JP2010004753A - Electrode unit for separating cell and cell separation device - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide an electrode unit for separating cells, constituted so as to have a small and compact volume while improving separation ability by securing the area, and to provide a cell separation device. <P>SOLUTION: The electrode unit 4 for separating cells includes a plurality of ring-shaped electrodes 6 arranged in a pathway 3 through which a cell suspension flows, having the axial directions coinciding with the flowing direction, and arranged in the axial direction at nearly parallel intervals so as to have alternately different polarities. When a high-frequency voltage is applied between the electrodes, a non-uniform high-frequency electric field with a changing intensity is formed around each of the electrodes, and dielectrophoretic forces having a magnitude and sign based on the dielectrophoretic characteristics acts on the cells in the cell suspension. As a result, the cells in the cell suspension are separated based on the electrophoretic characteristics. <P>COPYRIGHT: (C)2010,JPO&INPIT

Description

  The present invention relates to an electrode unit for cell separation and a cell separation device.

  Conventionally, a high-frequency unequal electric field is formed around the electrodes by electrodes arranged in a plane, and the cells are separated by type according to the magnitude of the dielectrophoretic force acting on the cells passing through the high-frequency unequal electric field. A cell separation device is known (see, for example, Patent Document 1).

JP-A-2005-227111

  However, the cell separation device of Patent Document 1 has electrodes arranged in a plane, and it is necessary to increase the area on the plane in order to increase the separation capability. In particular, since the distance from the electrode on which the dielectrophoretic force acts is about 500 μm, the conventional cell separation device has a disadvantage that the device must be extremely thin.

  The present invention has been made in view of the above-described circumstances, and provides a cell separation electrode unit and a cell separation device that can be configured to be small and compact while ensuring an area and enhancing separation performance. It is an object.

In order to achieve the above object, the present invention provides the following means.
The present invention is arranged in a flow path for circulating a cell suspension, the axial direction coincides with the flow direction, and a plurality of polarities alternately arranged at substantially parallel intervals in the axial direction An electrode unit for cell separation comprising an annular electrode is provided.

  According to the present invention, a high frequency unequal electric field whose intensity changes around each electrode is formed by applying a high frequency voltage between adjacent electrodes while being arranged in the flow path. In this state, when the cell suspension is circulated in the flow path, a dielectrophoretic force having a size and a sign based on the dielectrophoretic characteristics acts on the cells in the cell suspension. In the space surrounded by the annular electrode, a high-frequency unequal electric field with a lower gradient toward the center is formed, so that cells having negative dielectrophoretic characteristics move toward the center of the electrode by the dielectrophoretic force. It is drawn and allowed to flow along the central region of the flow path. On the other hand, cells having positive dielectrophoretic properties are attracted to the electrode and flow along the outer region of the channel. Thereby, the cells in the cell suspension are separated based on their dielectrophoretic properties.

  In this case, according to the present invention, by forming the electrode in an annular shape, it is possible to effectively use the space and secure an area with a small volume as compared with the conventional flat electrode. Thus, the separation capability can be enhanced while the device is made compact.

In the above invention, a plurality of the annular electrodes may be arranged at intervals in the radial direction.
By doing this, it is possible to apply high frequency voltages of different frequencies to the annular electrodes arranged in the radial direction to form different high frequency unequal electric fields and attract cells with different dielectrophoretic characteristics. it can. Thereby, cells having different dielectrophoretic characteristics can be collected in the central flow path in the radial direction and the tubular flow path outside thereof, and the cells in the cell suspension can be further finely separated.

  Further, the present invention is arranged in a flow path for circulating the cell suspension, and the axial direction is made coincident with the flow direction, and the polarities are alternately different at substantially parallel intervals in the circumferential direction along the axial direction. An electrode unit for cell separation comprising a plurality of linear electrodes arranged in a tubular shape is provided.

  According to the present invention, a high frequency unequal electric field whose intensity changes around each electrode is formed by applying a high frequency voltage between adjacent electrodes while being arranged in the flow path. In this state, when the cell suspension is circulated in the flow path, a dielectrophoretic force having a size and a sign based on the dielectrophoretic characteristics acts on the cells in the cell suspension. A high-frequency unequal electric field is formed in the other space surrounded by the linear electrodes arranged in a tubular shape, and the gradient increases toward the center of the other spaces. It is drawn toward the center of the electrode. On the other hand, cells having negative dielectrophoretic characteristics flow radially outward from the gaps between the electrodes. Thereby, the cells in the cell suspension are separated based on their dielectrophoretic properties.

  Even in this case, by arranging the linear electrodes in a tubular shape with an interval in the circumferential direction, it is possible to effectively use the space and to reduce the volume as compared with the conventional electrodes formed in a planar shape. Thus, it is possible to increase the separation capability while securing an area and reducing the size of the apparatus.

In the above-described invention, a plurality of the electrodes arranged in the tubular shape may be arranged at intervals in the radial direction.
By doing so, high frequency voltages of different frequencies are applied to linear electrodes arranged at intervals in the radial direction to form different high frequency unequal electric fields and attract cells with different dielectrophoretic characteristics. Can do. As a result, cells having different dielectrophoretic characteristics can be collected in a flow path near the center in the radial direction and an annular flow path in the vicinity of the radial position corresponding to each of the outer electrodes. Can be further finely separated.

The present invention also provides a cell separation apparatus comprising a flow path for circulating a cell suspension and any one of the cell separation electrode units disposed in the flow path.
According to the present invention, when a cell suspension is circulated through the flow path with a high frequency voltage applied between the electrodes, a high frequency unequal electric field is formed around the annular electrode, so that cells passing near the electrode A dielectrophoretic force in accordance with the dielectrophoretic characteristics acts on. As a result, cells that are retained in the annular electrode and cells that flow through the gap between the electrodes and flow outside the electrode are generated, and the cells in the cell suspension can be separated according to the dielectrophoretic characteristics. It becomes possible.

  According to the present invention, there is an effect that the volume can be reduced and the configuration can be made compact while securing the area and increasing the separation capability.

Hereinafter, an electrode unit for cell separation and a cell separation device according to a first embodiment of the present invention will be described with reference to FIGS.
As shown in FIG. 1 and FIG. 2, the cell separation device 1 according to this embodiment includes a container 2 for storing a cell suspension, a flow path 3 connected to the container 2, and a flow path 3. The cell separation electrode unit 4 and the cell collection unit 5 that collects the cells that have flowed through the flow path 3 are provided.

In the container 2, for example, a cell suspension containing adipose-derived cells isolated by stirring adipose tissue collected from a human together with a digestive enzyme solution is stored.
The flow path 3 is formed in a circular tube shape.

  As shown in FIG. 3, the cell separation electrode unit 4 includes an electrode body 4A formed in a cylindrical bowl shape by processing a wire, and a voltage supply unit 4B that applies a high-frequency voltage to the electrode body 4A. I have. The electrode body 4A has a plurality of annular annular electrodes 6 arranged at intervals in the longitudinal direction of the flow path, and a straight rod-like shape that is arranged at intervals in the circumferential direction and connects the annular electrodes to each other. It is comprised from the rod-shaped electrode 7. FIG. The diameter of the annular electrode 6 is about 1 mm in diameter. The distance between adjacent annular electrodes 6 and the distance between adjacent rod-shaped electrodes 7 are about 100 μm.

The voltage supply unit 4B applies high-frequency voltages of different polarities to the adjacent annular electrodes 6 and rod-shaped electrodes 7 of the electrode body 4A, respectively, and the high-frequency voltage decreases gradually as the distance from the electrodes 6 and 7 increases. An equal electrolysis is formed.
As a frequency of the high frequency voltage applied to the electrode body 4A, a frequency at which the positive dielectrophoretic characteristic or the negative dielectrophoretic characteristic becomes large is selected in accordance with the dielectrophoretic characteristic of the cell to be separated.

  For example, it is preferable that the first cell has a large negative dielectrophoretic characteristic at one frequency and the second cell is selected such that it has a large positive dielectrophoretic characteristic at the same frequency. Alternatively, the first cell has a large negative dielectrophoretic characteristic at the first frequency but a small dielectrophoretic characteristic at the second frequency, and the second cell has a small dielectrophoretic characteristic at the first frequency. In the case where a frequency having only a characteristic but having a large positive dielectrophoretic characteristic can be selected at the second frequency, it is preferable to apply a high frequency voltage including both frequencies.

  The cell recovery unit 5 includes recovery containers 8 and 9 connected to a radial center portion downstream of the flow path 3 and a peripheral portion, respectively. With each of the collection containers 8 and 9, the cell suspension that has flowed in the vicinity of the center in the radial direction of the flow path 3 and the cell suspension that has flowed in the vicinity of the electrode body 4A at the periphery of the flow path 3 are separately collected. Be able to.

The operation of the cell separation electrode unit 4 and the cell separation device 1 according to this embodiment configured as described above will be described below.
In order to separate the cells contained in the cell suspension using the cell separation device 1 according to the present embodiment, the cell suspension is accommodated in the container 2 and the voltage of the cell separation electrode unit 4 is stored. The cell suspension in the container 2 is caused to flow into the flow path 3 in a state where the supply unit 4B is operated and a high frequency voltage is applied to the electrode body 4A.

  In the electrode body 4A, a high-frequency unequal electric field is generated in which the gradient gradually decreases inward in the radial direction. Therefore, in the radially inward direction of the electrode body 4A, the gradient gradually decreases toward the center in the radial direction, and high frequency inequality that is lowest at the center occurs.

  Of the cell suspension that has flowed from the container 2 into the electrode body 4A of the cell separation electrode unit 4 through the flow path 3, the cell S1 having positive dielectrophoretic characteristics at the frequency of the applied high-frequency voltage is shown in FIG. As indicated by a circle filled with 4, it moves outward in the radial direction so as to be drawn in a direction in which the potential gradient increases, that is, in a direction close to the electrode body 4 </ b> A. On the contrary, the cell S2 having negative dielectrophoretic characteristics at the frequency of the applied high-frequency voltage is separated from the electrode body 4A as shown by a white circle in FIG. Held in the vicinity.

As a result, a cell suspension containing a large amount of cells S1 having negative dielectrophoretic characteristics flows in the center of the flow path 3 in the radial direction, and in the vicinity of the electrode body 4A disposed on the outer peripheral side of the flow path 3 in the radial direction. The cell suspension containing many cells S2 having positive dielectrophoretic properties flows.
Since the collection containers 8 and 9 are connected to the center and the outer periphery in the radial direction on the downstream side of the electrode body 4A of the cell separation electrode unit 4, different dielectrophoresis is performed on each of the collection containers 8 and 9. The cells S1 and S2 having characteristics are collected separately.

  In this case, according to the cell separation electrode unit 4 and the cell separation device 1 according to the present embodiment, the electrode body 4A is formed in a cylindrical bowl shape, which is compared with a conventional flat electrode. Thus, there is an advantage that the space can be used effectively, and the separation capacity can be enhanced while ensuring the area with a small volume and making the apparatus compact.

Next, a cell separation electrode unit 4 ′ and a cell separation device 1 ′ according to a second embodiment of the present invention will be described below with reference to FIG.
In the description of the present embodiment, the same reference numerals are given to the portions having the same configurations as those of the cell separation electrode unit 4 and the cell separation device 1 according to the first embodiment described above, and the description thereof is omitted.

  As shown in FIG. 5, the cell separation electrode unit 4 ′ according to the present embodiment includes two cylindrical bowl-shaped electrode bodies 4 </ b> A and 4 </ b> A ′ having different radial dimensions. The two electrode bodies 4A and 4A ′ have different diameter dimensions of the annular electrodes 6 and 6 ′ constituting the respective electrode bodies 4A and 4A ′, and are arranged coaxially with a space in the radial direction. Is formed.

  Thereby, in the flow path 3, the first flow path 3 </ b> A surrounded by the inner electrode body 4 </ b> A ′, and the second tubular tube disposed outside and surrounded by the two electrode bodies 4 </ b> A and 4 </ b> A ′. A flow path 3B is formed. The container 2 is connected to the first flow path 3A, and all the cell suspension flowing from the container 2 is allowed to flow into the first flow path 3A.

The two electrode bodies 4A and 4A ′ are respectively applied with high-frequency voltages of different polarities to the annular electrodes 6 and 6 ′ and the rod-like electrodes 7 and 7 ′ that are adjacent to each other by the voltage supply unit 4B. , 7, 7 ′, high-frequency unequal electrolysis is formed with the potential gradient gradually decreasing as the distance from 7, 7 ′ increases. Further, a high frequency voltage having a different frequency is applied to the two electrode bodies 4A and 4A ′.
As the frequency of the high frequency voltage applied to the electrode bodies 4A and 4A ′, a frequency at which the positive dielectrophoretic characteristic or the negative dielectrophoretic characteristic becomes large is selected in accordance with the dielectrophoretic characteristic of the cell to be separated. Yes.

  For example, a first cell has a large negative dielectrophoretic characteristic at a first frequency but a small dielectrophoretic characteristic at a second frequency, and a second cell is large at the same first frequency. Has positive dielectrophoretic properties but only small dielectrophoretic properties at the second frequency, and the third cell has small dielectrophoretic properties at the first frequency but large at the second frequency. Preferably, a frequency is selected that has positive dielectrophoretic properties.

  The cell recovery unit 5 includes three recovery containers 8 and 9 connected to the downstream of the center in the radial direction of the flow path 3 and the downstream of the radial position where the two electrode bodies 4A and 4A ′ are arranged. I have. With each of the collection containers 8 and 9, the cell suspension flowing in the vicinity of the center in the radial direction of the flow path 3 and the cell suspension flowing in the vicinity of each of the electrode bodies 4A and 4A ′ are separately collected. Can be done.

The operation of the cell separation device 1 ′ according to this embodiment configured as described above will be described below.
In order to separate the cells contained in the cell suspension using the cell separation device 1 ′ according to this embodiment, the cell suspension is accommodated in the container 2, and the cell separation electrode unit 4 In a state where a high frequency voltage is applied to the voltage supply unit 4B, the cell suspension in the container 2 is caused to flow into the flow path.

  A high-frequency unequal electric field is generated in the two electrode bodies 4A and 4A '. Accordingly, inwardly in the radial direction of the inner electrode body 4A, the gradient gradually decreases toward the center in the radial direction, and high-frequency inequality that is lowest at the center occurs.

In addition, high frequency voltages of different frequencies are applied to the two electrode bodies 4A and 4A ′, and different high frequency unequal electric fields are generated.
Of the cell suspension that has flowed inward in the radial direction of the electrode body 4A inside the electrode unit 4 for cell separation from the container 2, the first frequency and / or the second frequency of the high-frequency voltage applied to the electrode body 4A. Cells having positive dielectrophoretic characteristics in frequency move outward in the radial direction so as to be attracted in the direction in which the potential gradient increases, that is, in the direction approaching the two electrode bodies 4A and 4A ′.

Conversely, the cells S1 having negative dielectrophoretic characteristics at the first frequency and / or the second frequency of the high-frequency voltage applied to the electrode bodies 4A and 4A ′ are separated from the electrode bodies 4A and 4A ′. 4A and 4A ′ are held in the vicinity of the center position in the radial direction.
Furthermore, it has a large positive dielectrophoretic characteristic at the first frequency of the high-frequency voltage applied to the inner electrode body 4A, but has a small dielectric at the second frequency of the high-frequency voltage applied to the outer electrode body 4A ′. Cells having only electrophoretic properties are held at positions near the inner electrode body 4A.

  Furthermore, it has a large positive dielectrophoretic characteristic at the second frequency of the high frequency voltage applied to the outer electrode body, but has a small dielectrophoretic characteristic at the first frequency of the high frequency voltage applied to the inner electrode body 4A. The cells having only the permeation pass through the inner electrode body 4A and are held at a position near the outer electrode body 4A ′.

As a result, a cell suspension containing a large number of cells having negative dielectrophoretic properties flows in the center of the flow path 3 in the radial direction, and positive dielectrophoresis is performed near the two electrode bodies 4A and 4A ′ at different frequencies. Each of the cell suspensions containing many cells having characteristics flows.
The collection containers 8 and 9 are connected to the downstream side of the electrode bodies 4A and 4A 'on the downstream side of the electrode bodies 4A and 4A' of the cell separation electrode unit 4 ', respectively. In the collection containers 8 and 9, cells having different dielectrophoretic characteristics are collected separately.

That is, according to the cell separation electrode unit 4 ′ and the cell separation device 1 ′ according to the present embodiment, cells having negative dielectrophoretic characteristics are collected in the vicinity of the central position in the radial direction of the flow path 3, and different positive Since cells having dielectrophoretic properties are collected near the radial positions of the two electrode bodies 4A and 4A ′, there is an advantage that the cell suspension can be further finely separated.
In the present embodiment, the case where the number of electrode bodies 4A and 4A ′ is two has been described. However, the present invention is not limited to this, and three or more electrode bodies may be provided.

  In each of the above embodiments, as the electrode bodies 4A and 4A ′, a plurality of annular electrodes 6 and 6 ′ arranged in the axial direction and a plurality of rod-like electrodes 7 arranged in the circumferential direction at intervals, Although the case of having both 7 'has been described, any one of the electrodes 6', 6, 7, 7 'may be provided instead.

It is a typical longitudinal section showing the cell separation device concerning a 1st embodiment of the present invention. It is the front view which looked at the cell separation device of Drawing 1 from the direction of an axis. It is a perspective view which shows the electrode unit for cell separation which concerns on this embodiment with which the cell separation apparatus of FIG. 1 was equipped. It is a schematic diagram explaining the effect | action of the cell separation apparatus of FIG. It is the front view which looked at the cell separation device concerning a 2nd embodiment of the present invention from the axial direction.

Explanation of symbols

DESCRIPTION OF SYMBOLS 1,1 'Cell separation apparatus 3 Flow path 3A 1st flow path 3B 2nd flow path 4, 4' Cell separation electrode unit 5 Cell collection | recovery part 6, 6 'Annular electrode (electrode)
7,7 'Rod electrode (electrode)

Claims (6)

  A plurality of annular electrodes arranged in a flow path for circulating the cell suspension, aligned in the axial direction with the flow direction, and arranged with different polarities alternately at substantially parallel intervals in the axial direction. An electrode unit for cell separation provided.   The electrode unit for cell separation according to claim 1, wherein a plurality of the annular electrodes are arranged at intervals in the radial direction.   Arranged in a flow channel that circulates the cell suspension, aligned in the axial direction in the flow direction, and arranged in a tubular shape with different polarities alternately spaced substantially parallel in the circumferential direction along the axial direction. A cell separation electrode unit comprising a plurality of linear electrodes.   The electrode unit for cell separation according to claim 3, wherein a plurality of the electrodes arranged in a tubular shape are arranged at intervals in the radial direction. A flow path for circulating the cell suspension;
A cell separation apparatus comprising the cell separation electrode unit according to any one of claims 1 to 4 disposed in the flow path.   The cell separation device according to claim 5, further comprising a cell collection unit that collects cells that have circulated through the inner and outer spaces in the radial direction of the electrode.

Images