Induced pluripotent stem cell establishment apparatus and method

Abstract

To simply establish an induced pluripotent stem (iPS) cell.SOLUTION: According to one embodiment, an apparatus for the establishment of an induced pluripotent stem (iPS) cell includes a suspension liquid feeder, a trap, an inducing factor feeder, and an establisher. The suspension liquid feeder is adapted to feed a suspension liquid containing a target cell; the trap is adapted to trap the target cell contained in the suspension liquid fed from the suspension liquid feeder; the inducing factor feeder is adapted to feed an inducing factor to the trap; and the establisher is adapted to establish an iPS cell by introducing the inducing factor, which has been fed to the trap, into the target cell trapped by the trap.SELECTED DRAWING: Figure 1

Description

Technical Field

[0001] The embodiments disclosed in this specification and the drawings relate to an induced pluripotent stem cell establishment apparatus and method.

Background Art

[0002] In order to establish induced pluripotent stem cells (iPS (induced pluripotent stem) cells), it is necessary to perform a step of extracting mononuclear cells from blood, a step of adding factors for growing the extracted mononuclear cells into iPS cells, and a step of culturing the cells. These steps are performed by a combination of large equipment such as a centrifuge and techniques by technicians.

[0003] However, the above method has the following problems. The first is cost. The preparation and maintenance of large equipment are very expensive. The second is quality. In a series of steps, the operations are complex and delicate, and in the manual part, the quality of the cells varies depending on the skills of individuals. The third is time. In the above steps, each step requires about several days, and furthermore, in order to prevent cell contamination, only one person's worth of iPS cells can be established per production line. Therefore, it takes a very long time to produce iPS cells derived from many donors.

Prior Art Documents

Patent Documents

[0004]

Patent Document 1

Summary of the Invention

Problems to be Solved by the Invention

[0005] One of the problems to be solved by the embodiments disclosed in this specification and the drawings is to easily establish induced pluripotent stem cells. However, the problems to be solved by the embodiments disclosed in this specification and the drawings are not limited to the above problems. The problems corresponding to the respective effects of the respective configurations shown in the embodiments described later can also be regarded as other problems.

Means for Solving the Problems

[0006] The induced pluripotent stem cell establishment device according to the embodiment includes a suspension supply unit, a capture unit, an induction factor supply unit, and an establishment unit. The suspension supply unit supplies a suspension containing target cells. The capture unit captures the target cells contained in the suspension supplied by the suspension supply unit. The induction factor supply unit supplies an induction factor to the capture unit. The establishment unit introduces the induction factor supplied to the capture unit into the target cells captured by the capture unit to establish induced pluripotent stem cells.

Brief Description of the Drawings

[0007]

Figure 1

Figure 2

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Figure 8

Embodiments for Carrying Out the Invention

[0008] Hereinafter, embodiments of an induced pluripotent stem cell establishment apparatus and method will be described in detail with reference to the drawings.

[0009] FIG. 1 is a diagram showing a configuration example of an induced pluripotent stem cell establishment apparatus 1 according to this embodiment. As shown in FIG. 1, the induced pluripotent stem cell establishment apparatus 1 includes a suspension supply unit 11, an induction factor supply unit 12, a capture unit 13, an establishment unit 14, a cleaning liquid supply unit 15, a waste liquid storage unit 16, a culture unit 17, and a culture medium supply unit 18.

[0010] As shown in FIG. 1, the suspension supply unit 11, the induction factor supply unit 12, the capture unit 13, the establishment unit 14, the cleaning liquid supply unit 15, the waste liquid storage unit 16, the culture unit 17, and the culture medium supply unit 18 are provided in a flow path. The flow path has, for example, a first flow path 21, a second flow path 22, and a third flow path 23. The first flow path 21 is provided with the cleaning liquid supply unit 15, the suspension supply unit 11, the capture unit 13, the induction factor supply unit 12, and the waste liquid storage unit 16 in the direction of the flow path (from upstream to downstream). The first flow path 21 allows the suspension supplied by the suspension supply unit 11 and the induction factor supplied by the induction factor supply unit 12 to flow in. In the first flow path 21, the suspension flows in the first direction, and the induction factor flows in a second direction opposite to the first direction. The second flow path 22 is branched and provided upstream of the capture unit 13 of the first flow path 21, and the establishment unit 14 is provided on the downstream side. The second flow path 22 branches from the first flow path 21 at a position between the suspension inlet in the first flow path 21 and the capture unit 13. The third flow path 23 is connected downstream of the second flow path 22, the culture medium supply unit 18 is provided on the upstream side, and the culture unit 17 is provided on the downstream side.

[0011] The suspension supply unit 11 supplies a suspension 51 containing target cells. The suspension 51 may be blood in which the target cells collected from the human body are suspended, or a preservation solution in which the target cells are suspended. The target cells are cells to be initialized by an induction factor. The target cells may be blood-derived cells or somatic cells derived from sources other than blood, as long as they are somatic cells capable of being initialized. Mononuclear cells are used as blood-derived target cells.

[0012] The suspension supply unit 11 is composed of, for example, a syringe including a cylinder and a piston. The cylinder can accommodate the suspension 51 and has airtightness to suppress or prevent the suspension 51 from being exposed to the outside air. Also, the tip of the cylinder is connected to the first flow path 21 via a valve 31. The piston discharges the suspension 51 accommodated in the cylinder.

[0013] The induction factor supply unit 12 supplies a medium 52 containing an induction factor to the capture unit 13. The medium 52 containing an induction factor has an induction factor and a medium for establishing induced pluripotent stem cells. The induction factor is one that initializes the target cells, and specifically, it is an Oct family gene, a Klf family gene, and a Myc family gene, or their respective gene products. As an example, Oct3 / 4 is used as the Oct family gene, Klf4 is used as the Klf family gene, and c-Myc or L-Myc is used as the Myc family gene. Also, the induction factor may be a Sox family gene or its gene product. Sox2 is used as the Sox family gene.

[0014] The induction factor supply unit 12 is composed of, for example, a syringe including a cylinder and a piston. The cylinder can accommodate the medium 52 containing an induction factor and has airtightness to suppress or prevent the medium 52 from being exposed to the outside air. Also, the tip of the cylinder is connected to the first flow path 21 via a valve 33. The piston discharges the medium 52 accommodated in the cylinder.

[0015] The capture unit 13 is provided between the suspension supply unit 11 and the inducer supply unit 12 in the first flow path 21. That is, the capture unit 13 is provided between the inlet of the suspension and the inlet of the inducer in the first flow path 21. The capture unit 13 captures the target cells contained in the suspension 51 supplied from the suspension supply unit 11. The capture unit 13 is composed of a container including a filter 131 having openings that can capture the target cells and allow other substances with a particle size smaller than that of the target cells to pass through.

[0016] The establishment unit 14 is connected to the second flow path 22, and the target cells and the inducer are supplied through the second flow path 22. The establishment unit 14 introduces the inducer supplied to the capture unit 13 into the target cells captured by the capture unit 13 to establish induced pluripotent stem cells (iPS cells). In addition, the establishment unit 14 moves the established induced pluripotent stem cells to the culture unit 17.

[0017] The establishment unit 14 is composed of, for example, a syringe including a cylinder and a piston. The cylinder can accommodate the medium 52 containing the inducer supplied from the inducer supply unit 12 and passing through the capture unit 13, and the target cells captured by the capture unit 13, and has airtightness to suppress or prevent the medium 52 containing the inducer and the target cells from being exposed to the outside air. In addition, the cylinder can accommodate the iPS cells established by introducing the inducer into the target cells. The tip of the cylinder is connected to the second flow path 22 via a valve 34. The piston discharges the iPS cells accommodated in the cylinder.

[0018] The cleaning liquid supply unit 15 supplies the cleaning liquid 55 to the first flow path 21 and cleans the capture unit 13 with the cleaning liquid 55. The cleaning liquid 55 is, for example, physiological saline or the like, and a liquid that has little effect on damaging the target cells is used.

[0019] The cleaning liquid supply unit 15 is composed of, for example, a syringe including a cylinder and a piston. The cylinder can accommodate the cleaning liquid 55 and has airtightness to suppress or prevent the cleaning liquid 55 from being exposed to the outside air. Further, the tip of the cylinder is connected to the first flow path 21 via the valve 31. The piston discharges the cleaning liquid accommodated in the cylinder.

[0020] The waste liquid storage unit 16 is a container for storing waste liquid. The waste liquid storage unit 16 stores the suspension 51 and the cleaning liquid 55 that have passed through the capture unit 13 as waste liquid. The waste liquid storage unit 16 may be a container capable of storing waste liquid, such as a flask or a bag.

[0021] The culture unit 17 cultures the iPS cells established by the establishment unit 14. The culture unit 17 may be a container capable of culturing iPS cells, such as a well plate, a flask, a dish, or a bag.

[0022] The medium supply unit 18 supplies a medium for culturing iPS cells to the culture unit 17. The medium supply unit 18 has a medium storage container 181 and a pump 182. The medium storage container 181 is a container for storing the medium. The pump 182 sucks the medium stored in the medium storage container 181, discharges the sucked medium into the third flow path 23, and supplies it to the culture unit 17.

[0023] In addition, valves 31 to 33 are provided in the first flow path 21. Valve 31 is provided at one end (upstream side) of the first flow path 21, to which the suspension supply unit 11 and the cleaning liquid supply unit 15 are connected. Valve 31 is provided so that the supply and stop of the suspension supplied from the suspension supply unit 11 to the first flow path 21 and the supply and stop of the cleaning liquid supplied from the cleaning liquid supply unit 15 to the first flow path 21 can be switched respectively. Valve 32 is provided at the branch portion between the first flow path 21 and the second flow path 22. Valve 33 is provided at the other end (downstream side) of the first flow path 21, to which the inducer supply unit 12 is connected. Valve 33 is provided so that the supply and stop of the medium 52 containing the inducer to the first flow path 21 can be switched. In addition, a valve 34 is provided in the second flow path 22. Valve 34 is connected to the establishment unit 14. Valve 34 is provided so that the supply and stop of the medium 52 containing the inducer that has passed through the capture unit 13 and the target cells captured by the capture unit 13 to the second flow path 22 can be switched. In addition, a valve 35 is provided in the third flow path 23. Valve 35 is connected to the culture unit 17 and the medium supply unit 18, and is provided so that the supply and stop of the induced pluripotent stem cells established by the establishment unit 14 to the third flow path 23 and the supply and stop of the medium supplied from the medium supply unit 18 to the third flow path 23 can be switched respectively.

[0024] Valves 31 to 35 may be valves of any type as long as they can open and close the flow path, such as two-way valves or three-way valves. As an example, it is assumed that they are three-way valves that are flow path switching valves. A three-way valve is a mechanical part having a main body portion with three holes and a valve body that opens two holes and closes the remaining one hole. The valve bodies of valves 31 to 35 may be switched manually or electromagnetically.

[0025] The first flow path 21, the second flow path 22, and the third flow path 23 are composed of a tubular member having airtightness and flexibility, such as a vinyl or plastic tube. Thereby, exposure of various liquids passing through the first flow path 21, the second flow path 22, and the third flow path 23 to the outside air is suppressed or prevented.

[0026] An operation example of the induced pluripotent stem cell establishment device 1 configured as described above will be described. FIG. 2 is a diagram showing the flow of establishment and culture of iPS cells using the induced pluripotent stem cell establishment device 1. FIG. 3 is a diagram schematically showing the blood flow in step S2. FIG. 4 is a diagram schematically showing the flow of the washing liquid in step S4. FIG. 5 is a diagram schematically showing the flow of the medium containing the induction factor in step S6. FIG. 6 is a diagram schematically showing the flow of iPS cells in step S8. In FIG. 2, it is assumed that the suspension is blood collected from a provider. At the start point of FIG. 2, it is assumed that one portion of blood is stored in the suspension supply unit 11, one portion of the medium containing the induction factor is stored in the induction factor supply unit 12, and one portion of the washing liquid is stored in the washing liquid supply unit 15.

[0027] As shown in FIG. 2, first, valves 31, 32, and 33 are switched (step S1). Specifically, the operator operates valve 31 to move the valve body in order to secure the path from the suspension supply unit 11 to the capture unit 13 and block the path to the establishment unit 14, thereby opening the hole on the suspension supply unit 11 side (hereinafter referred to as the blood supply hole) and the hole on the first flow path 21 side (hereinafter referred to as the discharge hole), and closing the hole on the washing liquid supply unit 15 side (hereinafter referred to as the washing liquid supply hole). In addition, the operator operates valve 32 to move the valve body, thereby opening the hole on the suspension supply unit 11 side (hereinafter referred to as the blood washing liquid supply hole) and the hole on the capture unit 13 side (hereinafter referred to as the capture side hole), and closing the hole on the establishment unit 14 side (hereinafter referred to as the establishment side hole). Further, the operator operates valve 33 to move the valve body, thereby opening the hole on the capture unit 13 side (hereinafter referred to as the capture side hole) and the hole on the waste liquid recovery unit 16 side (hereinafter referred to as the waste liquid supply hole), and closing the hole on the induction factor supply unit 12 side (hereinafter referred to as the induction factor supply hole). Note that, initially, if the path from the suspension supply unit 11 to the capture unit 13 is secured and the path to the establishment unit 14 is blocked, step S1 can be omitted. In the drawings, each triangle representing a three-way valve corresponding to each of valves 31, 32, 33, 34, and 35 represents a hole, the white triangle represents the hole opened by the valve body, and the black triangle represents the hole closed by the valve body.

[0028] When step S1 is performed, a suspension 51 (blood) containing target cells is supplied to the capture unit 13 (step S2). In step S2, the suspension supply unit 11 supplies the suspension 51 to the capture unit 13 along the forward direction via the first flow path 21. The forward direction is defined as the direction in which the suspension 51 goes toward the waste liquid storage unit 16. Specifically, the operator operates the suspension supply unit 11 to discharge the suspension 51 stored in the suspension supply unit 11 into the first flow path 21. The suspension supply unit 11 stores one portion of the suspension 51 containing the target cells necessary for producing iPS cells. Therefore, all of the suspension 51 stored in the suspension supply unit 11 may be discharged. The suspension 51 discharged into the first flow path 21 flows into the capture unit 13. Among the various components (substances) contained in the suspension 51 that has flowed into the capture unit 13, the components having a particle size larger than the aperture of the filter 131 are captured, and the components having a particle size smaller than the aperture of the filter 131 pass through the filter 131. As the filter 131, for example, a filter having an aperture capable of capturing components of the same size as white blood cells may be used. Since the target cells 53 have a particle size larger than the aperture, they will be captured by the filter 131. Red blood cells and the like that are not target cells pass through the filter 131 because their particle sizes are smaller than the aperture. The blood components that have passed through the capture unit 13 are stored in the waste liquid storage unit 16 as waste liquid 56.

[0029] When step S2 is performed, the valve 31 is switched (step S3). Specifically, the operator operates the valve 31 to move the valve body in order to secure the path from the cleaning liquid supply unit 15 to the capture unit 13 and block the path to the establishing unit 14, thereby opening the cleaning liquid supply hole and the discharge hole and closing the blood supply hole.

[0030] When step S3 is performed, the capture unit 13 is washed with the washing liquid 55 (step S4). In step S4, the washing liquid supply unit 15 flushes the components other than the target cells remaining in the first flow path 21 and the capture unit 13 into the waste liquid storage unit 16 with the washing liquid 55. Specifically, in step S4, the operator operates the washing liquid supply unit 15 to discharge the washing liquid 55 stored in the washing liquid supply unit 15 into the first flow path 21. Since the washing liquid supply unit 15 stores the washing liquid 55 for one time, all the washing liquid 55 stored in the washing liquid supply unit 15 may be discharged. The washing liquid 55 discharged into the first flow path 21 flushes the unnecessary blood components other than the target cells remaining in the first flow path 21 and the capture unit 13 into the waste liquid storage unit 16. The unnecessary blood components are mainly plasma components, red blood cell components and platelet components. By pushing and pulling the piston of the washing liquid supply unit 15 and repeating the discharge and suction of the washing liquid 55, it is possible to separate the unnecessary blood components entangled in the first flow path 21 and the capture unit 13 and efficiently wash them away. Further, by tilting the induced pluripotent stem cell establishment device 1 itself in parallel with the discharge and suction of the washing liquid 55, it is possible to efficiently wash away the unnecessary blood components.

[0031] When step S4 is performed, valves 32, 33 and 34 are switched (step S5). Specifically, the operator operates valve 32 to move the valve body to open the hole on the capture unit 13 side and the hole on the establishment unit 14 side and close the hole on the washing liquid supply unit 15 side in order to secure the path from the induction factor supply unit 12 to the establishment unit 14. Further, the operator operates valve 33 to move the valve body to open the hole on the induction factor supply unit 12 side and the hole on the capture unit 13 side and close the waste liquid supply hole, and operates valve 34 to move the valve body to open the hole on the valve 32 side (hereinafter referred to as the branch valve side hole) and the hole on the establishment unit 14 side (hereinafter referred to as the establishment side hole) and close the hole on the culture unit 17 side (hereinafter referred to as the culture side hole).

[0032] When step S5 is performed, the medium 52 containing the induction factor is supplied to the establishment unit 14 via the capture unit 13 (step S6). In step S6, the induction factor supply unit 12 supplies the medium 52 containing the induction factor to the capture unit 13 along the direction opposite to the forward direction via the first flow path 21, and supplies the medium 52 containing the induction factor and the target cells 53 to the establishment unit 14 via the second flow path 22. Specifically, in step S6, the operator operates the induction factor supply unit 12 to discharge the medium 52 containing the induction factor stored in the induction factor supply unit 12 into the first flow path 21. Since the medium 52 containing the induction factor for one dose is stored in the induction factor supply unit 12, all the medium 52 containing the induction factor stored in the induction factor supply unit 12 may be discharged. The discharged medium 52 containing the induction factor flows into the capture unit 13 from the direction opposite to the flow direction of the suspension 51 in step S2. The medium 52 containing the induction factor that has flowed into the capture unit 13 passes through the filter 131 and flows into the establishment unit 14 via the second flow path 22 together with the target cells 53 captured by the filter 131. In order to accommodate the medium 52 containing the induction factor and the target cells 53 in the cylinder of the establishment unit 14, the operator may pull the piston of the establishment unit 14 to the limit in advance.

[0033] In the establishment unit 14, the induction factor is introduced into the target cells 53, and iPS cells are established from the target cells. The induction factor can be supplied in various forms. For example, the induction factor may be incorporated into various vectors for supply. The vector is not particularly limited, and may be a viral vector such as a Sendai virus vector or a retrovirus vector, or a non-viral vector such as a plasmid. For example, when using a Sendai virus vector incorporated with the induction factor, the induction factor is introduced into the target cells when the Sendai virus vector comes into contact with the target cells. Thereafter, the gene product of the induction factor is synthesized. By the action of the gene product, the initialization of the target cells is induced, and iPS cells, which are undifferentiated cells having pluripotency and proliferation ability, are generated. The iPS cells are cultured for about one hour in the presence of the medium. Thereby, iPS cells are established. During the establishment operation, the establishment unit 14 may be maintained at about 37 degrees and 5% CO2.

[0034] In order to improve the establishment accuracy of iPS cells in the establishment unit 14, a container with a relatively small volume may be used as the cylinder of the establishment unit 14. This is because the probability of contact between the target cells and the induction factor or the vector incorporated with the induction factor increases, and as a result, the probability of establishing iPS cells increases.

[0035] When step S6 is performed, valves 34 and 35 are switched (step S7). Specifically, the operator operates valve 34 to move the valve body to open the establishment side hole and the culture side hole, close the branch valve side hole, and operates valve 35 to move the valve body to open the hole on the valve 34 side (hereinafter referred to as the establishment side hole) and the hole on the culture unit 17 side (referred to as the culture side hole), and close the hole on the medium supply unit 18 side (referred to as the medium supply hole) in order to secure the path from the establishment unit 14 to the culture unit 17.

[0036] When step S7 is performed, the iPS cells are transferred to the culture unit 17 (step S8). More specifically, in step S7, the operator operates the establishment unit 14 to discharge the iPS cell suspension 54 accommodated in the establishment unit 14 into the second flow path 22. The iPS cell suspension 54 discharged into the second flow path 22 moves to the culture unit 17. The iPS cells 60 transferred to the culture unit 17 are cultured in the culture unit 17. The culture unit 17 may be maintained at a room temperature of about 37 degrees during the culture operation. During the culture, if necessary, the medium 58 can be appropriately supplied from the medium supply unit 18. For example, the operator may operate valve 35 to move the valve body to open the medium supply hole and the culture side hole, close the establishment side hole, and operate the pump 182 to supply an appropriate amount of the medium 58 from the medium storage container 181 to the culture unit 17.

[0037] In order to adhere the iPS cells to the culture unit 17, a coating agent may be added to the culture unit 17 in the stage before step S8. Specifically, the coating agent is added to the bottom surface of the culture dish of the culture unit 17. As the coating agent, cell adhesion molecules such as laminin protein and vitronectin may be used. Thereby, a scaffold for the iPS cells supplied from the establishment unit 14 is formed in the culture unit 17, and it becomes possible to stably culture the iPS cells.

[0038] Note that the coating agent may not be added to the culture unit 17 in advance. For example, when supplying iPS cells from the establishment unit 14 to the culture unit 17, the operator may supply the coating agent to the culture unit 17 using a syringe. The supply of the coating agent may be performed in parallel with the supply of iPS cells from the establishment unit 14 to the culture unit 17, or may be performed before or after that.

[0039] The induced pluripotent stem cell establishment device 1 may be configured to be detachable between the suspension supply unit 11, the induction factor supply unit 12, the establishment unit 14, and the culture unit 17 and the culture medium supply unit 18. In this case, after the iPS cells are transferred to the culture unit 17, it is possible to separate the suspension supply unit 11, the induction factor supply unit 12, the establishment unit 14, and the culture unit 17 and the culture medium supply unit 18. For example, by pulling out the second flow path 22 from the valve 35, it becomes possible to form a culture system having the culture unit 17 and the culture medium supply unit 18 connected to the third flow path 23. By separating the culture unit 17 and the culture medium supply unit 18 from the induced pluripotent stem cell establishment device 1, it becomes possible to culture the iPS cells in a more space-saving manner.

[0040] Thus, the establishment and culture of iPS cells using the induced pluripotent stem cell establishment device 1 are completed.

[0041] Next, the separation accuracy of blood components by the capture unit 13 according to the present embodiment will be described.

[0042] FIG. 7 is a diagram showing the measurement results of flow cytometry by a flow cytometer device regarding a comparative example and the present embodiment. The comparative example shows the measurement results by a method of separating blood components by a general density gradient centrifugation method. Comparative example (A) shows the measurement results of flow cytometry of separated blood cells after separating the blood contained in a BD Vacutainer blood collection tube by the density gradient centrifugation method. Comparative example (B) shows the measurement results of flow cytometry of separated blood cells after separating the blood contained in a SepMate, which is a density gradient centrifuge dedicated tube of Veritas, by the density gradient centrifugation method. The present embodiment shows the measurement results of flow cytometry of the blood components (separated blood components) captured by the filter 131 of the capture unit 13 as described above.

[0043] Each measurement result shown in FIG. 7 is represented by a dot plot in which the vertical axis is defined as the side scatter signal (SSC) and the horizontal axis is defined as the forward scatter signal (FSC). The side scatter signal is related to the complexity of the cell internal structure, and the forward scatter signal is related to the size of the cell. As can be seen by comparing the present embodiment with the comparative example, it can be confirmed that the same separation accuracy as that of the comparative example is obtained by the separation by the filter 131 according to the present embodiment. The present inventor has also confirmed that iPS cells having a morphology and a proliferation ability can be established using the induced pluripotent stem cell establishment device 1 according to the present invention.

[0044] The configuration of the induced pluripotent stem cell establishment device 1 described above is an example, and various modifications are possible.

[0045] (Modification example 1) In the above embodiment, it is assumed that the target cells and the medium containing the inducer flow into the establishment unit 14, and iPS cells are established from the target cells in the establishment unit 14. The induced pluripotent stem cell establishment device 1 according to modification example 1 can also serve as both the capture unit 13 and the establishment unit 14.

[0046] FIG. 8 is a diagram schematically showing the flow of the medium containing an inducer according to Modification 1. As shown in FIG. 8, after step S4 in FIG. 2 is executed, the operator operates valve 32 to move the valve body in order to secure the path from the inducer supply unit 12 to the capture unit 13 and block the path from the capture unit 13 to the establishment unit 14, thereby opening the blood washing liquid supply hole and the establishment side hole, closing the capture side hole, operating valve 33 to move the valve body, opening the inducer supply hole and the capture side hole, and closing the waste liquid supply hole.

[0047] Next, the operator operates the inducer supply unit 12 to discharge the medium 52 containing an inducer stored in the inducer supply unit 12 into the first flow path 21. The discharged medium 52 containing an inducer flows into the capture unit 13 from the direction opposite to the blood flow direction in step S2. In the capture unit 13, the medium 52 containing an inducer is blocked. The means for blocking is not particularly limited. For example, the operator may block the hole on the valve 32 side of the capture unit 13 with a stopper or the like, or may block the first flow path 21 between the capture unit 13 and the valve 32 by pinching it with a clip or the like. In the capture unit 13, the inducer is introduced into the target cells, and iPS cells are established from the target cells. After the iPS cells are established in the capture unit 13, the iPS cell suspension will be moved to the culture unit 17.

[0048] According to Modification 1, it becomes possible to form the capture unit 13 and the establishment unit 14 with a single container. Thereby, it becomes possible to configure the induced pluripotent stem cell establishment apparatus 1 more simply.

[0049] (Modification 2) In the above embodiment, the suspension supply unit 11 is configured to supply blood collected from a provider. However, the suspension supply unit 11 according to Modification 2 supplies a preservation solution in which target cells are suspended. For example, in step S2 or S4, a target cell suspension preservation solution may be generated by suspending the target cells captured by the capture unit 13 in the preservation solution.

[0050] To produce a target cell suspension preservation solution, instead of the inducer supply unit 12, a preservation solution supply unit for storing the preservation solution may be connected to the second flow path 22. In this case, in step S6, the preservation solution is supplied from the preservation solution supply unit to the capture unit 13. The preservation solution supplied to the capture unit 13 flows into the establishment unit 14 together with the target cells captured by the capture unit 13. In the establishment unit 14, a preservation solution (target cell suspension preservation solution) in which the target cells are suspended will be stored. The target cell suspension preservation solution stored in the establishment unit 14 may be stored in the suspension supply unit 11. The target cell suspension preservation solution is more suitable for storing target cells than blood.

[0051] The suspension supply unit 11 storing the target cell suspension preservation solution can be used in the same manner as the suspension supply unit 11 storing blood. That is, in step S2, the target cell suspension preservation solution is supplied from the suspension supply unit 11 to the capture unit 13, and the target cells are captured from the target cell suspension preservation solution by the capture unit 13. Thereafter, a medium containing an inducer is supplied from the inducer supply unit 12 to the capture unit 13, and the target cells flow into the establishment unit 14 together with the medium containing the inducer, and iPS cells are established from the target cells in the establishment unit 14. In addition, also in Modification 1, the target cells captured by the capture unit 13 are those of the same size as white blood cells, similar to the above-described embodiment.

[0052] According to Modification 2, the target cells contained in the preservation solution can also be captured by the capture unit 13. Therefore, the convenience of the induced pluripotent stem cell establishment apparatus 1 is improved. In addition, it becomes possible to generate a target cell suspension preservation solution using the induced pluripotent stem cell establishment apparatus 1.

[0053] (Modification 3) The capture unit 13 according to Modification 3 may culture the target cells captured in step S2. For example, after the target cells are captured in step S2 and the capture unit 13 is washed in step 4, a medium for culturing the target cells is supplied to the capture unit 13, and the target cells are cultured in the capture unit 13.

[0054] The medium for culturing the target cells may be supplied to the capture unit 13 depending on the method. For example, it may be directly supplied to the capture unit 13 by an operator. Further, the suspension supply unit 11, the inducer supply unit 12, or the cleaning liquid supply unit 15 may be replaced with a medium supply unit that stores the medium for culturing the target cells, and the medium may be supplied from the medium supply unit to the capture unit 13.

[0055] According to Modification 3, it becomes possible to increase the target cells that are the material cells of iPS cells, and as a result, it becomes possible to establish a large amount of iPS cells.

[0056] (Modification 4) In the above embodiment, the suspension supply unit 11, the inducer supply unit 12, the establishment unit 14, and the cleaning liquid supply unit 15 are configured by a cylinder and a piston, and the piston is manually pushed and pulled. However, the piston may be configured to be automatically pushed and pulled by a machine. For example, the suspension supply unit 11, the inducer supply unit 12, the establishment unit 14, and / or the cleaning liquid supply unit 15 can be configured by an electric cylinder capable of pushing and pulling a piston. Specifically, the electric cylinder has a cylinder, a piston, and a motor. The piston is supported by a linear motion mechanism that can slide inside the cylinder. The linear motion mechanism is connected to the motor and operates in conjunction with the drive of the motor to push and pull the piston. The motor, triggered by the pressing of the supply button, operates the linear motion mechanism to push out the piston and discharge the suspension, the medium containing the inducer, the iPS cell suspension, and the cleaning liquid stored in the cylinder.

[0057] The motor of the electric cylinder may activate the linear motion mechanism to push out the piston when a predetermined time has elapsed. In this case, the electric cylinder is provided with a processor, and the processor measures the elapsed time from the reference time. For example, when the establishment unit 14 is constituted by an electric cylinder, the processor measures the elapsed time from the reference time and detects that the elapsed time has reached the culture time (for example, 1 hour). The reference time may be set, for example, at the time when the medium containing the inducer is discharged from the inducer supply unit 12 or when it flows into the establishment unit 14. Then, when the elapsed time reaches the culture time, the processor may drive the motor to push out the piston via the linear motion mechanism. Thereby, it becomes possible to automatically move the iPS cell suspension to the culture unit 17. Note that the amount of each blood component per unit volume is known to vary depending on the provider and also vary depending on the physical condition, etc. even for the same provider. When strictly determining the culture time in the establishment unit 14, the culture time may be determined according to the number of blood component amounts such as white blood cells.

[0058] (Modification Example 5) In the establishment unit 14 according to Modification Example 5, an inducer may be introduced into target cells using electroporation. For example, a pair of electrodes is provided in the cylinder of the establishment unit 14, and the pair of electrodes is connected to a power supply device. The power supply device applies an electric pulse between the pair of electrodes when instructed by an operator, or when the medium containing the inducer is discharged from the inducer supply unit 12 or when it flows into the establishment unit 14. The target cells accommodated in the cylinder receive the application of the electric pulse, and micropores are formed in their cell membranes. An inducer or a vector incorporating the inducer is introduced through the micropores.

[0059] According to Modification Example 5, since the inducer is introduced into the target cells using electroporation, the efficiency of introducing the inducer into the target cells can be increased compared to the case where electroporation is not used as in the above-described embodiment. As a result, it becomes possible to increase the establishment efficiency of iPS cells.

[0060] (Modification Example 6) In the above-described embodiment, the valves 31, 32, 33, 34, and / or 35 are three-way valves, but they may be two-way valves such as pinch valves, gate valves, ball valves, diaphragm valves, etc. The two-way valve may be manually switchable or electromagnetically switchable.

[0061] (Modification Example 7) The suspension supply unit 11, the inducer supply unit 12, the establishment unit 14, and the cleaning liquid supply unit 15 are constituted by a cylinder and a piston, and the piston is manually pushed and pulled. However, the suspension supply unit 11, the inducer supply unit 12, the establishment unit 14, and / or the cleaning liquid supply unit 15 may be constituted by a flexible container. As such a container, for example, a plastic or vinyl bag having a connection portion for connecting to a tube forming a flow path is suitable. In this case, by the operator pressing the bag, it is possible to discharge the target cell suspension, the medium containing the inducer, the iPS cell suspension, the cleaning liquid, etc. accommodated in the bag from the bag.

[0062] (Modification Example 8) In the above-described embodiment, a coating agent is added to the culture unit 17. However, the present embodiment is not limited to this. In order to adhere the iPS cells to the establishment unit 14, a coating agent may be added to the establishment unit 14 in the stage before step S6. Specifically, the coating agent is added to the bottom surface of the culture dish of the establishment unit 14. Thereby, a scaffold for the target cells supplied from the capture unit 13 is formed in the establishment unit 14, and it becomes possible to stably establish iPS cells.

[0063] Note that the coating agent does not necessarily have to be added to the seeding section 14 in advance. For example, when supplying the target cell suspension and the medium containing the induction factor from the capture section 13 to the seeding section 14, the operator may supply the coating agent to the seeding section 14 using a syringe. The supply of the coating agent may be performed in parallel with the supply of the target cell suspension and the medium containing the induction factor from the seeding section 14 to the culture section 17, or may be performed before or after that. The coating agent supplied to the seeding section 14 may be supplied from the seeding section 14 to the culture section 17 in step S8.

[0064] As another example, in order to supply a coating to the seeding section 14, the induction factor supply section 12 may contain a coating agent. In this case, the induction factor supply section 12 contains a mixture of the medium containing the induction factor and the coating agent. In step S6, a mixture of the medium containing the induction factor and the coating agent is supplied from the induction factor supply section 12 to the capture section 13, and the medium containing the induction factor and the coating agent pass through the filter 131 and are supplied to the seeding section 14 together with the target cells. Thereby, the coating agent is supplied to the seeding section 14. Thereby, a scaffold for the target cells supplied from the capture section 13 is formed in the seeding section 14, and it becomes possible to stably establish iPS cells.

[0065] Note that the coating agent does not necessarily have to be accommodated in the inducer supply unit 12. For example, when supplying the medium containing the inducer from the inducer supply unit 12 to the capture unit 13, the operator may supply the coating agent to the capture unit 13 using another injector. The supply of the coating agent may be performed in parallel with the supply of the medium containing the inducer from the inducer supply unit 12 to the capture unit 13, or may be performed before or after that. Further, the coating agent may be accommodated in the empty inducer supply unit 12 after the supply of the medium containing the inducer, and the coating agent may be supplied from the inducer supply unit 12 to the capture unit 12. Conversely, first, the coating agent may be accommodated in the empty inducer supply unit 12, the coating agent may be supplied to the capture unit 12, and then the medium containing the inducer may be accommodated in the empty inducer supply unit 12, and the medium containing the inducer may be supplied from the inducer supply unit 12 to the capture unit 12. The coating agent supplied to the establishment unit 14 may be supplied from the establishment unit 14 to the culture unit 17 in step S8.

[0066] (Modification Example 9) The capture unit 13 according to Modification Example 9 may include a first filter having a first aperture and a second filter having a second aperture. The first aperture is an aperture for capturing a substance having a first particle size larger than the target cells and allowing a substance having a particle size equal to or smaller than the first particle size to pass through. The third aperture is a second aperture for capturing a substance having a second particle size equal to the size of the target cells among the substances that have passed through the first aperture and allowing a substance having a particle size equal to or smaller than the second particle size to pass through. The first filter and the second filter are provided in this order from upstream to downstream in the capture unit 13. A gap (space) is provided between the first filter and the second filter. The target cells are accommodated in the gap.

[0067] In the capture unit 13 according to Modification Example 9, similar to the above-described embodiment, a medium containing an inducer is supplied from the inducer supply unit 12. The medium containing the inducer passes through the second opening of the second filter and is supplied to the gap between the first filter and the second filter. Then, the medium containing the inducer passes through the first opening of the first filter together with the target cells accommodated in the gap and is supplied to the establishment unit 14 via the second flow path 22. At this time, in order to suppress the medium containing the inducer and the target cells from flowing upstream (toward the suspension supply unit 11 and the cleaning liquid supply unit 15 side) through the first opening of the first filter, it is preferable that the holes on the upstream side of the capture unit 13 are blocked. Further, an opening / closing mechanism for selectively opening and closing the openings of the first filter may be provided. The opening / closing mechanism should be electrically and mechanically controllable. In this case, when supplying the suspension from the suspension supply unit 11, by opening the openings of the first filter, substances having a size equal to or smaller than the first particle size, which is larger than the target cells, are allowed to pass through. On the other hand, when supplying the medium containing the inducer from the inducer supply unit 12, by closing the openings of the first filter, it is possible to suppress the medium containing the inducer and the target cells from flowing upstream (toward the suspension supply unit 11 and the cleaning liquid supply unit 15 side) through the first opening of the first filter.

[0068] Further, a second flow path 22 and another flow path (hereinafter referred to as a fifth flow path) may be branched and connected to the gap between the first filter and the second filter of the capture unit 13 according to Modification Example 9. In the capture unit 13, the first flow path 21 intersects with the flow paths composed of the second flow path 22 and the fifth flow path. In this case, the inducer supply unit 12 is connected to the fifth flow path, and the inducer supply unit 12 is not connected to the first flow path 21 downstream from the capture unit 13. That is, the flow path into which the medium containing the inducer supplied from the inducer supply unit 12 flows and the flow path into which the medium containing the inducer and the target cells flow into the establishment unit 14 may be connected. When the medium containing the inducer is supplied to the gap between the first filter and the second filter by the inducer supply unit 12, the target cells captured in the gap portion of the capture unit 13 are supplied to the establishment unit 14 together with the medium containing the inducer supplied to the gap portion.

[0069] In this case, an opening / closing mechanism for selectively opening and closing the openings of the first filter and the second filter may be provided. The opening / closing mechanism shall be electrically and mechanically controllable. In this case, when supplying the suspension from the suspension supply unit 11, by opening the openings of the first filter and the second filter, substances with a size not larger than the first particle size, which is larger than the target cells, are allowed to pass through, substances having the second particle size equal to the size of the target cells are captured, and substances with a size not larger than the second particle size are allowed to pass through. On the other hand, when supplying the medium containing the inducer from the inducer supply unit 12, by closing the openings of the first filter and the second filter, it is possible to suppress the medium containing the inducer and the target cells from passing through the first opening of the first filter and the opening of the second filter, and the medium containing the inducer and the target cells can be made to flow into the establishment unit 14.

[0070] (Modification Example 10) In the above embodiment, the capture unit 13 is assumed to have a filter 131 for capturing the target cells. However, as long as the capture unit 13 can capture the target cells, it does not necessarily have to have the filter 131. The induced pluripotent stem cell establishment device 1 according to Modification Example 10 has a control device for controlling the capture unit 13. When the suspension is supplied by the suspension supply unit 11, the control device controls the capture unit 13 to capture the target cells contained in the suspension. When the medium containing the inducer is supplied by the inducer supply unit 12, the control device controls the capture unit 13 to release the capture of the target cells. The control device is configured to be able to capture the target cells electrically, magnetically, and / or optically. The control device captures the target cells in the capture unit 13 by applying electrical energy, magnetic energy, and / or optical energy to the capture unit 13, and releases the capture of the target cells by canceling the application of electrical energy, magnetic energy, and / or optical energy.

[0071] In at least one of the above embodiments, the induced pluripotent stem cell establishment device 1 includes a suspension supply unit 11, a capture unit 13, an induction factor supply unit 12, and an establishment unit 14. The suspension supply unit 11 supplies a suspension containing target cells. The capture unit 13 captures the target cells contained in the suspension supplied by the suspension supply unit 11. The induction factor supply unit 12 supplies an induction factor to the capture unit 13. The establishment unit 14 introduces the induction factor supplied to the capture unit 13 into the target cells captured by the capture unit 13 to establish iPS cells.

[0072] According to the above configuration, it is possible to capture target cells from a suspension and establish iPS cells from the captured target cells without providing large-scale equipment such as a centrifuge. According to the above configuration, when establishing iPS cells, the operator does not need to perform operations that require special skills, so it is possible to suppress variations in the quality of iPS cells. Specifically, the operator only needs to operate the suspension supply unit 11, the induction factor supply unit 12, and various valves 31 to 35, and these operations can be said to be simple operations that do not require special skills. The induced pluripotent stem cell establishment device 1 has airtightness against the outside air for each component necessary from the supply of the suspension to the establishment of iPS cells, and cleanliness is maintained. In addition, since a plurality of components necessary from the supply of the suspension to the establishment of iPS cells in the induced pluripotent stem cell establishment device 1 are integrally formed, it is possible to suppress cross-contamination of target cells. Further, by using a plurality of induced pluripotent stem cell establishment devices 1 in parallel for a plurality of suspensions, it is possible to establish iPS cells from a plurality of providers in parallel, and it is possible to significantly shorten the establishment time of iPS cells.

[0073] According to at least one of the embodiments described above, induced pluripotent stem cells can be easily established.

[0074] The term "processor" used in the above description means, for example, a CPU, a GPU, or a circuit such as an Application Specific Integrated Circuit (ASIC), a Programmable Logic Device (for example, a Simple Programmable Logic Device (SPLD), a Complex Programmable Logic Device (CPLD), and a Field Programmable Gate Array (FPGA)). The processor realizes its function by reading and executing a program stored in a storage circuit. Note that instead of storing the program in the storage circuit, the program may be directly incorporated into the circuit of the processor. In this case, the processor realizes its function by reading and executing the program incorporated in the circuit. Also, instead of executing the program, the function corresponding to the program may be realized by a combination of logic circuits. Note that each processor of the present embodiment is not limited to being configured as a single circuit for each processor, and a plurality of independent circuits may be combined to form one processor to realize its function. Further, a plurality of components may be integrated into one processor to realize its function.

[0075] Although several embodiments have been described, these embodiments are presented as examples and are not intended to limit the scope of the invention. These embodiments can be implemented in various other forms, and various omissions, replacements, changes, and combinations of the embodiments can be made without departing from the gist of the invention. These embodiments and their modifications are included in the scope and gist of the invention, and are also included in the invention described in the claims and its equivalent scope.

Explanation of Reference Numerals

[0076] 1 Induced pluripotent stem cell establishment device 11 Suspension supply unit 12 Inducing factor supply unit 13 Capture unit 14 Establishment unit 15 Cleaning liquid supply unit 16 Waste liquid storage unit 17 Culture unit 18 Culture medium supply unit 21 First flow path 22 Second flow path 23 Third flow path 31, 32, 33, 34, 35 Valves

Claims

1. A suspension supply unit that supplies a suspension containing target cells; A capture unit having a filter capable of capturing the target cells contained in the suspension supplied by the suspension supply unit; An induction factor supply unit that supplies an induction factor to the capture unit; An establishment unit that introduces the induction factor supplied to the capture unit into the target cells captured by the capture unit to establish induced pluripotent stem cells; Comprising: The suspension supply unit and the induction factor supply unit are connected via a first flow path, and the capture unit is provided between the suspension supply unit and the induction factor supply unit in the first flow path; The establishment unit is connected to a second flow path branched from the first flow path at a position between the suspension supply unit and the capture unit in the first flow path; In the capture unit, the suspension supplied by the suspension supply unit flows into the first flow path from a first direction, and after the target cells are captured by the capture unit, the induction factor supplied by the induction factor supply unit flows into the first flow path from a second direction opposite to the first direction; In the establishment unit, the target cells captured by the capture unit, together with the induction factor supplied to the capture unit, flow out of the capture unit from the second direction and into the first flow path and the second flow path; An induced pluripotent stem cell establishment device.

2. The capture unit has openings capable of capturing the target cells, captures substances having a particle size larger than the openings, and allows substances having a particle size smaller than the openings to pass through; The induced pluripotent stem cell establishment device according to Claim 1.

3. The capture unit includes a first opening that captures substances having a first particle size larger than the target cells and allows substances having a particle size equal to or smaller than the first particle size to pass through, and a second opening that captures substances having a second particle size equal to the size of the target cells among the substances that have passed through the first opening and allows substances having a particle size equal to or smaller than the second particle size to pass through; The induced pluripotent stem cell establishment device according to Claim 1.

4. The induction factor supply unit supplies the induction factor to the space between the first opening and the second opening; The induced pluripotent stem cell establishment device according to Claim 3.

5. Further comprising a cleaning liquid supply unit that supplies a cleaning liquid to the capture unit; The cleaning liquid supply unit flushes substances other than the target cells remaining in the capture unit with the cleaning liquid; The induced pluripotent stem cell establishment device according to any one of Claims 1 to 4.

6. After the suspension is supplied by the suspension supply unit, the cleaning liquid supply unit supplies the cleaning liquid to the capture unit before the induction factor is supplied by the induction factor supply unit. The induced pluripotent stem cell establishment device according to claim 5.

7. The cleaning liquid supply unit is configured to be able to suck and discharge the cleaning liquid supplied to the capture unit, and repeatedly performs the sucking and discharging to wash away substances other than the target cells remaining in the capture unit with the cleaning liquid. The induced pluripotent stem cell establishment device according to claim 5 or 6.

8. The induction factor supply unit supplies a medium necessary for the establishment of the induced pluripotent stem cells together with the induction factor. The induced pluripotent stem cell establishment device according to any one of claims 1 to 7.

9. It includes a culture unit for culturing the induced pluripotent stem cells established by the establishment unit. The culture unit receives the induced pluripotent stem cells supplied from the establishment unit and cultures the induced pluripotent stem cells. The induced pluripotent stem cell establishment device according to any one of claims 1 to 8.

10. It includes a medium supply unit that supplies a medium necessary for culturing the induced pluripotent stem cells to the culture unit. The induced pluripotent stem cell establishment device according to any one of claims 1 to 9.

11. The suspension supply unit supplies blood or a preservation solution as the suspension. The induced pluripotent stem cell establishment device according to any one of claims 1 to 10.

12. A preservation solution supply unit that supplies a preservation solution to the capture unit, and A preservation solution recovery unit that recovers the preservation solution containing the target cells captured by the capture unit. The induced pluripotent stem cell establishment device according to any one of claims 1 to 11.

13. The capture unit and the establishment unit are constituted by a single container. The induced pluripotent stem cell establishment device according to any one of claims 1 to 12.

14. It includes a control unit that controls the capture unit. When the suspension is supplied by the suspension supply unit, the control unit controls the capture unit to capture the target cells contained in the suspension, and when the induction factor is supplied by the induction factor supply unit, the control unit controls the capture unit to release the capture of the target cells. The induced pluripotent stem cell establishment device according to claim 1.

15. A suspension supply step of supplying a suspension containing target cells from a suspension supply unit. A capturing step of capturing the target cells contained in the suspension supplied in the suspension supply step with a capturing unit having a filter capable of capturing the target cells; An inducer supply step of supplying an inducer from an inducer supply unit to the capturing unit; An establishing step of introducing the inducer supplied to the capturing unit into the target cells captured by the capturing unit and establishing induced pluripotent stem cells in an establishing unit; An induced pluripotent stem cell establishing method comprising: The suspension supply unit and the inducer supply unit are connected via a first flow path, and the capturing unit is provided between the suspension supply unit and the inducer supply unit in the first flow path. The establishing unit is connected to a second flow path branched from the first flow path at a position between the suspension supply unit and the capturing unit in the first flow path. In the capturing unit, the suspension supplied by the suspension supply unit flows into the first flow path from a first direction, and after the target cells are captured by the capturing unit, the inducer supplied by the inducer supply unit flows into the first flow path from a second direction opposite to the first direction. In the establishing unit, the target cells captured by the capturing unit together with the inducer supplied to the capturing unit flow into the establishing unit after exiting the capturing unit from the second direction and passing through the first flow path and the second flow path. Induced pluripotent stem cell establishing method.

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