JP2014117220A - Cell separator - Google Patents

Abstract

PROBLEM TO BE SOLVED: To collect healthy cells in high yield under distributed state while ensuring sterility.SOLUTION: To provide a cell separator 1 comprising: a suspension container 3 for storing cell suspension; a cell capturing filter 2 which is connected to the suspension container 3 and capturing cell in cell suspension injected from the suspension container 3; a collected solution introducing part 5 which is connected to the cell capturing filter 2 on the opposite side to the suspension container 3 and introducing the collected solution into the cell capturing filter 2; a cell distributing part 7 which is connected to the cell capturing filter 2 on the same side with the suspension container 3 and distributing cell in the collected solution flown out from the cell capturing filter 2; and deceleration means 8 decelerating flow speed of cells flown from the cell capturing filter 2 into the cell distributing part 7.

Description

  The present invention relates to a cell separation device.

  Conventionally, in regenerative medicine, a cell separation device that separates cells effective for treatment such as stem cells from adipose tissue collected from a living body is known (for example, see Patent Document 1). According to Patent Document 1, cells are released from adipose tissue by decomposing adipose tissue with an enzyme, and the cells are captured by the filter by passing the released cell suspension through the filter. The cells are released from the filter and collected by vigorously flowing in the opposite direction. At this time, the cells captured by the filter tend to aggregate with each other. Since cells cannot be transplanted to a living body at a uniform density if the cells are aggregated with each other, it is preferable that the cells are dispersed in a single state during transplantation.

  On the other hand, there is known a dispersion apparatus that mixes two kinds of liquids by colliding the liquid and the wall surface of the flow path or the liquids to generate turbulent flow, thereby further reducing the fine particles contained in the liquid (for example, (See Patent Document 2). Since the dispersion device using such turbulent flow can disperse particles in a closed system, it can be suitably applied to disperse cells for transplantation that must ensure sterility.

JP 2007-289076 A Japanese Patent No. 2788010

  However, when the dispersing device of Patent Literature 2 is applied to the cell separation device of Patent Literature 1 and the cells released from the filter are dispersed by the dispersing device and then collected, the following problems occur. In order to reliably release the cells from the filter, it is necessary to vigorously introduce the recovery solution into the filter. On the other hand, the turbulent flow rate required to disperse cells is sufficiently slow, and when turbulent flow with an excessively high flow rate acts on the cells, a large shear force is generated between the cells that are aggregating with each other. Affects the cells. That is, the cells released from the filter vigorously flow into the dispersion device, and the cells are exposed to excessive turbulence inside the dispersion device, so that the recovery rate of healthy cells decreases.

  The present invention has been made in view of the above-described circumstances, and an object thereof is to provide a cell separation device capable of recovering healthy cells in a high yield and dispersed state while ensuring sterility. And

In order to achieve the above object, the present invention provides the following means.
The present invention relates to a suspension container that contains a cell suspension, and a cell capture filter that is connected to the suspension container and captures cells contained in the cell suspension flowing from the suspension container. And a recovery liquid inlet that is connected to the cell capture filter on the side opposite to the suspension container and introduces a recovery liquid into the cell capture filter, and the cell capture filter on the same side as the suspension container. A cell dispersion part that disperses the cells contained in the recovery solution that has flowed out of the cell trapping filter, and a speed reducer that reduces the flow rate of the cells flowing into the cell dispersion part from the cell trapping filter. A cell separation device is provided.

  According to the present invention, the cell suspension is flowed from the suspension container into the cell trapping filter, and then the recovery liquid is introduced from the recovery liquid introduction unit to the cell trapping filter in the opposite direction to the previous one. The cells captured by the capture filter are pushed out of the cell capture filter together with the recovery liquid, and dispersed in the cell dispersion unit. Thereby, the cell of the state disperse | distributed ensuring sterility in a closed system can be obtained.

  In this case, the flow rate of the cells pushed out from the cell trapping filter and flowing into the cell dispersion portion is reduced by the speed reduction means. That is, while the cell is reliably released by introducing the collection liquid at a high flow rate into the cell trapping filter, the influence of the cells due to the cells flowing at a low flow rate in the cell dispersion portion is reduced. Thereby, healthy cells can be recovered in a high yield and dispersed state.

In the above-described invention, a pipe line that connects the cell trapping filter and the cell dispersion part is provided, and the speed reduction means is on the cell dispersion part side that is formed with a smaller diameter than the other part of the pipe line. It may consist of ends.
By doing in this way, flow path resistance increases in an edge part with a small flow path diameter rather than other parts, and the flow rate of the recovery liquid and the cell contained in this recovery liquid decreases. Thereby, the flow rate of the cells can be reduced with a simple configuration and operation.

Moreover, in the said invention, the said deceleration means may be provided with the movable body which receives the fluid pressure of the said collection | recovery liquid and moves between the said cell capture filter and the said cell dispersion | distribution part.
By doing so, a part of the momentum of the recovery liquid is converted into the momentum of the movable body and consumed, thereby reducing the flow rate of the recovery liquid and the cells contained in the recovery liquid. Thereby, the flow rate of the cells can be reduced with a simple configuration and operation.

Moreover, in the said invention, the said movable body may be comprised between the said cell capture | acquisition filter and the said cell dispersion | distribution part, and may consist of an elastic pipe line which can be elastically expanded-contracted in the circumferential direction. Further, in the above invention, a pipe line that connects the cell trapping filter and the cell dispersion part is provided, and the speed reduction means is a valve body as the movable body arranged at a midway position of the pipe line, The valve body may be supported on the side opposite to the cell trapping filter, and may include an elastic body that can be elastically expanded and contracted in the flow direction of the collected liquid in the conduit.
By doing so, the momentum of the recovered liquid can be consumed as elastic deformation of the elastic pipe line or elastic body.

Moreover, in the said invention, the said cell dispersion | distribution part may be provided with the dispersion | distribution flow path which generates a turbulent flow in the said collection | recovery liquid.
By doing in this way, a collection | recovery liquid can be disperse | distributed efficiently by stirring by a turbulent flow.

In the above invention, the cell dispersion part includes an inlet through which the recovered liquid flows, and a plurality of the dispersion channels connected in parallel to the inlet and having equal channel resistance, and the deceleration The means may comprise the plurality of dispersion channels.
In this way, when the recovered liquid flowing in from the inlet is distributed to the dispersion flow path, the flow rate of the recovered liquid is also equally divided by the number of dispersion flow paths and reduced. Thereby, the flow rate of the cells can be reduced with a simple configuration and operation.

Moreover, in the said invention, the said deceleration means may be equipped with the cell capture mechanism which makes a capture force act on the cell which flows through the said dispersion | distribution flow path.
By doing in this way, the flow rate of a cell can be reduced because a cell counters the flow of a collection | recovery liquid by a capture | acquisition force in a dispersion | distribution flow path.

Moreover, in the said invention, the said cell capture | acquisition mechanism may make the said cell act on the said cell as a capture force, dielectrophoretic force, electrophoretic force, an ultrasonic wave, optical tweezers, or affinity.
By doing in this way, capture force can be made to act on a cell without contact.

In the above invention, the cell trapping mechanism may trap the cells in the vicinity of the wall surface of the dispersion channel.
By doing so, it is possible to effectively reduce the cell flow rate by capturing the cell in the vicinity of the wall surface having a relatively low flow rate.

  According to the present invention, it is possible to collect healthy cells in a high yield and dispersed state while ensuring sterility.

It is a whole block diagram of the cell separation device concerning one embodiment of the present invention. It is a block diagram which shows the deceleration means with which the cell separation apparatus of FIG. 1 is provided. It is a block diagram which shows the modification of the deceleration means with which the cell separation apparatus of FIG. 1 is provided. It is a block diagram which shows another modification of the deceleration means with which the cell separation apparatus of FIG. 1 is provided. (A) The side view of a cell dispersion | distribution part which shows another modification of the deceleration means with which the cell separation apparatus of FIG. 1 is equipped, (b) Sectional drawing in II line, and (c) Sectional drawing in II-II line is there. It is a block diagram which shows another modification of the deceleration means with which the cell separation apparatus of FIG. 1 is provided.

Below, cell separation device 1 concerning one embodiment of the present invention is explained with reference to drawings.
As shown in FIG. 1, the cell separation device 1 according to the present embodiment is aseptically connected to a cell trapping filter 2 that traps cells contained in a cell suspension, and to the cell trapping filter 2 by a conduit 9. The suspension container 3, the recovery container 4, the recovery liquid introduction part 5, and the drainage container 6 are provided. In FIG. 1, the vertical direction of the paper surface corresponds to the vertical vertical direction.

  The cell trapping filter 2 has a pore size smaller than that of the cells, and can selectively capture cells contained in the cell suspension. The filter medium of the cell trapping filter 2 is made of a woven fabric, a nonwoven fabric or a porous body. When the filter medium is made of a nonwoven fabric, the fiber diameter is preferably 20 μm to 100 μm. When the filter medium is made of a porous material, the pore diameter is preferably 20 μm to 100 μm.

  The material of the filter medium is a synthetic polymer such as polyethylene, polypropylene, polystyrene, acrylic resin, nylon, polyester, polycarbonate, polyacrylamide, polyurethane, agarose, cellulose, cellulose acetate from the viewpoint of ease of molding and sterilization and biocompatibility. Natural polymers such as chitin, chitosan and alginate, inorganic materials such as hydroxyapatite, glass, alumina and titania, or metals such as stainless steel, titanium and aluminum are preferred.

  The suspension container 3 and the collection container 4 are connected in parallel to the upper part of the cell trapping filter 2 via a valve V1, and can selectively communicate with the cell trapping filter 2 by switching the valve V1.

  The suspension container 3 contains a cell suspension. This cell suspension is, for example, a digestive enzyme solution containing adipose tissue-derived cells obtained by stirring and decomposing adipose tissue collected from a living body in the digestive enzyme solution. The suspension container 3 is disposed at a position higher than the cell trapping filter 2 and has an air vent valve that aseptically opens the inside to atmospheric pressure. By opening the air vent valve while communicating with the cell trapping filter 2 by the valve V1, the cell suspension is lowered from the suspension container 3 toward the cell trapping filter 2 by gravity.

  The collection container 4 is, for example, a culture container, and the collected cells can be cultured as they are. The collection container 4 may contain a scaffold such as a transplant material, and cells may be seeded on the scaffold in the collection container 4.

The collection liquid introduction unit 5 and the drainage container 6 are connected in parallel to the lower part of the cell trapping filter 2 via a valve V2, and can selectively communicate with the cell trapping filter 2 by switching the valve V2.
The collection liquid introduction unit 5 includes, for example, a syringe containing the collection liquid, and press-fits the collection liquid into the cell capture filter 2 communicated via the valve V2. As a result, the cells captured by the cell trapping filter 2 are released from the cell trapping filter 2 by the fluid pressure of the recovery liquid from the bottom to the top, and are pushed out of the cell trapping filter 2 together with the recovery liquid.

  In addition, the cell separation device 1 includes a cell dispersion unit 7 connected between the cell trapping filter 2 and the collection container 4, and a speed reducing unit 8 that reduces the flow rate of the collected liquid flowing into the cell dispersion unit 7. ing.

  The cell dispersion unit 7 has a structure for generating a turbulent flow in the collected liquid flowing in from the cell trapping filter 2. Specifically, in the cell dispersion portion 7, a dispersion channel having a channel diameter of about 50 μm to 500 μm is formed. The dispersion flow path is bent and branched at an intermediate position, and the branched dispersion flow paths merge again. The recovery liquid collides with the wall surface of the dispersion flow path at the position where the dispersion flow path is bent or branched, and the recovery liquids flowing from different directions collide with each other at the position where the dispersion flow path joins. This effectively generates turbulence in the recovered liquid. The collected liquid flowing through the dispersion flow path is agitated by such turbulent flow, so that the aggregated cells are dispersed apart.

  As shown in FIG. 2, the speed reduction means 8 includes an end portion (hereinafter also referred to as a small diameter portion) 81 on the cell dispersion portion 7 side of the conduit 9 that connects the cell trapping filter 2 and the cell dispersion portion 7. The diameter is smaller than the other part on the cell trapping filter 2 side. The small-diameter portion 81 having a smaller pipe diameter than other portions has a larger flow resistance than the other portions. Therefore, the flow rate of the recovery liquid A flowing from the cell trapping filter 2 to the cell dispersion portion 7 is reduced in the narrow diameter portion 81 where the pipe diameter is reduced.

  Specifically, the pressure loss Δp that the narrow diameter portion 81 gives to the recovered liquid A is expressed by the Fanning equation of the following equation (1). Here, L is the length of the small-diameter portion 81, D is the inner diameter of the small-diameter portion 81, ρ is the fluid density of the recovered liquid A, V is the cross-sectional average flow velocity of the small-diameter portion 81, and f is the friction loss coefficient.

  The friction loss coefficient is expressed by the Blasius equation shown in the following equation (2). Here, Re is the Reynolds number.

  For example, in a mesenchymal stem cell separation device (manufactured by Kaneka Corporation) commercially available as a cell trapping filter, the recommended value of the recovery liquid introduction rate is 16.7 mL / sec. When the inner diameter of the narrow diameter part 81 is 1 mm and the recovery liquid A flows from the cell trapping filter 2 into the small diameter part 81 at a flow rate of 16.7 mL / sec, the fluid pressure of the recovery liquid A flows during 1 cm through the small diameter part 81. 60 kPa.

Next, the operation of the cell separation device 1 configured as described above will be described below.
In order to separate and collect cells from the cell suspension using the cell separation device 1 according to the present embodiment, the suspension is communicated from the suspension container 3 to the drainage container 6 via the cell capture filter 2 by switching the valve V1. Then, the air vent valve of the suspension container 3 is opened. As a result, the cell suspension in the suspension container 3 descends due to gravity and flows through the cell trapping filter 2 from top to bottom. In the cell capture filter 2, the cells in the cell suspension are captured, and other components of the cell suspension flow out to the drainage container 6.

  Next, by switching the valves V <b> 1 and V <b> 2, the recovery liquid introduction unit 5 communicates with the recovery container 4 through the cell capture filter 2, and the recovery liquid is pressed into the cell capture filter 2 from the recovery liquid introduction unit 5. Thereby, the cells captured by the cell trapping filter 2 are pushed out toward the recovery container 4 together with the recovery liquid.

  The pushed-out recovery liquid is decelerated in the narrow diameter part 81 constituting the speed reduction means 8 and then flows into the cell dispersion part 7 and flows through the dispersion channel while being stirred in the cell dispersion part 7. The contained aggregated cells are dispersed. Thereby, in the collection | recovery container 4, the cell of the state disperse | distributed to single can be collect | recovered aseptically.

  In addition, after the cells captured by the cell trapping filter 2 are washed with a washing liquid, the collection liquid may be introduced into the cell trapping filter 2. In this case, the cleaning liquid may be flowed from the cleaning liquid container (not shown) connected in parallel with the suspension container 3 to the upper part of the cell trapping filter 2 toward the drainage container 6.

  Thus, according to the present embodiment, the flow rate of the collected liquid that has been vigorously introduced into the cell trapping filter 2 is reduced by the speed reduction means 8 and then flows into the cell dispersion unit 7. Therefore, it is possible to suppress the influence on the cells by introducing the collection liquid into the cell dispersion portion 7 at a flow rate necessary and sufficient for cell dispersion while reliably releasing the cells in the cell trapping filter 2. Thereby, healthy cells can be recovered in a high yield and dispersed state.

  In the present embodiment, the speed reducing means 8 reduces the flow rate of the recovered liquid by the narrow diameter portion 81 connected to the inlet of the cell dispersion portion 7. A movable body that is moved by receiving the fluid pressure of the collected liquid is provided at an intermediate position with respect to the cell dispersion unit 7, and a part of the momentum of the collected liquid is converted into a momentum of the movable body. May be reduced.

For example, as shown in FIG. 3, the speed reduction means 8 includes a valve body 82 as a movable body that is movably disposed in the middle of the conduit 9, and the valve body 82 on the side opposite to the cell trapping filter 2. And a damper valve comprising an elastic member 83 that can be expanded and contracted in the flow direction of the recovered liquid A. When the valve body 82 is pressed by the fluid pressure of the recovered liquid A from the cell trapping filter 2, the elastic member 83 contracts to open the conduit 9 (see the two-dot chain line). .) At this time, a part of the momentum of the recovery liquid A is consumed as the contraction motion of the elastic member 83, so that the flow rate of the recovery liquid A is reduced.
Even if it does in this way, the flow rate of the collection | recovery liquid A can be reduced by a simple structure, without requiring special operation.

  Moreover, the deceleration means 8 may be provided with the elastic pipe line 84 as a movable body, as FIG. 4 shows. The elastic conduit 84 is made of an elastic member such as rubber that can expand and contract in the circumferential direction, and can be expanded in the radial direction by the fluid pressure of the recovered liquid A. The elastic conduit 84 may be formed by constituting only a part of the conduit 9 in the circumferential direction from an elastic member.

  In this case, it is preferable that the cell dispersion portion 7 has an orifice 7a that restricts the inflow amount of the recovered liquid A at the inlet, and the elastic conduit 84 is provided immediately before the orifice 7a. In this way, the pressure of the recovered liquid A that has risen locally before the orifice 7a is effectively converted into the circumferential extension of the elastic conduit 84, so that the recovery flowing into the cell dispersion portion 7 is performed. The flow rate of the liquid A can be effectively reduced.

Moreover, in this embodiment, the deceleration means 8 may be comprised from the some dispersion | distribution flow path 71 provided in the cell dispersion | distribution part 7, as FIG.5 (a)-(c) shows. The plurality of dispersion channels 71 are provided in parallel to the inlet 7 b of the cell dispersion unit 7. The plurality of dispersion channels 71 are equal in inner diameter and length, and have the same channel resistance. The recovered liquid flowing in from the inlet 7b is evenly distributed to the plurality of dispersion channels 71 and merges at the outlet 7c. At this time, the flow rate of the recovered liquid flowing through the dispersion channel 71 is equal to the flow rate at the inlet 7b divided by the number of the dispersion channels 71.
Even if it does in this way, the flow rate of the collection | recovery liquid A can be reduced by a simple structure, without requiring special operation.

In the present embodiment, the speed reduction means 8 may include a cell capturing mechanism that captures cells flowing through the cell dispersion unit 7.
FIG. 6 is a cross-sectional view of the dispersion flow path 71 of the cell dispersion unit 7 and shows a configuration for capturing cells using the dielectrophoretic force. In FIG. 6, the cell trapping mechanism includes two electrodes 10 a and 10 b that are arranged to face each other with a gap in the direction intersecting the flow direction of the collected liquid (direction perpendicular to the paper surface) in the dispersion channel 71, and these electrodes 10 a. , 10b, and an AC power source 12 for applying an AC voltage. One of the two electrodes 10a and 10b is covered with an insulating film 11 having a large number of pores.

  When an AC voltage is applied from the AC power supply 12 between the two electrodes 10a and 10b, an AC electric field is formed in the dispersion channel 71. At this time, an electric field gradient is formed in the opposing direction of the electrodes 10a and 10b, and the closer to the electrodes 10a and 10b, the stronger the electric field. A dielectrophoretic force according to the dielectrophoretic characteristics of the cell acts on the cell, and the cell is attracted to and captured by one of the electrodes 10a and 10b.

  On the other hand, the recovered liquid forms a laminar flow in the sufficiently narrow dispersion flow path 71. The laminar flow velocity increases at the center of the dispersion channel 71 and decreases near the wall surface of the dispersion channel 71. That is, since the flow of the recovery liquid is slow in the vicinity of the wall surface of the dispersion flow path 71 where the electrodes 10a and 10b are arranged, the cells attracted to the electrodes 10a and 10b are sufficiently captured by the dielectrophoretic force, and the flow velocity of the cells Can be reduced.

  When cells are dispersed apart by agitation, the shearing force generated between the aggregated cells greatly affects the cells. That is, by capturing the cells in the collected liquid flowing relatively fast and reducing the flow rate of the cells, the shearing force generated between the cells can be reduced and the influence on the cells can be reduced. Furthermore, by setting the frequency of the alternating electric field so that the dielectrophoretic force acting on the desired cell is maximized, the desired cell, for example, a living cell can be selectively recovered.

  The cell trapping mechanism only needs to be able to act on the cell in a non-contact manner. In addition to the dielectrophoretic force, the cell trapping mechanism uses affinity such as electrophoretic force, ultrasonic wave, magnetic force, optical tweezers or antibody. Cells may be captured.

DESCRIPTION OF SYMBOLS 1 Cell separator 2 Cell capture filter 3 Suspension container 4 Recovery container 5 Recovery liquid introduction part 6 Drainage container 7 Cell dispersion part 7a Orifice 8 Deceleration means 81 Narrow diameter part 82 Valve body 83 Elastic member 84 Elastic pipe | tube 9 Pipe Path 10a, 10b Electrode 11 Insulating film 12 AC power supply A Recovery liquid V1, V2 Valve

Claims (10)

A suspension container containing the cell suspension;
A cell capturing filter connected to the suspension container and capturing cells contained in the cell suspension flowing from the suspension container;
A recovery liquid inlet that is connected to the cell capture filter on the side opposite to the suspension container and introduces a recovery liquid into the cell capture filter;
A cell dispersion unit that is connected to the cell trapping filter on the same side as the suspension container and disperses the cells contained in the collected liquid that has flowed out of the cell trapping filter;
A cell separation apparatus comprising: deceleration means for reducing a flow rate of the cells flowing into the cell dispersion part from the cell trapping filter. Comprising a conduit connecting the cell trapping filter and the cell dispersion part,
The cell separation device according to claim 1, wherein the speed reducing unit includes an end portion on the cell dispersion portion side that is formed to have a smaller diameter than the other portion of the conduit.   3. The cell separation device according to claim 1, wherein the speed reduction unit includes a movable body that is moved by receiving the fluid pressure of the collected liquid between the cell trapping filter and the cell dispersion unit.   The cell separation device according to claim 3, wherein the movable body is connected between the cell trapping filter and the cell dispersion portion, and is composed of an elastic conduit that can be elastically expanded and contracted in a circumferential direction. Comprising a conduit connecting the cell trapping filter and the cell dispersion part,
The decelerating means supports the valve body as the movable body disposed in the middle of the pipeline, and supports the valve body on the side opposite to the cell trapping filter, and the flow of the recovered liquid in the pipeline The cell separation device according to claim 3 or 4, comprising an elastic body elastically stretchable in a direction.   The cell separation device according to any one of claims 1 to 5, wherein the cell dispersion unit includes a dispersion channel that generates a turbulent flow in the collected liquid. The cell dispersion unit includes an inlet through which the recovery liquid flows and a plurality of the dispersion channels connected in parallel to the inlet and having equal channel resistance.
The cell separation device according to claim 6, wherein the speed reducing unit includes the plurality of dispersion channels.   The cell separation device according to claim 6 or 7, wherein the speed reduction unit includes a cell trapping mechanism that applies a trapping force to cells flowing in the dispersion flow path.   The cell separation device according to claim 8, wherein the cell capturing mechanism captures the cells by dielectrophoretic force, electrophoretic force, ultrasonic wave, magnetic force, optical tweezers, or affinity.   The cell separation device according to claim 8 or 9, wherein the cell trapping mechanism traps the cells near the wall surface of the dispersion channel.

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