JP2015188381A - Separation device and separation method - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a separation device and a separation method for efficiently removing megakaryocytes from culture solution in which platelets and megakaryocytes exist in a mixed manner, without adversely affecting the platelets.SOLUTION: There is provided a separation device 1 for removing megakaryocytes from culture solution in which megakaryocytes M and platelets P exist in a mixed manner, the separation device comprising: a culture solution storage tank 2 storing culture solution SI therein; culture solution feeding means which has a function of sending the culture solution SI in the culture solution storage tank 2; and a recovery liquid storage tank 10 which supplies electric charge having polarity opposite to electric charge electrified onto a surface of the megakaryocytes M to a wall surface of an electric charge supply flow passage 7 by electric charge supply means 8 to make the megakaryocytes M in the culture solution SI adhere onto the wall surface of the electric charge supply flow passage 7 and remove the megakaryocytes, recovers recovery liquid SO, from which the megakaryocytes M have been removed, from the culture solution SI, and stores the recovery liquid.

Description

  The present invention relates to a separation apparatus and a separation method for removing a specific component from a culture solution. Specifically, the present invention relates to a separation apparatus and a separation method for removing megakaryocytes from a culture solution containing megakaryocytes and platelets.

  Blood cells are transfused in the treatment of blood-related diseases. Since platelets are essential cells for hemostasis, platelets are the only treatment for leukemia, bone marrow transplantation, anticancer treatment, etc., as the only treatment for the prevention and treatment of bleeding based on the quantitative and qualitative decrease of platelets. A blood transfusion is taking place. Platelet preparations used for blood transfusions are covered by blood donation, and recently, there is also a phenomenon that the number of blood donors contaminated with viral infections increases while the total number of blood donors decreases, and stable platelets instead of blood donation A source is sought and research is actively underway.

  Among them, the method described in Non-Patent Document 1 has succeeded in inducing megakaryocytes capable of self-replication from human iPS cells. Since megakaryocytes are cells that produce platelets, it may be possible to supply platelets stably and in large quantities.

  In the method of Non-Patent Document 1, it is described that megakaryocytes mature to produce platelets by forcibly stopping self-replication of megakaryocytes in the culture medium. By the way, megakaryocytes in the culture solution do not mature and produce platelets at the same time, but it takes time for all megakaryocytes to produce platelets. On the other hand, since the characteristics of platelets deteriorate with time, at the stage where all megakaryocytes produce platelets, there is a possibility that degradation of platelets produced in the early stage has progressed.

  In addition, it is not preferable to use a platelet preparation in a state where platelets and megakaryocytes are mixed because the immune system is activated. For this reason, it is necessary to remove megakaryocytes from a culture solution containing platelets and megakaryocytes.

Nakamura et al., “Expandable Megakalyte Cell Lines Enable Clinically-Appliable Generation of Privates, 27th Heisei, Cell 2” URL; http: // www. cell. com / cell-stem-cell / newarts>

  Centrifugation is generally used as a method for removing specific cell components from the culture solution. However, megakaryocytes and platelets are close in specific gravity, and therefore it is necessary to apply a large centrifugal acceleration. For this reason, in the centrifugal separation method, even if megakaryocytes and platelets can be separated, there is a problem that the platelets destroyed by a large centrifugal acceleration increase and the platelet recovery rate decreases.

  The present invention has been made in view of such circumstances, and provides a separation apparatus and a separation method that efficiently remove megakaryocytes from a culture solution in which platelets and megakaryocytes coexist and do not adversely affect platelets. Is.

  In order to solve the above-mentioned problem, the invention described in claim 1 is a separation device that removes megakaryocytes from a culture solution in which megakaryocytes and platelets are mixed, a culture solution storage tank for storing the culture solution, and the culture solution A separator comprising a culture solution feeding means having a function of feeding a culture solution in a storage tank, a collected solution storage tank for collecting and storing a collected solution obtained by removing megakaryocytes from the culture solution, and a charge supply means.

  With the separation device of the present invention, a recovered solution from which megakaryocytes have been removed from the culture solution can be obtained.

The invention according to claim 2 is the separation apparatus according to claim 1, further comprising a charge supply channel functioning as a charge supply means between the culture solution storage tank and the recovery solution storage tank, wherein the charge supply channel is recovered. An outflow destination switching valve having a function of switching the connection destination on the liquid storage tank side to either the recovery liquid storage tank or the return flow path to the culture liquid storage tank is provided, and the surface of the megakaryocyte is charged in the charge supply flow path The separation device according to the present invention has a function of attaching megakaryocytes in a flowing culture solution to the wall surface of the charge supply channel by supplying a charge having a polarity opposite to the charge. In the separation device, megakaryocytes adhere to the wall surface in the charge supply flow path, so the ratio of megakaryocytes in the fluid decreases. For this reason, finally, megakaryocytes can be removed from the culture solution.

  Invention of Claim 3 is the separation apparatus of Claim 2, Comprising: The buffer solution storage tank which stores a buffer solution, The buffer solution sending means which sends the buffer solution in the said buffer solution tank, The said Equipped with an inflow destination switching valve having a function of switching the connection destination of the charge supply channel inlet to either the culture solution feeding means or the buffer solution feeding means, and the deposits adhering to the wall surface are removed with the buffer solution. Thus, the separator has a function of cleaning the charge supply channel.

  According to the separation device of the present invention, it is possible to prevent a decrease in separation performance due to deposits adhering to the wall surface of the charge supply channel.

  In the invention according to claim 4, the surface of the megakaryocyte is charged on the wall surface of the culture solution storage tank and the flow channel in the process of flowing the culture solution containing megakaryocytes and platelets from the culture solution storage tank to the flow channel. The separation method is characterized in that the megakaryocytes are removed from the culture solution by supplying a charge having a polarity opposite to the charge and attaching the megakaryocytes to the wall surface.

  According to the separation method of the present invention, megakaryocytes adhere to the wall surface of the channel along the flow direction of the channel, so that the ratio of megakaryocytes in the fluid decreases. For this reason, finally, megakaryocytes can be removed from the culture solution.

  Invention of Claim 5 is the separation method of Claim 4, Comprising: It adheres to the said wall surface by supplying the electric charge of the same polarity as the electric charge currently charged to the megakaryocyte to the wall surface of a flow path The separation method is characterized in that the attached matter is peeled off and the flow path is washed.

  According to the separation method of the present invention, megakaryocytes, which are main deposits adhering to the wall surface of the flow path, can be easily peeled off, and the flow path can be washed efficiently.

  It is possible to efficiently remove megakaryocytes from a culture medium in which platelets and megakaryocytes are mixed, and to separate them without adversely affecting the platelets.

It is a figure explaining the structure of the separation apparatus in one Embodiment of this invention. It is a figure explaining the function of the electric charge supply flow path in one Embodiment of this invention. It is a figure explaining the function of the electric charge supply channel in one embodiment of the present invention by a section. It is a figure explaining the structure of the separation apparatus in another embodiment of this invention. It is a figure explaining the cylindrical module in another embodiment of this invention. It is a figure explaining isolation | separation of the megakaryocyte using another embodiment of this invention. It is a figure explaining the example of a function using another embodiment of the present invention. It is a figure for demonstrating embodiment to which a megakaryocyte is made to adhere in a culture solution storage tank.

  Embodiments of the present invention will be described below with reference to the drawings.

  FIG. 1 shows a configuration of a separation apparatus 1 that removes megakaryocytes M from a culture solution SI in which megakaryocytes M and platelets P are mixed. The culture solution SI is stored in the culture solution storage tank 2, and the pump 3 is a culture solution supply means for supplying the culture solution SI in the culture solution storage tank 2. Moreover, the buffer solution B is stored in the buffer solution storage tank 4, and the pump 5 is a buffer solution supply means for supplying the buffer solution B in the buffer solution storage tank 4. There is a switching valve 6 on the liquid feed side of the pump 3 and the pump 5, and there is a charge supply channel 7 at the tip of the switch valve 6, and charge is supplied to either the liquid fed from the pump 3 or the pump 5. The valve 6 has an inflow destination switching function for passing through the flow path 7. The charge supply channel 7 is a channel through which liquid flows, and is configured to supply charges to the wall surface. The charge supply means 8 has a function of supplying charges to the wall surface of the charge supply channel 7. A switching valve 9 is provided on the outflow side of the charge supply flow path 7, and the switching valve 9 has an outflow destination switching function for switching the outflow destination to either the recovery liquid storage tank 10 or the return flow path 11. The recovery liquid storage tank 10 is configured to store the recovery liquid SO from which the megakaryocytes M are removed by the charge supply channel 7. The return channel 11 is connected to the culture medium storage tank 2.

  Next, a method for removing the megakaryocytes M from the culture solution SI using the separation device 1 will be described. First, when the pump 3 is driven in a state where the inflow destination of the switching valve 6 is the pump 3 and the outflow destination of the switching valve 9 is the recovery liquid storage tank 10, the culture liquid SI in the culture liquid storage tank 2 is transferred to the charge supply channel 7. Flows inside. At this stage, the charge supply means 8 supplies a charge having a polarity opposite to the charge charged on the megakaryocyte surface to the wall surface of the charge supply channel 7. Then, as shown in FIG. 2A, the megakaryocyte M in the culture solution SI comes to adhere to the wall surface of the charge supply channel 7. That is, taking the case where the cross section of the flow path is cylindrical as an example, the Coulomb force acting on the wall surface side of the main flow path 7 acts on the megakaryocyte M in the culture solution SI as shown in FIG. Adhere to. For this reason, when the culture solution SI flows through the charge supply channel 7, the ratio of megakaryocytes decreases from the culture solution SI to the wall surface and from upstream to downstream. Accordingly, by appropriately setting the length of the charge supply channel 7 and the amount of charge supplied to the wall surface of the charge supply channel 7, the recovery liquid SO that does not contain the megakaryocytes M flows into the recovery liquid storage tank 10. When the length of the charge supply channel 7 and the amount of charge supplied to the wall surface are insufficient, the megakaryocytes M remain in the recovered solution SO, and it is necessary to return the recovered solution SO to the culture solution storage tank 2 and perform separation again. In addition, when it is clear that all megakaryocytes cannot be separated only by flowing the culture solution through the charge supply channel 7 once, the flow-out destination of the switching valve 9 may be the return channel 11. In this case, if the pump 3 is driven, the culture medium storage tank 3 → the charge supply flow path 7 → the return flow path 11 → the circulation of the culture liquid storage tank 3 is performed for a predetermined time, and then the outlet of the switching valve 9 is changed to the recovery liquid storage tank 11. As a result, it is possible to obtain a recovered liquid SO that does not contain megakaryocytes.

  As described above, when the megakaryocyte M is removed from the culture solution SI by the separation device 1, the megakaryocyte M is attached to the wall surface of the charge supply channel 7. For this reason, if this megakaryocyte removal operation is continued, a large number of megakaryocytes M adhere to the wall surface of the charge supply channel 7. In such a state, the megakaryocyte M may be detached from the wall surface of the charge supply channel 7 due to the flow of the culture solution SI. Further, when not used for a long period of time, there is a concern that impurities accumulate on the wall surface of the charge supply channel 7. Therefore, it is desirable that the separation device 1 has a function of cleaning the charge supply channel 7.

  In order to wash the charge supply flow path 7, if the pump 5 is driven using the pump 5 as the inflow destination of the switching valve 6 and the return flow path 11 as the outflow destination of the switching valve 9 in the separation device 1 of FIG. Buffer B flows through the flow path 7. By increasing the flow rate of the buffer solution B, the megakaryocytes M and impurities adhering to the wall surface of the charge supply channel 7 are separated and flow into the culture solution storage tank 2 side, so that the charge supply channel 7 is washed.

  Further, in the process of flowing the buffer solution B into the charge supply channel 7, if a charge having the same polarity as the charge charged on the megakaryocyte surface is supplied to the wall surface of the charge supply channel 7, the megakaryocyte attached to the wall surface M can be easily peeled off.

  This will be described using the separation device 1. First, when the pump 5 is driven in a state where the inflow destination of the switching valve 6 is the pump 5 and the outflow destination of the switching valve 9 is the return flow path 11, the buffer solution B in the buffer solution storage tank 4 becomes the charge supply flow path 7. Flows inside. At this stage, charges having the same polarity as the charges charged on the megakaryocyte surface are supplied to the wall surface of the charge supply channel 7 by the charge supply means 8. Then, as shown in FIG. 2B, the megakaryocytes M attached to the wall surface of the main flow path 7 are separated from the wall surface and mixed with the buffer solution B. That is, taking the case where the cross section of the channel is cylindrical as an example, the Coulomb force acting away from the wall surface acts on the megakaryocyte M attached to the wall surface of the charge supply channel 7 as shown in FIG. Peel from. The peeled megakaryocytes M and platelets P flow into the culture medium storage tank 2 through the return channel 11 due to the flow of the buffer B.

  FIG. 4 shows a configuration of a separation apparatus 100 which is another embodiment of the present invention. The difference from the separation device 1 of FIG. 1 is that the charge supply flow path 7 is constituted by a plurality of cylindrical modules. The relationship between the charge supply channel 7 in FIG. 1 and the cylindrical module 71 in FIG. 4 (the same applies to the cylindrical modules 72, 73 and 74) is the same as that shown in FIGS. 5 (a) and 5 (b). The length is shortened. In FIG. 5, the charge supply channel 7 and the cylindrical module are cylindrical, but the cross-sectional shape is not limited to a circle, and may be a polygonal shape or the like as long as the channel can be formed. Further, in FIG. 4, the main flow path 7 is constituted by the cylindrical modules 71, 72, 73, 74. However, the number of the cylindrical modules is not limited to four. It is also possible to determine the length of the charge supply channel 7 (the number of cylindrical modules) as necessary.

  The megakaryocyte M when the culture fluid flows in a state in which the cylindrical module 71, 72, 73, 74 is supplied with a charge having a polarity opposite to that charged on the megakaryocyte surface by the separation device 100 of FIG. FIG. 6 illustrates an example of the mixed state of the platelets P and the platelets P, and shows how the megakaryocytes M are gradually removed by adhering to the wall surface. Since the number of cylindrical modules to be connected is changed as described above, the separation module 74 may be omitted in the separation operation of the culture solution SI having a low concentration of the megakaryocyte M as shown in FIG.

  In the embodiment described so far, the charge is supplied to the wall surface of the charge supply channel 7 and the megakaryocytes M are attached, but the charge is supplied to the culture solution storage tank 2 and the meganucleus is applied to the wall surface of the culture solution storage tank. It is also possible to adsorb the sphere M. An example of such a case will be described with respect to the separation device 101 of FIG. 8 in which charges can be supplied from the charge supply means 8 to the wall surface of the culture medium storage tank 2 of the separation device 1 of FIG. In the separation device 101, the inflow destination of the switching valve 6 is switched to the pump 3 while the charge supply means 8 supplies the wall surface of the culture solution storage tank 2 with the charge having the opposite polarity to the charge charged on the megakaryocyte surface. The pump 3 is driven with the flow-out destination of the valve 9 as the return flow path 11. In this way, the culture solution SI is circulated through the culture solution storage tank 2 → the charge supply channel 7 → the return flow channel 11 → the culture solution storage tank 2, so that the megakaryocytes M in the circulating liquid are transferred to the culture solution storage tank 2. It will be adsorbed on the wall. Thus, if the flow-out destination of the switching valve 9 is the recovery liquid storage tank 10 after a predetermined circulation condition (flow velocity, time, etc.), the recovery liquid SO that does not contain the megakaryocytes M is obtained. At this stage, when the recovered liquid SO that does not contain the megakaryocyte M is introduced into the recovered liquid storage tank 10, a flow path and a valve are provided so that the outflow destination of the pump 3 becomes the recovered liquid storage tank 10. May be.

  In addition, when supplying a charge to the wall surface of the culture solution storage tank 2 to remove the megakaryocytes M, the charge supply channel 7 may be a normal channel without a charge supply function. The megakaryocyte M may be attached to both the storage tank 2 and the charge supply channel 7 by supplying a charge having a polarity opposite to the charge charged on the megakaryocyte surface.

  The present invention is suitable for use in removing megakaryocytes from a culture solution in which megakaryocytes and platelets are mixed. However, the present invention is not limited to this, and specific microparticles or cells are removed from a suspension containing microparticles and cells. It can also be applied to such general purposes.

DESCRIPTION OF SYMBOLS 1,100 Separation apparatus 2 Culture solution storage tank 3 Pump 4 Buffer solution storage tank 5 Pump 6 Switching valve 7 Charge supply flow path 8 Charge supply means 9 Switching valve 10 Recovery liquid storage tank 11 Return flow path 71, 72, 73, 74 Tubular module B buffer M megakaryocyte P platelet SI culture solution SO recovery solution

Claims (5)

A separator for removing megakaryocytes from a culture solution containing megakaryocytes and platelets,
A culture solution storage tank for storing the culture solution;
A culture solution feeding means having a function of feeding the culture solution in the culture solution storage tank;
A recovery liquid storage tank for recovering and storing the recovery liquid from which the megakaryocytes have been removed from the culture liquid;
A separation device comprising charge supply means. The separation device according to claim 1,
A charge supply channel functioning as a charge supply means is provided between the culture solution storage tank and the recovery solution storage tank,
An outlet switching valve having a function of switching the connection destination of the charge supply channel on the recovery liquid storage tank side to either the recovery liquid storage tank or the return flow path to the culture liquid storage tank;
By supplying a charge of the opposite polarity to the charge charged on the megakaryocyte surface to the charge supply channel, the megakaryocyte in the flowing culture solution is attached to the wall surface of the charge supply channel. Separation device characterized by that. The separation device according to claim 2,
A buffer storage tank for storing the buffer solution;
A buffer solution feeding means for feeding a buffer solution in the buffer solution storage tank;
An inflow destination switching valve having a function of switching the connection destination of the charge supply channel inlet to either the culture solution feeding means or the buffer solution feeding means;
A separation device having a function of separating the deposits adhering to the wall surface with a buffer solution and washing the charge supply channel. In the process of flowing the culture solution containing megakaryocytes and platelets from the culture solution storage tank to the flow path,
Removing the megakaryocytes from the culture solution by supplying a charge of the opposite polarity to the charge on the megakaryocyte surface to the wall of the culture medium reservoir and the flow path, and attaching the megakaryocytes to the wall surface Separation method characterized by.   5. The separation method according to claim 4, wherein a charge having the same polarity as a charge charged on a megakaryocyte is supplied to a wall surface of a total charge supply flow channel, thereby peeling off the attached matter attached to the wall surface. And the separation method characterized by washing | cleaning an electric charge supply flow path.

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