blood. The mononuclear cell collector 15 may have a structure capable of sealing its interior relative to external air. The enclosed space including the interior of the mononuclear cell collector 15 may be configured to prevent exchange of gases, viruses, microorganisms, and impurities with the outside. The mononuclear cell collector 15 may be embedded and encased in a gas-impermeable material. At least a portion of the mononuclear cell collector 15 may be formed by etching into a component. At least a portion of the mononuclear cell collector 15 may be formed by etching into a component and overlapping recesses. The volume of the mononuclear cell collector 15 may be variable.

[0178] like Figure 2 As shown, for example, a first opening 115 is provided at the bottom of the mononuclear cell recycler 15, and a second opening 116 is provided on the side of the mononuclear cell recycler 15. The position of the first opening 115 is lower than that of the second opening 116 in the direction of gravity.

[0179] A flow path 19 is connected to the first opening 115 of the mononuclear cell recycler 15. The flow path 19 may have a structure capable of sealing the interior relative to the outside air. The enclosed space including the interior of the flow path 19 may be configured to prevent the exchange of gases, viruses, microorganisms, and impurities with the outside. The flow path 19 may be embedded and encased in a gas-impermeable material. At least a portion of the flow path 19 may be formed by etching it into a component. At least a portion of the flow path 19 may be formed by etching it into a component and overlapping the recesses.

[0180] A flow path 117 is connected to the second opening 116 of the mononuclear cell recycler 15. The flow path 117 can have a structure capable of sealing the interior relative to the outside air. The enclosed space including the flow path 117 can be configured to prevent exchange of gases, viruses, microorganisms, and impurities with the outside. The flow path 117 can be embedded and encased in a gas-impermeable material. At least a portion of the flow path 117 can be formed by etching it into a component. At least a portion of the flow path 117 can be formed by etching it into a component and overlapping the recesses. Figure 1 As shown, a fluid machine 21, such as a pump, is provided in the flow path 117 for moving fluid within the flow path 117.

[0181] like Figure 2 As shown, the bottom of the mononuclear cell collector 15 can 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 mononuclear cell collector 15, and a second opening 116 is provided on the side of the funnel-shaped bottom. A filter that prevents mononuclear cells from passing through can be provided in the second opening 116.

[0182] The mononuclear cell recoverer 15 can internally contain diluents such as buffer solutions. The diluent can be drawn from the container holding the diluent. Figure 1 The diluent container 61 shown is introduced into the mononuclear cell recoverer 15 via flow path 60. The volume of the diluent container 61 can be varied. Additionally, flow paths 19 and 117 are filled with diluent, for example.

[0183] At least one of the dilution container 61 and the flow path 60 may have a structure capable of sealing the interior relative to the outside air. The enclosed space including the dilution container 61 and the flow path 60 may be configured to prevent exchange of gases, viruses, microorganisms, and impurities with the outside. The dilution container 61 and the flow path 60 may be embedded and encased in a gas-impermeable material. At least a portion of the dilution container 61 and the flow path 60 may be formed by etching into a component. At least a portion of the dilution container 61 and the flow path 60 may be formed by etching into a component and overlapping the recesses.

[0184] A flow path 17 is provided between the red blood cell remover 11 and the mononuclear cell collector 15 for conveying processed blood, from the red blood cell remover 11 to the mononuclear cell collector 15, after at least partial removal of red blood cells. The flow path 17 may have a structure capable of sealing its interior relative to external air. The enclosed space including the interior of the flow path 17 can be configured to prevent exchange of gases, viruses, microorganisms, and impurities with the outside. The flow path 17 may be embedded and encased in a gas-impermeable material. At least a portion of the flow path 17 may be formed by etching it into a component. At least a portion of the flow path 17 may be formed by etching it into a component and overlapping the recesses.

[0185] Additionally, a flow path 16 is provided between the red blood cell remover 11 and the mononuclear cell collector 15 for conveying fluids such as air or gas from the mononuclear cell collector 15 to the red blood cell remover 11. The flow path 16 may have a structure capable of sealing its interior relative to the external air. The enclosed space including the interior of the flow path 16 can be configured to prevent exchange of gases, viruses, microorganisms, and impurities with the outside. The flow path 16 may be embedded and encased in a gas-impermeable material. At least a portion of the flow path 16 can be formed by etching it into a component. At least a portion of the flow path 16 can be formed by etching it into a component and overlapping the recesses.

[0186] A fluid machine 18, such as a pump, is provided in flow path 17 to move the fluid within flow path 17. It should be noted that fluid machine can be provided in flow path 16, or in both flow path 16 and flow path 17.

[0187] With the mononuclear cell reclaimer 15 pre-filled with gas and diluent, if the fluid machinery 18 aspirates processed blood from the red blood cell remover 11 after at least partial removal of red blood cells via flow path 17, and supplies the aspirated processed blood with at least partial removal of red blood cells to the mononuclear cell reclaimer 15, the gas in the mononuclear cell reclaimer 15 is pressurized and transported to the red blood cell remover 11 via flow path 16. Thus, by transporting the processed blood from the red blood cell remover 11 with at least partial removal of red blood cells to the mononuclear cell reclaimer 15, and by transporting the gas from the mononuclear cell reclaimer 15 to the red blood cell remover 11, the pressure in the red blood cell remover 11 and the mononuclear cell reclaimer 15 can be averaged. The diluent can be repeatedly supplied from the dilution liquid container 61.

[0188] It should be noted that the fluid machinery 18 can draw gas from the mononuclear cell reclaimer 15 via the flow path 17 and supply the drawn gas to the red blood cell remover 11. In this case, the processed blood obtained by removing at least some red blood cells from the red blood cell remover 11 is propelled by gas pressure and transported to the mononuclear cell reclaimer 15 via the flow path 16. Thus, by removing gas from the mononuclear cell reclaimer 15, it is also possible to transport the processed blood obtained by removing at least some red blood cells from the red blood cell remover 11 to the mononuclear cell reclaimer 15.

[0189] While red blood cells are sedimented in the red blood cell remover 11, the supernatant in the red blood cell remover 11 is delivered to the mononuclear cell recoverer 15 in the form of processed blood with at least partial removal of red blood cells.

[0190] Processed blood obtained by at least partially removing red blood cells after being fed into the mononuclear cell recycler 15, such as... Figure 2 As shown in (a), the blood is diluted with a diluent. In the diluted processed blood solution, platelets are suspended, and monocytes settle to the bottom of the monocyte collector 15. It should be noted that the diluent may contain a red blood cell remover. In this case, the remaining red blood cells in the processed blood solution undergo hemolysis.

[0191] like Figure 2 As shown in (b), the settled mononuclear cells accumulate at the front end of the funnel-shaped bottom of the mononuclear cell collector 15. After settling in the diluted processed blood solution, as... Figure 2 As shown in (c), the flow path 117 is provided in connection with the second opening 116 of the mononuclear cell recycler 15. Figure 1 The fluid machinery 21 shown aspirates a diluted blood solution used as a supernatant. The suction force for aspirating the supernatant is set to be difficult to aspirate. Figure 2 (c) shows the sedimented monocytes. The supernatant contains platelets and hemolyzed red blood cells. Therefore, monocytes can be separated from platelets and red blood cells by aspirating and removing the supernatant from the monocyte recycler 15. The aspirated supernatant can be transported to... Figure 1 The red blood cell remover 11 or blood container 10 is shown. Additionally, a volume of gas, the same as the supernatant drawn from the mononuclear cell recoverer 15, can be transferred from the red blood cell remover 11 or blood container 10 to the mononuclear cell recoverer 15.

[0192] A mononuclear cell suction device 20 is provided in the flow path 19 to suction mononuclear cells accumulated at the bottom of the mononuclear cell collector 15. Fluid machinery such as a pump can be used as the mononuclear cell suction device 20. Figure 2The size of the first opening 115 shown is set, for example, in such a way that when the mononuclear cell aspiration device 20 is not aspirating mononuclear cells, the mononuclear cells block the first opening 115; when the mononuclear cell aspiration device 20 is aspirating mononuclear cells, the mononuclear cells can pass through the first opening 115. If the mononuclear cell aspiration device 20 aspirates mononuclear cells, the mononuclear cells move from the mononuclear cell collector 15 into the flow path 19.

[0193] It should be noted that the mononuclear cells in the mononuclear cell collector 15 can also be moved to the flow path 19 by pressurizing the mononuclear cell collector 15. In this case, a mononuclear cell suction device 20 can be provided in the flow path 19, or it can be omitted.

[0194] like Figure 1 As shown, the cell culture apparatus 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 its interior relative to the outside air. The sealed space including the interior of the cell culture vessel 22 may be configured to prevent exchange of gases, viruses, microorganisms, and impurities with the outside. The cell culture vessel 22 may be embedded and encapsulated in a gas-impermeable material. At least a portion of the cell culture vessel 22 may be formed by etching it into a component. At least a portion of the cell culture vessel 22 may be formed by etching it into a component and overlapping the recesses.

[0195] Cell culture vessel 22 can be used for both adhesion culture and suspension culture. For adhesion culture, cell adhesion coating agents such as matrix gel, collagen, polylysine, fibronectin, hyalin, and laminin can be used to coat the inside of the cell culture vessel 22. The following explanation uses suspension culture as an example. The interior of the cell culture vessel 22 can be divided by permeable components that allow culture medium components and waste to pass through, but which are impermeable to cells. Alternatively, cell-nonadhesive substances such as poly-HEMA (poly2-hydroxyethyl methacrylate) can be coated onto the inner wall of the cell culture vessel 22 in a cell-nonadhesive manner, making the inner wall of the cell culture vessel 22 cell-nonadhesive. Windows that allow observation of the interior can also be provided in the cell culture vessel 22. Materials for the windows include, for example, glass and resin.

[0196] The cell culture vessel 22 may be equipped with a temperature regulating unit for heating and cooling the window. This temperature regulating unit may be a transparent heater, such as a transparent conductive film disposed on the window and used to heat it. Alternatively, the cell culture vessel 22 may include 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 within the cell culture vessel 22 can be regulated. The cell culture vessel 22 may also include a thermometer for measuring the temperature of the culture medium within it. 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 it may directly measure the temperature of the culture medium by contacting it. In this case, feedback control of the temperature regulating unit may be implemented to ensure that the temperature of the culture medium reaches a predetermined temperature. For example, the temperature of the culture medium may be adjusted to 20°C to 45°C.

[0197] A flow path 19 is connected to the cell culture vessel 22. Cells are transported into the cell culture vessel 22 via the flow path 19. A flow path 23 is connected to the flow path 19. The flow path 23 may have a structure capable of sealing the interior relative to the external air. The enclosed space including the interior of the flow path 23 may be configured to prevent the exchange of gases, viruses, microorganisms, and impurities with the outside. The flow path 23 may be embedded and encased in a gas-impermeable material. At least a portion of the flow path 23 may be formed by etching it into a component. At least a portion of the flow path 23 may be formed by etching it into a component and overlapping the recesses. A fluid machine 24, such as a pump, is provided in the flow path 23 for moving fluid within the flow path 23.

[0198] A first culture medium container 25 is connected to the flow path 23 as a fluid container for holding somatic cell culture media, such as differentiation cell culture medium. The somatic cell culture medium can be a gel or a liquid.

[0199] When the culture medium is in gel form, it may contain polymeric compounds. These polymeric compounds may be, for example, at least one selected from gellan gum, deacylated gellan gum, hyaluronic acid, neutral rhamn gum, diutangum, xanthan gum, carrageenan, fucoidan, pectin, pectic acid, pectin esters, heparan sulfate, heparin, heparan sulfate, keratin sulfate, chondroitin sulfate, dermatan sulfate, rhamnose sulfate, and their salts. Additionally, the culture medium may contain methylcellulose. The inclusion of methylcellulose further inhibits cell aggregation.

[0200] Alternatively, the culture medium may contain poly(glycerolmonomethacrylate) (PGMA), poly(2-hydroxypropylmethacrylate) (PHPMA), poly(N-isopropylacrylamide) (PNIPAM), amine-terminated, carboxylic acid-terminated, maleimide-terminated, N-hydroxysuccinimide (NHS) ester-terminated, triethoxysilane-terminated poly(N-isopropylacrylamide-co-acrylamide) (Poly(N-isopropylacrylamide-co-acrylamide)), poly(N-isopropylacrylamide-co-acrylic acid) (Poly(N-isopropylacrylamide-co-acrylic acid)), selected from poly(N-isopropylacrylamide-co-acrylic acid) (PGMA), poly(2-hydroxypropylmethacrylate) (PHMA), poly(N-isopropylacrylamide-co-acrylic acid ... A small amount of thermosensitive gel selected from N-isopropylacrylamide (acid), poly(N-isopropylacrylamide-co-butylacrylate), poly(N-isopropylacrylamide-co-methacrylic acid), poly(N-isopropylacrylamide-co-methacrylic acid-co-octadecyl acrylate), and N-isopropylacrylamide.

[0201] It should be noted that, in this invention, the gel-like culture medium or gel culture medium includes a polymer culture medium.

[0202] When the cells transported from flow path 19 to cell culture vessel 22 are mononuclear cells, a somatic cell culture medium, such as blood cell culture medium, can be used. The first culture medium container 25 may have a structure capable of sealing its interior relative to external air. The sealed space including the interior of the first culture medium container 25 may be configured to prevent exchange of gases, viruses, microorganisms, and impurities with the outside. The first culture medium container 25 may be embedded and encased in a gas-impermeable material. At least a portion of the first culture medium container 25 may be formed by etching into a component. At least a portion of the first culture medium container 25 may be formed by etching into a component and overlapping recesses. The first culture medium container 25 may be able to change its volume. In this case, for example, the first culture medium container 25 includes a syringe for containing somatic cell culture medium and a plunger inserted into the syringe and movable within the syringe; by moving the plunger, the volume of somatic cell culture medium within the syringe can be changed. Alternatively, the first culture medium container 25 may also be a flexible bellows or bag.

[0203] If mononuclear cells are delivered from the mononuclear cell reclaimer 15 to the flow path 19, the fluid dynamics 24 delivers 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 capable of holding the somatic cell culture medium. It should be noted that the first culture medium container 25 can actively shrink its volume, or it can passively shrink its volume using suction force from within the flow path 23. The somatic cell culture medium delivered via the flow path 23 to the flow path 19 mixes with the mononuclear cells in the flow path 19 and is then delivered to the cell culture vessel 22.

[0204] A temperature control device can be installed in the first culture medium container 25 to regulate the temperature of the culture medium inside the first culture medium container 25.

[0205] A first variable-volume container 27 is connected to the cell culture vessel 22, for example, via a flow path 26. The flow path 26 may have a structure capable of sealing the interior relative to the external air. The enclosed space including the interior of the flow path 26 may be configured to prevent exchange of gases, viruses, microorganisms, and impurities with the outside. The flow path 26 may be embedded and encased in a gas-impermeable material. At least a portion of the flow path 26 may be formed by etching into a component. At least a portion of the flow path 26 may be formed by etching into a component and overlapping the recesses. Fluid machinery 28, such as a pump, may be provided in the flow path 26 for moving fluid within the flow path 26.

[0206] The first variable-volume container 27 can have a structure capable of sealing its interior relative to the outside air. The enclosed space including the interior of the first variable-volume container 27 can be configured to prevent exchange of gases, viruses, microorganisms, and impurities with the outside. The first variable-volume container 27 can be embedded and encased in a gas-impermeable material. At least a portion of the first variable-volume container 27 can be formed by engraving it into a component. At least a portion of the first variable-volume container 27 can be formed by engraving it into a component and overlapping the recesses. The first variable-volume container 27 can have its volume changed. In this case, for example, the first variable-volume container 27 includes a syringe for containing fluid and a plunger inserted into the syringe and movable within the syringe; by moving the plunger, the volume of fluid contained within the syringe can be changed. Alternatively, the first variable-volume container 27 can also be a flexible bellows or bag.

[0207] A second variable-volume container 30 is connected to the cell culture vessel 22, for example, via a flow path 29. The flow path 29 may have a structure capable of sealing the interior relative to the outside air. The enclosed space including the interior of the flow path 29 may be configured to prevent exchange of gases, viruses, microorganisms, and impurities with the outside. The flow path 29 may be embedded and encased in a gas-impermeable material. At least a portion of the flow path 29 may be formed by etching into a component. At least a portion of the flow path 29 may be formed by etching into a component and overlapping recesses. Fluid machinery such as a pump for moving fluid within the flow path 29 may be provided in the flow path 29.

[0208] The second variable-volume container 30 can have a structure capable of sealing its interior relative to the outside air. The enclosed space including the interior of the second variable-volume container 30 can be configured to prevent exchange of gases, viruses, microorganisms, and impurities with the outside. The second variable-volume container 30 can be embedded and encased in a gas-impermeable material. At least a portion of the second variable-volume container 30 can be formed by engraving it into a component. At least a portion of the second variable-volume container 30 can be formed by engraving it into a component and overlapping the recesses. The second variable-volume container 30 can have its volume changed. In this case, for example, the second variable-volume container 30 includes a syringe for containing fluid and a plunger inserted into the syringe and movable within the syringe; by moving the plunger, the volume of fluid contained within the syringe can be changed. Alternatively, the second variable-volume container 30 can also be a flexible bellows or bag.

[0209] If mononuclear cell and somatic cell culture media are introduced into the cell culture vessel 22 through flow path 19, gases such as air within the cell culture vessel 22 will move into the second variable-volume container 30, which will then expand to receive the gases moving from the cell culture vessel 22. It should be noted that the second variable-volume container 30 can expand actively or passively under pressure.

[0210] The first variable-volume container 27 may contain substances such as inducing factors that induce cells in state 1 to become cells in state 2. The inducing factor may be RNA, protein, or a compound. The RNA may be modified RNA or unmodified RNA. The first variable-volume container 27 may, for example, contain lipid transfection reagents. The inducing factor may be contained in a plasmid vector, or a retroviral vector, lentiviral vector, or Sendai virus vector, or other viral vectors or viruses. In this invention, induction refers to reprogramming, initialization, transformation, transdifferentiation or lineage reprogramming, differentiation induction, and cell fate reprogramming. Reprogramming factors may include, for example, OCT3 / 4, SOX2, KLF4, and c-MYC. When introducing inducing factors such as reprogramming factors into monocytes to create iPS cells, the fluid dynamics 28 move the somatic cell culture medium containing monocytes in the cell culture vessel 22 to the first variable-volume container 27 via the flow path 26. Additionally, the first variable-volume container 27 expands to receive somatic cell culture medium containing monocytes. It should be noted that the first variable-volume container 27 can expand actively or passively under pressure. The second variable-volume container 30, containing gas, contracts, and the contained gas is introduced into the cell culture vessel 22. It should be noted that the second variable-volume container 30 can contract actively or passively under suction from within the cell culture vessel 22.

[0211] Monocytes move from cell culture vessel 22 into the first variable-volume container 27, coming into contact with the inducing factor within the first variable-volume container 27, thus introducing the inducing factor into the monocytes. It should be noted that the first variable-volume container 27 can repeatedly expand and contract to agitate the somatic cell culture medium containing monocytes and the inducing factor.

[0212] After a predetermined period, the fluid mechanism 28 moves the somatic cell culture medium containing mononuclear cells infused with inducing factors within the first variable-volume container 27 through the flow path 26 into the cell culture vessel 22. The first variable-volume container 27 then contracts. Simultaneously, the second variable-volume container 30 expands, receiving gas from the cell culture vessel 22.

[0213] Alternatively, when creating iPS cells by introducing reprogramming factors or other inducing factors into monocytes, the fluid dynamics 28 can move the inducing factors in the first variable-volume container 27 to the cell culture vessel 22 via the flow path 26. In this case, the first variable-volume container 27 can shrink, and the second variable-volume container 30 can expand. The inducing factors move 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 inducing factors into the monocytes. It should be noted that the fluid dynamics 28 can also move the inducing factors in the first variable-volume container 27 into the cell culture vessel 22 in multiple stages via the flow path 26. This results in the inducing factors being introduced into the monocytes in multiple stages.

[0214] The cell culture apparatus 22 is connected, for example, via a flow path 31, to a second culture medium container 32, which serves as a fluid container for holding culture media such as stem cell culture medium or somatic cell culture medium. Hereinafter, an example of the second culture medium container 32 containing stem cell culture medium will be described. The stem cell culture medium can be a gel or a liquid. Induction medium, expansion medium, and maintenance medium can be used as stem cell culture media.

[0215] The flow path 31 can have a structure capable of sealing the interior relative to the external air. The enclosed space including the interior of the flow path 31 can be configured to prevent the exchange of gases, viruses, microorganisms, and impurities with the outside. The flow path 31 can be embedded and encased in a gas-impermeable material. At least a portion of the flow path 31 can be formed by etching it into a component. At least a portion of the flow path 31 can be formed by etching it into a component and overlapping the recesses. Fluid machinery 33, such as a pump, for moving fluid within the flow path 31 can be provided in the flow path 31.

[0216] The second culture medium container 32 can have a structure capable of sealing the interior relative to the external air. The enclosed space including the interior of the second culture medium container 32 can be configured to prevent exchange of gases, viruses, microorganisms, and impurities with the outside. The second culture medium container 32 can be embedded and encased in a gas-impermeable material. At least a portion of the second culture medium container 32 can be formed by engraving it into a component. At least a portion of the second culture medium container 32 can be formed by engraving it into a component and overlapping the recesses. The second culture medium container 32 can have its volume varied. In this case, for example, the second culture medium container 32 includes a syringe for containing fluid and a plunger inserted into the syringe and movable within the syringe; by moving the plunger, the volume of fluid contained within the syringe can be changed. Alternatively, the second culture medium container 32 can also be a flexible bellows or bag.

[0217] A temperature control device can be installed in the second culture medium container 32 to regulate the temperature of the culture medium inside the second culture medium container 32.

[0218] The cell culture vessel 22 is connected, for example, to a third variable-volume container 35 via a flow path 34. The flow path 34 may have a structure capable of sealing the interior relative to the outside air. The enclosed space including the interior of the flow path 34 may be configured to prevent exchange of gases, viruses, microorganisms, and impurities with the outside. The flow path 34 may be embedded and encased in a gas-impermeable material. At least a portion of the flow path 34 may be formed by etching into a component. At least a portion of the flow path 34 may be formed by etching into a component and overlapping recesses.

[0219] The third variable volume container 35 can have a structure capable of sealing its interior relative to the outside air. The enclosed space including the interior of the third variable volume container 35 can be configured to prevent exchange of gases, viruses, microorganisms, and impurities with the outside. The third variable volume container 35 can be embedded and encased in a gas-impermeable material. At least a portion of the third variable volume container 35 can be formed by engraving it into a component. At least a portion of the third variable volume container 35 can be formed by engraving it into a component and overlapping the recesses. The third variable volume container 35 can change its volume. In this case, for example, the third variable volume container 35 includes a syringe for containing fluid and a plunger inserted into the syringe and movable within the syringe; by moving the plunger, the volume of fluid contained within the syringe can be changed. Alternatively, the third variable volume container 35 can also be a flexible bellows or bag.

[0220] After a predetermined period following the introduction of inducing factors into monocytes, fluid dynamics 33 moves the stem cell culture medium in the second culture medium container 32 to the cell culture vessel 22 via flow path 31. The stem cell culture medium can contact the cell-containing sections within the cell culture vessel 22, defined by the culture medium component permeability component, and be added to cell-free sections. The second culture medium container 32, having internally aspirated the stem cell culture medium, shrinks in volume. It should be noted that the second culture medium container 32 can shrink in volume actively or passively. The third variable-volume container 35 expands in volume, receiving the remaining fluid within the cell culture vessel 22 due to the inflow of stem cell culture medium via flow path 34. Flow path 34 can contact the cell-containing sections within the cell culture vessel 22, defined by the culture medium component permeability component, and connect to cell-free sections. It should be noted that the third variable-volume container 35 can expand in volume actively or passively under pressure.

[0221] Alternatively, flow path 34 can contact the cell compartments within the cell culture vessel 22 that contain cells, defined by the culture medium components permeating the compartments. In this case, remaining cells within the cell culture vessel 22 can be delivered via flow path 34 to the third variable-volume container 35.

[0222] Within the cell culture apparatus 22, the culture medium in the cell-containing zone is separated from the cell-free zone by a culture medium component permeability component, for example, by osmotic pressure to exchange culture medium components and waste. The culture component permeability component can be, for example, a semi-permeable membrane, a sieve, or a hollow fiber membrane. Semi-permeable membranes include dialysis membranes.

[0223] When the component through which the cultured components permeate is a semipermeable membrane, the molecular weight cutoff of the semipermeable membrane is, for example, 0.1 kDa or higher, 10 kDa or higher, or 50 kDa or higher. The semipermeable membrane is formed, for example, from cellulose esters, ethyl cellulose, cellulose esters, regenerated cellulose, polysulfone, polyacrylonitrile, polymethyl methacrylate, ethylene-vinyl alcohol copolymer, polyester polymer alloys, polycarbonate, polyamide, cellulose acetate, cellulose diacetate, cellulose triacetate, cuprammonium rayon, saponified cellulose, blood formaldehyde membrane, phosphatidylcholine membrane, and vitamin E coating.

[0224] When the culture component permeable member is a sieve, the sieve has pores smaller than those of the cells cultured in the cell culture vessel 22. The material of the sieve is, for example, resin and metal, but is not particularly limited. The surface of the culture component permeable member can be cell-non-adhesive.

[0225] In the case where the culture component permeation member is a hollow fiber membrane, the hollow fiber membrane has pores smaller than those of the cells cultured within the cell culture vessel 22. For example, cells can be cultured on the inside of the hollow fiber membrane.

[0226] During cell culture in cell culture vessel 22, at predetermined times, fluid dynamics 33 moves stem cell culture medium from the second culture medium container 32 into the cell culture vessel 22 via flow path 31. The third variable-volume container 35 expands to receive the remaining used stem cell culture medium in the cell culture vessel 22, which is then filled with fresh stem cell culture medium. Fluid dynamics 33 can, for example, control the flow rate of culture medium or initiate and terminate the flow of culture medium based on changes in 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.

[0227] Cells can be present in partitions within the cell culture apparatus 22, defined by a culture medium component permeable component, and connected to fluid machinery 37 such as a pump via a flow path 36. The flow path 36 can have a structure capable of sealing the interior relative to external air. The enclosed space including the interior of the flow path 36 can be configured to prevent exchange of gases, viruses, microorganisms, and impurities with the outside. The flow path 36 can be embedded and encased in a gas-impermeable material. At least a portion of the flow path 36 can be formed by etching into a component. At least a portion of the flow path 36 can be formed by etching into a component and overlapping recesses.

[0228] For example, to control cell aggregation, the fluid mechanism 37 circulates the culture medium between the cell culture vessel 22 and the flow path 36, within the partitions defined by the culture medium component permeability component. The fluid mechanism 37 can circulate the culture medium continuously or at any time. Alternatively, the fluid mechanism 37 can agitate the culture medium by reciprocating between the cell culture vessel 22 and the cell culture vessel 22, within the partitions defined by the culture medium component permeability component and the flow path 36. The fluid mechanism 37 can agitate the culture medium continuously or at any time. The fluid mechanism 37 can, for example, control the volume of culture medium delivered, or initiate and terminate the delivery of culture medium, based on changes in 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 of the culture medium.

[0229] Cells can be passaged and expanded by aspirating cells from cell culture vessel 22 into flow path 36 and then returning them to cell culture vessel 22. Flow path 36 may have structures that divide cell clusters. For example, by having meandering structures or structures with increasing or decreasing diameters within flow path 36, cell clusters flowing within flow path 36 can be divided.

[0230] Within the cell culture apparatus 22, partitions defined by the culture medium components can be formed, with no cells present in the partitions. These partitions are connected to fluid machinery 39, such as pumps, via flow paths 38. Flow paths 38 can have a structure capable of sealing the interior relative to external air. The enclosed space, including the interior of the flow path 38, can be configured to prevent exchange of gases, viruses, microorganisms, and impurities with the outside. Flow paths 38 can be embedded and encased in a gas-impermeable material. At least a portion of the flow path 38 can be formed by etching into a component. At least a portion of the flow path 38 can be formed by etching into a component and overlapping recesses.

[0231] For example, to increase the opportunity for contact between the culture medium and its components through the permeation mechanism, the fluid mechanism 39 circulates the culture medium within the cell culture vessel 22 between the cell culture vessel 22, where there are no cells in the partitions defined by the permeation mechanism, and the flow path 38. The fluid mechanism 39 can circulate the culture medium continuously or at any time. Alternatively, the fluid mechanism 39 can agitate the culture medium by causing it to reciprocate between the cell culture vessel 22, where there are no cells in the partitions defined by the permeation mechanism, and the flow path 38. The fluid mechanism 39 can agitate the culture medium continuously or at any time. The fluid mechanism 39 can, for example, control the volume of culture medium delivered, or initiate and terminate the delivery of culture medium, based on 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 changes in pH.

[0232] For example, iPS cells can be created from monocytes in which inducing factors have been introduced into cell culture vessel 22. After expansion culture, the iPS cells can be recovered from cell culture vessel 22. iPS cells can form cell clusters (colonies) within cell culture vessel 22.

[0233] According to the inventors' understanding, cells can be cultured in a completely closed, sealed space, therefore, it is not necessary to actively supply carbon dioxide, nitrogen, and oxygen to the cell culture vessel 22. Therefore, it is also not necessary to place the cell culture vessel 22 in a CO2 incubator. Furthermore, cells, microorganisms, viruses, and dust existing outside the cell culture vessel 22 will not enter the sealed cell culture vessel 22, thus maintaining the cleanliness within the cell culture vessel 22. Therefore, it is not necessary to place the cell culture vessel 22 in a cleanroom. However, this does not necessarily preclude the supply of carbon dioxide, nitrogen, and oxygen to the closed system in which the cells reside.

[0234] The cell culture apparatus 200 according to the embodiment, for example, culturees cells in a completely closed system, thus reducing the risk of cross-contamination caused by cell leakage from the culture apparatus. Furthermore, even if the cells are infected with viruses such as HIV or hepatitis viruses, the risk of infection to operators due to cell leakage can be reduced. In addition, the risk of the culture medium inside the cell culture apparatus being contaminated by bacteria, viruses, and molds in the air outside the cell culture apparatus can be reduced. Furthermore, the cell culture apparatus according to the embodiment can also culture cells without using a CO2 incubator.

[0235] (Second Implementation)

[0236] 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 treatment agent container 53 for containing red blood cell sedimentation agent or red blood cell removal agent.

[0237] Blood container 50 contains blood internally. Blood container 50 may have a structure capable of sealing the interior relative to external air. The enclosed space including the interior of blood container 50 may be configured to prevent exchange of gases, viruses, microorganisms, and impurities with the outside. Blood container 50 may be embedded and encased in a gas-impermeable material. At least a portion of blood container 50 may be formed by engraving into a component. At least a portion of blood container 50 may be formed by engraving into a component and overlapping recesses. Blood container 50 may be capable of changing its volume. In this case, for example, blood container 50 includes a syringe containing fluid and a plunger inserted into the syringe and movable within the syringe; by moving the plunger, the volume of fluid contained within the syringe can be changed. Alternatively, blood container 50 may also be a flexible bellows or bag.

[0238] The red blood cell treatment container 53 contains a red blood cell settling agent or a red blood cell removal agent. The red blood cell treatment container 53 may have a structure capable of sealing the interior relative to the outside air. The enclosed space including the interior of the red blood cell treatment container 53 may be configured to prevent exchange of gases, viruses, microorganisms, and impurities with the outside. The red blood cell treatment container 53 may be embedded and encased in a gas-impermeable material. At least a portion of the red blood cell treatment container 53 may be formed by etching into a component. At least a portion of the red blood cell treatment container 53 may be formed by etching into a component and overlapping recesses. The red blood cell treatment container 53 may be able to change its volume. In this case, for example, the red blood cell treatment container 53 includes a syringe containing fluid and a plunger inserted into the syringe and movable within the syringe; by moving the plunger, the volume of fluid contained within the syringe can be changed. Alternatively, the red blood cell treatment container 53 may also be a flexible bellows or bag.

[0239] The red blood cell removal device 101 of the second embodiment also includes a mixer 57, for example, for mixing blood with a red blood cell settling agent or a red blood cell removal agent. The mixer 57, for example, has a zigzag flow path for the flow of the mixture of blood and the red blood cell settling agent or red blood cell removal agent. The zigzag flow path can be zigzag-shaped. The flow path can be meandering. The cross-sectional area can be repeatedly increased or decreased in the zigzag flow path. The mixer 57 can have a structure capable of sealing the interior relative to the external air. The enclosed space including the interior of the mixer 57 can be configured to prevent exchange of gases, viruses, microorganisms, and impurities with the outside. The mixer 57 can be embedded and encased in a gas-impermeable material. At least a portion of the mixer 57 can be formed by etching it into a component. At least a portion of the mixer 57 can be formed by etching it into a component and overlapping the recesses.

[0240] A flow path 51 is connected to the blood container 50 for conveying at least blood from the blood container 50 to the mixer 57. A flow path 54 is connected to the red blood cell treatment agent container 53 for conveying at least red blood cell settling agent or red blood cell removal agent from the red blood cell treatment agent container 53 to the mixer 57. Flow paths 51 and 54 merge with flow path 56. Flow path 56 is connected to the mixer 57. A flow path 58 is connected to the mixer 57 for conveying the mixture of blood and red blood cell settling agent or red blood cell removal agent mixed in the mixer 57 to the red blood cell removal device 11.

[0241] Fluid machinery such as a pump 52 for moving fluid within the flow path 51 can be installed in the flow path 51. Fluid machinery such as a pump 55 for moving fluid within the flow path 54 can be installed in the flow path 54.

[0242] Flow paths 51, 54, 56, and 58 can have a structure capable of sealing the interior relative to the external air. The enclosed space within flow paths 51, 54, 56, and 58 can be configured to prevent exchange of gases, viruses, microorganisms, and impurities with the outside. Flow paths 51, 54, 56, and 58 can be embedded and encased in a gas-impermeable material. At least a portion of flow paths 51, 54, 56, and 58 can be formed by etching into a component. At least a portion of flow paths 51, 54, 56, and 58 can be formed by etching into a component and overlapping the recesses.

[0243] When delivering a mixture of blood and erythrocyte sedimentation agent or erythrocyte removal agent to the erythrocyte remover 11, fluid machinery 52 moves the blood in the blood container 50 through flow paths 51 and 56 into the mixer 57. Additionally, fluid machinery 55 moves the erythrocyte sedimentation agent or erythrocyte removal agent in the erythrocyte treatment agent container 53 through flow paths 54 and 56 into the mixer 57. It should be noted that fluid machinery may not be installed in flow paths 51 and 54, but instead in flow path 56. The fluid machinery in flow path 56 moves the blood in the blood container 50 and the erythrocyte sedimentation agent or erythrocyte removal agent in the erythrocyte treatment agent container 53 into the mixer 57. In the mixer 57, the blood is mixed with the erythrocyte sedimentation agent or erythrocyte removal agent. The mixture of blood and erythrocyte sedimentation agent or erythrocyte removal agent in the mixer 57 is then delivered to the erythrocyte remover 11 through flow path 58. In the case of red blood cell sedimentation or hemolysis within the red blood cell remover 11, the situation is the same as in the first embodiment. Furthermore, other components of the red blood cell remover 101 in the second embodiment may also be the same as those in the red blood cell remover 100 of the first embodiment.

[0244] (Third Implementation)

[0245] like Figure 4 As shown, the red blood cell removal apparatus 101 of the third embodiment includes a vacuum container 70, which is provided in a flow path 51 for conveying at least blood from the blood container 50 to the mixer 57, and is capable of making the interior a vacuum.

[0246] The vacuum container 70 can have a structure capable of sealing the interior relative to the outside air. The enclosed space including the interior of the vacuum container 70 can be configured to prevent exchange of gases, viruses, microorganisms, and impurities with the outside. The vacuum container 70 can be embedded and encased in a gas-impermeable material. At least a portion of the vacuum container 70 can be formed by etching into a component. At least a portion of the vacuum container 70 can be formed by etching into a component and overlapping recesses. The vacuum container 70 can have its volume varied. The vacuum container 70 can also be a flexible bellows or bag.

[0247] The red blood cell removal apparatus 101 of the third embodiment includes a vacuum container 71, which is provided in a flow path 54 for conveying at least a red blood cell settling agent or a red blood cell removal agent from the red blood cell treatment agent container 53 to the mixer 57, and is capable of making the interior a vacuum.

[0248] The vacuum container 71 can have a structure capable of sealing the interior relative to the outside air. The enclosed space including the interior of the vacuum container 71 can be configured to prevent exchange of gases, viruses, microorganisms, and impurities with the outside. The vacuum container 71 can be embedded and encased in a gas-impermeable material. At least a portion of the vacuum container 71 can be formed by etching it into a component. At least a portion of the vacuum container 71 can be formed by etching it into a component and overlapping the recesses. The vacuum container 71 can have its volume varied. The vacuum container 71 can also be a flexible bellows or bag.

[0249] If the blood container 50 is connected to the flow path 51 while the vacuum container 70 is already under vacuum, the blood in the blood container 50 moves into the vacuum container 70, and then flows through the flow paths 51 and 56 into the mixer 57. Alternatively, if the red blood cell treatment agent container 53 is connected to the flow path 54 while the vacuum container 71 is already under vacuum, the red blood cell settling agent or red blood cell removal agent in the red blood cell treatment agent container 53 moves into the vacuum container 71, and then flows through the flow paths 54 and 56 into the mixer 57.

[0250] Other components of the red blood cell removal device 101 in the third embodiment can be the same as those in the second embodiment.

[0251] (Fourth implementation)

[0252] It can also be omitted Figure 4 The vacuum containers 70 and 71 shown are pre-vacuumed in the red blood cell remover 11. If the blood container 50 is connected to the flow path 51 and the red blood cell treatment agent container 53 is connected to the flow path 54 while the red blood cell remover 11 is pre-vacuumed, the blood in the blood container 50 moves to the mixer 57 via the flow paths 51 and 56, and the red blood cell settling agent or red blood cell remover in the red blood cell treatment agent container 53 moves to the mixer 57 via the flow paths 54 and 56. Furthermore, the blood and red blood cell settling agent or red blood cell remover mixed in the mixer 57 move back to the red blood cell remover 11 via the flow path 58.

[0253] Alternatively, if flow paths 51 and 54 are blocked by valves or the like to create a vacuum inside the red blood cell remover 11, and then the valves in flow paths 51 and 54 are opened, the blood in the blood container 50 moves through flow paths 51 and 56 into the mixer 57, and the red blood cell settling agent or red blood cell remover in the red blood cell treatment agent container 53 moves through flow paths 54 and 56 into the mixer 57. Furthermore, the blood and red blood cell settling agent or red blood cell remover mixed in the mixer 57 move through flow path 58 into the red blood cell remover 11.

[0254] (Other implementation methods)

[0255] As described above, the present invention has been illustrated through embodiments, but it should not be construed as limiting the invention to the descriptions and drawings that constitute a part of this disclosure. According to the present invention, those skilled in the art will understand various alternative embodiments, implementation methods, and techniques employed. For example, conveying to... Figure 1 The cells in the cell culture vessel 22 shown are not limited to monocytes. Cells delivered to the cell culture vessel 22 can be stem cells, fibroblasts, or other somatic cells. The cells delivered to the cell culture vessel 22 are arbitrary.

[0256] Furthermore, in the first embodiment, an example of creating iPS cells from mononuclear cells within the cell culture vessel 22 was described, but differentiated cells such as neural cells can also be created from stem cells within the cell culture vessel 22. The stem cells can be iPS cells, embryonic stem cells (ES cells), adult stem cells, or other artificially induced stem cells, etc. In this case, for example, the first variable-volume container 27 contains differentiation-inducing factors. Thus, it should be understood that the present invention includes various embodiments, etc.

[0257] Example

[0258] (Example 1)

[0259] This embodiment illustrates an example of culturing cells in a completely enclosed environment without changing the culture medium or gas. Growth factors were added to the culture medium (StemSpan H3000, registered trademark, STEMCELL Technologies Inc.), and deacylated gellan gel was further added to the culture medium to prepare a gel medium.

[0260] Place the prepared gel culture medium into a 15 mL tube, and inoculate 2 × 10⁶ cells / mL into the gel culture medium. 5 A number of blood cells were cultured. Then, 15 mL tubes were placed in a CO2 incubator and the blood cells (monocytes) were cultured for 7 days. Then, Sendai virus vector carrying OCT3 / 4, SOX2, KLF4, and cMYC was added to gel medium at a multiplicity of infection (MOI) of 10.0 to infect the blood cells with Sendai virus.

[0261] After adding Sendai virus to the gel medium, 15 mL of gelled stem cell culture medium (DMEM / F12 containing 20% ​​KnockOut SR (registered trademark, ThermoFisher SCIENTIFIC)) was added to the gel medium. 15 mL of this medium, containing cells infected with Sendai virus, was then placed into a sealable cell culture vessel, and the gel medium was injected into the vessel. The cell culture vessel was then sealed to completely prevent gas exchange between the inside and outside of the vessel.

[0262] Suspension culture of cells infused with initialization factors was initiated in a cell culture vessel. Then, 2 mL of gel culture medium in the culture medium holder 40 was replaced with 2 mL of fresh gel culture medium every 2 days.

[0263] Fifteen days later, the cells were observed under a microscope, and the results were as follows: Figure 5 As shown, ES cell-like colonies were confirmed to have formed. Furthermore, cells were fixed using 4% paraformaldehyde, and the expression level of the cell surface antigen TRA-1-60 in the fixed cells was determined by flow cytometry. The results are as follows: Figure 6 As shown, over 90% were TRA-1-60 positive, confirming almost complete reprogramming. This indicates that iPS cells can be induced from somatic cells other than stem cells in a completely closed environment without culture medium or gas replacement.

[0264] (Example 2)

[0265] Blood was treated with a erythrocyte sedimentation agent to obtain treated blood with at least partial removal of red blood cells. The treated blood was then treated with surface cell marker antibodies and analyzed by fluorescence-activated cell sorting (FACS). The results are presented in [the table / document / etc.]. Figure 7 The processed blood contains 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.

[0266] The processed blood, from which at least some red blood cells have been removed, is placed into... Figure 2 In a mononuclear cell recoverer as shown, the cells are diluted with buffer to remove the supernatant. Then, the mononuclear cells are recovered from the recoverer. Figure 8 As shown in (a), the processed blood before being placed into the mononuclear cell recoverer contains a large number of platelets. On the other hand, as Figure 8As shown in (b), platelets in the mononuclear cell-containing solution recovered from the mononuclear cell recoverer are almost completely removed. A graph representing the number of platelets in the treated blood before being placed into the mononuclear cell recoverer and the number of platelets in the mononuclear cell-containing solution recovered from the mononuclear cell recoverer is shown in [the figure]. Figure 9 .

[0267] If processed blood containing platelets is placed into culture medium before being placed into the mononuclear cell recoverer, then... Figure 10 As shown in (a), agglutination occurred. In contrast, if the solution containing platelet-free monocytes recovered from the monocyte recycler is placed into the culture medium, then as shown in (a), aggregation occurs. Figure 10 As shown in (b), no aggregation occurred.

[0268] (Example 3)

[0269] Add deacylated gellan gel to blood culture medium to prepare gel medium. Place the prepared gel medium into 6-well dishes coated with laminin, and inoculate 2 × 10⁶ cells / well. 5 Blood cells (monocytes) were collected. Six-well dishes were then placed in a CO2 incubator at 37°C and cultured for 7 days. Then, Sendai virus vector (CytoTune-iPS2.0, ThermoFisher SCIENTIFIC) carrying OCT3 / 4, SOX2, KLF4, and cMYC was added to blood proliferation medium at a multiplicity of infection (MOI) of 5 to infect the blood cells with Sendai virus.

[0270] With the cells placed in 6-well dishes, Sendai virus was added to the blood proliferation medium for two days, and then the medium was replaced with 500 μL of stem cell medium (DMEM / F12 containing 20% ​​KnockOut SR (registered trademark, ThermoFisher SCIENTIFIC)) or Stem Fit (Japanese original: ステムフィット).

[0271] After adding Sendai virus to the blood proliferation culture medium for 15 days, the cells were observed under a microscope, and the results were as follows: Figure 11 As shown, ES cell-like colonies were confirmed to have formed. Furthermore, the cells were fixed with 4% paraformaldehyde, and the expression level of the cell surface antigen TRA-1-60 in the fixed cells was measured using flow cytometry. The results are as follows: Figure 12 As shown, the induced cells were confirmed to be almost 100% TRA-1-60 positive, indicating near-complete reprogramming. Therefore, this demonstrates that introducing reprogramming factors into cells within a cell culture vessel and culturing the introduced cells in the same vessel enables cell reprogramming.

[0272] (Example 4)

[0273] Add deacylated gellan gel to blood culture medium to prepare gel medium. Place the prepared gel medium into laminin-coated flasks and inoculate at 5 × 10⁶ cells / day. 5 Blood cells (monocytes) were cultured for 7 days in a CO2 incubator at 37°C. Then, Sendai virus vector (CytoTune-iPS2.0, ThermoFisherSCIENTIFIC) carrying OCT3 / 4, SOX2, KLF4, and cMYC was added to blood proliferation medium at a multiplicity of infection (MOI) of 5 to infect the blood cells with Sendai virus.

[0274] Two days after adding Sendai virus to the blood proliferation medium, the flask is completely filled with stem cell culture medium (DMEM / F12 containing 20% ​​KnockOut SR (registered trademark, ThermoFisher SCIENTIFIC)) or StemFit in a way that leaves no air inside. The flask is then capped to prevent gas exchange with the outside, thus sealing the interior of the flask in a way that prevents cells, microorganisms, and impurities from passing through.

[0275] After adding Sendai virus to the blood proliferation culture medium for 15 days, the cells were observed under a microscope, and the results were as follows: Figure 13 As shown, ES cell-like colonies were confirmed to have formed. Furthermore, the cells were fixed with 4% paraformaldehyde, and the expression level of the cell surface antigen TRA-1-60 in the fixed cells was measured using flow cytometry. The results are as follows: Figure 14 As shown, the induced cells were almost 100% TRA-1-60 positive, indicating near-complete reprogramming. Therefore, this demonstrates that introducing reprogramming factors into cells within a cell culture vessel and culturing the cells with the introduced reprogramming factors in the same closed cell culture vessel enables cell reprogramming.

[0276] (Example 5)

[0277] Non-gel-like liquid blood proliferation medium was placed into 6-well dishes coated with laminin, and 2 × 10⁶ cells were inoculated. 5 Blood cells (monocytes) were collected. Six-well dishes were then placed in a CO2 incubator at 37°C and cultured for 7 days. Then, Sendai virus vector (CytoTune-iPS2.0, ThermoFisher SCIENTIFIC) carrying OCT3 / 4, SOX2, KLF4, and cMYC was added to blood proliferation medium at a multiplicity of infection (MOI) of 5 to infect the blood cells with Sendai virus.

[0278] Two days after adding Sendai virus to blood proliferation medium while the cells were placed in 6-well dishes, the medium was replaced with 500 μL of stem cell medium (DMEM / F12 containing 20% ​​KnockOut SR (registered trademark, ThermoFisher SCIENTIFIC)) or Stem Fit.

[0279] Fifteen days after adding Sendai virus to the blood proliferation culture medium, the cells were observed under a microscope, and the results were as follows: Figure 15 As shown, ES cell-like colonies were confirmed to have formed. Furthermore, the cells were fixed with 4% paraformaldehyde, and the expression level of the cell surface antigen TRA-1-60 in the fixed cells was measured using flow cytometry. The results are as follows: Figure 16 As shown, the induced cells were confirmed to be almost 100% TRA-1-60 positive, indicating near-complete reprogramming. Therefore, this demonstrates that introducing reprogramming factors into cells within a cell culture vessel and culturing the introduced cells in the same vessel enables cell reprogramming.

[0280] (Example 6)

[0281] Non-gel-like liquid blood proliferation medium was placed into a laminin-coated flask and inoculated at 5 × 10⁶ cells / year. 5 A number of blood cells (monocytes) were collected. The flasks were then placed in a CO2 incubator at 37°C and cultured for 7 days. Then, Sendai virus vector (CytoTune-iPS2.0, ThermoFisher SCIENTIFIC) carrying OCT3 / 4, SOX2, KLF4, and cMYC was added to the blood proliferation medium at a multiplicity of infection (MOI) of 5, allowing the Sendai virus to infect the blood cells.

[0282] Two days after adding Sendai virus to the blood proliferation medium, the flask is completely filled with stem cell culture medium (DMEM / F12 containing 20% ​​KnockOut SR (registered trademark, ThermoFisher SCIENTIFIC)) or StemFit in a way that leaves no air inside. The flask is then capped to prevent gas exchange with the outside, thus sealing the interior of the flask in a way that prevents cells, microorganisms, and impurities from passing through.

[0283] After adding Sendai virus to the blood proliferation culture medium for 15 days, the cells were observed under a microscope, and the results were as follows: Figure 17 As shown, ES cell-like colonies were confirmed to have formed. Furthermore, the cells were fixed with 4% paraformaldehyde, and the expression level of the cell surface antigen TRA-1-60 in the fixed cells was measured using flow cytometry. The results are as follows: Figure 18As shown, the induced cells were confirmed to be almost 100% TRA-1-60 positive, indicating near-complete reprogramming. Therefore, this demonstrates that introducing reprogramming factors into cells within a cell culture vessel and culturing the introduced cells in the same closed cell culture vessel enables cell reprogramming.

Claims

1. A method for recovering monocytes, characterized by, comprises: processing blood to produce treated blood from which at least red blood cells are removed; diluting the treated blood; adding the diluted treated blood into a container to allow mononuclear cells contained in the diluted treated blood to settle and platelets to suspend; removing supernatant of the diluted treated blood containing the suspended platelets; and recovering the mononuclear cells by suctioning the mononuclear cells accumulated at the bottom of the container from an opening provided at the bottom of the container. the treated blood is produced in a red blood cell remover, 2. The method for recovering monocytes according to claim 1, wherein the dilution of the treated blood, the settlement of the mononuclear cells, and the removal of the supernatant are performed in a mononuclear cell recycler, and the red blood cell remover and the mononuclear cell recycler are closed. the blood is processed with a red blood cell settling agent or a red blood cell removing agent.

3. The method of recovering monocytes according to claim 1 or 2, wherein at least one of platelets and red blood cells is at least partially removed from the recovered mononuclear cells.

4. The method of recovering monocytes according to any one of claims 1 to 3, wherein ​

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