JP6815307B2 - Filtration filter for nucleated cells and filtration method using it - Google Patents

Description

The present invention relates to a filter for filtering nucleated cells and a filtration method using the same.

Patent Document 1 discloses a method for concentrating mononuclear cells and platelets from a liquid containing erythrocytes, nucleated cells and platelets using a cell capture filter material. The cell capture filter material of Patent Document 1 captures nucleated cells and platelets and allows unnecessary cells such as erythrocytes to pass through.

JP-A-2009-284860

However, the cell capture filter material of Patent Document 1 still has room for improvement in terms of improving the recovery rate of nucleated cells.

An object of the present invention is to provide a filter for filtering nucleated cells, which can improve the recovery rate of nucleated cells, and a filtration method using the same.

The filter of one aspect of the present invention
A filter for filtering nucleated cells
Main component is at least one of metal and metal oxide
Multiple through holes (excluding squares) are formed,
The major axis of the inscribed ellipse of the through hole is smaller than the size of the nucleus of the nucleated cell, and the inscribed ellipse of the through hole is an ellipse tangent to all sides defining the opening of the through hole.

The filtration method of one aspect of the present invention
A method for filtering nucleated cells
A plurality of through holes (excluding squares) are formed mainly containing at least one of a metal and a metal oxide, and the major axis of the inscribed ellipse of the through holes is smaller than the size of the nucleus of the nucleated cell. The step of preparing a filter, wherein the inscribed ellipse of the through hole is an ellipse tangent to all sides defining the opening of the through hole.
A step of passing a liquid containing the nucleated cells through the filter,
including.

According to the present invention, it is possible to provide a filter for filtering nucleated cells, which can improve the recovery rate of nucleated cells, and a filtration method using the same.

It is a schematic block diagram of the filter of Embodiment 1 which concerns on this invention. It is an enlarged perspective view of a part of the filter of Embodiment 1 which concerns on this invention. It is the schematic which saw a part of the filter of FIG. 2 from the thickness direction. It is the schematic which provided the support base material in the filter of Embodiment 1 which concerns on this invention. It is the schematic of the filter of the modification of Embodiment 1 which concerns on this invention. It is an enlarged cross-sectional view of a part of the filter of another modification of Embodiment 1 which concerns on this invention. It is an enlarged cross-sectional view of a part of the filter of another modification of Embodiment 1 which concerns on this invention. It is an enlarged cross-sectional view of a part of the filter of another modification of Embodiment 1 which concerns on this invention.

(Background to the present invention)
In Patent Document 1, mononuclear cells are separated from blood by capturing nucleated cells and platelets using a cell capture filter material composed of a non-woven fabric and passing unnecessary cells such as erythrocytes. There is. However, in the cell capture filter of Patent Document 1, the recovery rate of mononuclear cells is about 74%, and there is still room for improvement in terms of improving the recovery rate of cells to be captured.

As a result of diligent research, the present inventors have conducted a filter on a liquid containing nucleated cells using a filter containing at least one of a metal and a metal oxide as a main component, so that the liquid can be captured. We have found that the recovery rate of nuclear cells can be improved, and have reached the present invention.

The filter of one aspect of the present invention
A filter for filtering nucleated cells
Main component is at least one of metal and metal oxide
Multiple through holes (excluding squares) are formed,
The major axis of the inscribed ellipse of the through hole is smaller than the size of the nucleus of the nucleated cell, and the inscribed ellipse of the through hole is an ellipse tangent to all sides defining the opening of the through hole.

With such a configuration, the recovery rate of nucleated cells can be improved.

In the filter, the shape of the through hole may be polygonal.

With such a configuration, the recovery rate of nucleated cells can be improved.

In the filter, the shape of the through hole may be rectangular.

With such a configuration, the filtration time can be shortened and the recovery rate of nucleated cells can be improved.

In the filter, a filter portion having the plurality of through holes penetrating the first main surface and the second main surface facing each other, and
A frame portion arranged so as to surround the outer circumference of the filter portion and
With
In the filter portion, the film thickness of the filter portion in the central region away from the frame portion may be smaller than the film thickness of the filter portion in the peripheral region closer to the frame portion than the central region. ..

With such a configuration, a concave surface can be formed on a part of at least one surface of the first main surface and the second main surface in the filter portion. In the case where cells are captured on the side where the concave surface is formed, the surface tension of the fluid makes it easier for fluid pools to be formed on the concave surface, so that drying of the captured cells can be suppressed. Therefore, the handleability of the cells captured by the filter unit can be improved.

In the filter, at least one of the metal and the metal oxide has a reference electrode-referenced immersion potential of 0.03 V, which is composed of silver chloride immersed in a saturated potassium chloride solution in phosphate buffered physiological saline. It may be.

With such a configuration, it is possible to prevent the metal or metal oxide which is a component of the filter from being eluted into the liquid containing the nucleated cells.

In the filter, at least one of the metal and the metal oxide contains at least one selected from the group consisting of gold, silver, copper, platinum, nickel, palladium, alloys thereof and oxides thereof. You may be.

With such a configuration, the recovery rate of nucleated cells can be further improved.

The filtration method of one aspect of the present invention
A method for filtering nucleated cells
A plurality of through holes (excluding squares) are formed mainly containing at least one of a metal and a metal oxide, and the major axis of the inscribed ellipse of the through holes is smaller than the size of the nucleus of the nucleated cell. The step of preparing a filter, wherein the inscribed ellipse of the through hole is an ellipse tangent to all sides defining the opening of the through hole.
A step of passing a liquid containing the nucleated cells through the filter,
including.

With such a configuration, the recovery rate of nucleated cells can be improved.

In the filtration method, the shape of the through hole may be polygonal.

With such a configuration, the recovery rate of nucleated cells can be improved.

In the filtration method, the shape of the through hole may be rectangular.

With such a configuration, the filtration time can be shortened and the recovery rate of nucleated cells can be further improved.

In the filtration method
The filter is
A filter unit having the plurality of through holes penetrating the first main surface and the second main surface facing each other,
A frame portion arranged so as to surround the outer circumference of the filter portion and
With
In the filter portion, the film thickness of the filter portion in the central region away from the frame portion may be smaller than the film thickness of the filter portion in the peripheral region closer to the frame portion than the central region. ..

With such a configuration, cells can be captured by a concave surface formed on a part of at least one surface of the first main surface and the second main surface in the filter portion. Since the surface tension of the fluid facilitates the formation of a fluid pool on the concave surface, it is possible to suppress the drying of the captured cells. Therefore, the handleability of the cells captured by the filter unit can be improved.

In the filtration method, at least one of the metal and the metal oxide has a reference electrode-referenced immersion potential of 0.03 V, which is composed of silver chloride immersed in a saturated potassium chloride solution in phosphate buffered saline. You may be noble.

With such a configuration, it is possible to prevent the metal or metal oxide which is a component of the filter from being eluted into the liquid containing the nucleated cells.

In the filtration method, at least one of the metal and the metal oxide contains at least one selected from the group consisting of gold, silver, copper, platinum, nickel, palladium, alloys thereof and oxides thereof. You may be doing it.

With such a configuration, the recovery rate of nucleated cells can be further improved.

In the filtration method, the step of passing the liquid containing the nucleated cells through the filter may include a step of separating live cells and dead cells.

With such a configuration, live cells and dead cells can be separated.

The kit of one aspect of the present invention
A kit for carrying out the filtration method, which comprises a filter for filtering nucleated cells.
The filter contains at least one of a metal and a metal oxide as a main component, and a plurality of through holes (excluding squares) are formed, and the major axis of the inscribed ellipse of the through holes is the nucleus of the nucleated cell. The inscribed ellipse of the through hole, which is smaller than the size of, is an ellipse tangent to all sides defining the opening of the through hole.

With such a configuration, the recovery rate of nucleated cells can be improved.

Hereinafter, Embodiment 1 according to the present invention will be described with reference to the accompanying drawings. Further, in each figure, each element is exaggerated for the sake of easy explanation.

(Embodiment 1)
[Filter configuration]
FIG. 1 is a schematic configuration diagram of the filter 10 of the first embodiment according to the present invention. FIG. 2 is an enlarged perspective view of a part of the filter 10 according to the first embodiment of the present invention. The X, Y, and Z directions in FIGS. 1 and 2 indicate the vertical direction, the horizontal direction, and the thickness direction of the filter 10, respectively. As shown in FIG. 1, the filter 10 includes a filter portion 11 and a frame portion 15 provided on the outer periphery of the filter portion 11. As shown in FIG. 2, the filter 10 has a first main surface PS1 and a second main surface PS2 facing each other. The filter unit 11 includes a filter base unit 14 in which a plurality of through holes 12 penetrating the first main surface PS1 and the second main surface PS2 are formed.

The filter 10 filters the nucleated cells by passing a liquid (cell suspension) containing the nucleated cells through the filter unit 11.

As used herein, a "nucleated cell" is a cell in which a nucleolus and a cytoplasm are separated by a nuclear envelope.

<Material>
The material constituting the filter base portion 14 forming the base portion of the filter 10 is mainly composed of a metal and / or a metal oxide. The filter substrate portion 14 may be, for example, gold, silver, copper, platinum, nickel, palladium, alloys thereof and oxides thereof.

The outermost layer of the filter 10 may be composed of a metal and / or a metal oxide that is difficult to elute into the cell suspension. For example, the outermost layer of the filter 10 is covered with a metal having a reference electrode-referenced immersion potential of silver chloride immersed in a saturated potassium chloride solution in phosphate buffered saline, which is noble than 0.03 V. In this case, it is possible to prevent the material constituting the filter 10 from eluting into the cell suspension. As a result, stress on the cells can be reduced. Alternatively, the outermost layer of the filter 10 may be made of a hydrophilic material. For example, when treating an aqueous cell suspension, the treatment time can be shortened, so that stress on cells can be reduced.

<Outer shape>
The outer shape of the filter 10 is, for example, circular, rectangular, or elliptical. In the first embodiment, the outer shape of the filter 10 is substantially circular. By making the outer shape of the filter 10 substantially circular, the fluid can flow uniformly to the main surface of the filter 10 (for example, the first main surface PS1 of the filter unit 11). In addition, in this specification, "substantially circular" means that the ratio of the length of a major axis to the length of a minor axis is 1.0 or more and 1.2 or less.

<Filter part>
The filter portion 11 is a plate-like structure in which a plurality of through holes 12 are formed. The shape of the filter portion 11 is, for example, a circle, a rectangle, or an ellipse. In the first embodiment, the shape of the filter portion 11 is substantially circular. By making the shape of the filter portion 11 substantially circular, the fluid can flow uniformly to the first main surface PS1 of the filter portion 11.

FIG. 3 is a schematic view of a part of the filter unit 11 as viewed from the thickness direction (Z direction). As shown in FIG. 3, the plurality of through holes 12 are periodically arranged on the first main surface PS1 and the second main surface PS2 of the filter unit 11. Specifically, the plurality of through holes 12 are provided in the filter portion 11 in a matrix at equal intervals.

In the first embodiment, the through hole 12 has a rectangular shape when viewed from the first main surface PS1 side of the filter portion 11, that is, the Z direction. As shown in FIG. 3, the shape of the through hole 12 is a rectangle with a long side d1 in the X direction and a short side d2 in the Y direction. The through hole 12 has a shape other than a square when viewed from the Z direction. The through hole 12 may have a shape such as a rhombus or a polygon when viewed from the Z direction. The polygon may include a regular polygon other than a square.

In the first embodiment, the long side d1 of the through hole 12 is formed, for example, 1.2 times or more and 1.8 times or less the short side d2.

In the first embodiment, the shape (cross-sectional shape) of the through hole 12 projected on the plane perpendicular to the first main plane PS1 of the filter portion 11 is rectangular. Specifically, the cross-sectional shape of the through hole 12 is a rectangle in which the length of one side in the radial direction of the filter 10 is longer than the length of one side in the thickness direction of the filter 10. The cross-sectional shape of the through hole 12 is not limited to a rectangle, and may be a tapered shape such as a parallelogram or a trapezoid, a symmetrical shape, or an asymmetrical shape. ..

In the first embodiment, the plurality of through holes 12 are arranged in two arrangement directions parallel to each side of the rectangle when viewed from the first main surface PS1 side (Z direction) of the filter unit 11, that is, the X direction in FIG. They are provided at equal intervals in the Y direction. By providing the plurality of through holes 12 in a rectangular lattice arrangement in this way, it is possible to increase the aperture ratio and reduce the passage resistance of the fluid to the filter 10. With such a configuration, the processing time can be shortened and the stress on the cells can be reduced. Further, since the symmetry of the arrangement of the plurality of through holes 12 is improved, the observation of the filter becomes easy.

The arrangement of the plurality of through holes 12 is not limited to the rectangular lattice arrangement, and may be, for example, a quasi-periodic arrangement or a periodic arrangement. As an example of the periodic array, if it is a rectangular array, it may be a rectangular array in which the intervals in the two array directions are not equal, or it may be a triangular lattice array or a regular triangular lattice array. It should be noted that a plurality of through holes 12 may be provided in the filter portion 11, and the arrangement is not limited.

The corner portion to which each side defining the opening of the through hole 12 is connected may be R-processed. That is, the corner portion of the through hole 12 may be formed to be round. With such a configuration, it is possible to suppress the damage to the cells at the corners of the through holes 12 and further maintain the activity of the cells.

The spacing between the through holes 12 is appropriately designed according to the type (size, morphology, properties, elasticity) or amount of cells to be separated. Here, as shown in FIG. 3, the distance between the through holes 12 means that the through holes 12 are adjacent to the center of an arbitrary through hole 12 when viewed from the first main surface PS1 side of the filter portion 11. It means the distances b1 and b2 from the center. Specifically, the interval b1 means the distance between the center of the through hole 12 and the center of the adjacent through hole 12 in the short side direction (X direction) of the through hole 12. The interval b2 means the distance between the center of the through hole 12 and the center of the adjacent through hole 12 in the long side direction (Y direction) of the through hole 12. In the first embodiment, the center of the arbitrary through hole 12 means the intersection of the diagonal lines of the rectangular through hole 12.

In the case of a periodic array structure, the spacing b1 of the through holes 12 is, for example, greater than 1 times the long side d1 of the through holes 12 and 10 times or less, preferably 3 times or less the long side d1 of the through holes 12. is there. The distance b2 of the through holes 12 is, for example, larger than 1 times of the short side d2 of the through hole 12 and 10 times or less, preferably 3 times or less of the short side d2 of the through hole 12. Alternatively, for example, the aperture ratio of the filter unit 11 is 10% or more, and preferably the aperture ratio is 25% or more. With such a configuration, the passage resistance of the fluid to the filter unit 11 can be reduced. Therefore, the processing time can be shortened and the stress on the cells can be reduced. The aperture ratio is calculated by (the area occupied by the through hole 12) / (the projected area of the first main surface PS1 when it is assumed that the through hole 12 is not open).

The major axis of the inscribed ellipse of the through hole 12 is designed to be smaller than the size of the nucleus of a nucleated cell. In the present specification, the "inscribed ellipse of the through hole 12" is an ellipse having a major axis and a minor axis, and is an ellipse that can be drawn in the through hole 12 when viewed from the first main surface PS1 side of the filter portion 11. It means the ellipse with the largest major axis. That is, the "inscribed ellipse of the through hole 12" means the ellipse having the largest major axis among the ellipses in contact with the inner wall of the filter base portion 14 constituting the through hole 12. In other words, the "inscribed ellipse of the through hole 12" means an ellipse that touches all the sides that define the opening of the through hole 12 when viewed from the first main surface PS1 side of the filter portion 11. Further, the major axis and the minor axis of the "inscribed ellipse of the through hole 12" may have different lengths or may have the same length. In the present specification, the "inscribed ellipse of the through hole 12" may include a perfect circle.

Further, in the present specification, "the size of the nucleus of a nucleated cell" means that the nucleated cell is placed in a liquid and observed with a microscope, and any two points on the outer periphery of the nucleus of the nucleated cell are connected. When the longest of the grid lines is the nuclei length of nucleated cells, it means the average value of the nuclei lengths of a plurality of nucleated cells.

The dimensions of the plurality of through holes 12 are designed to be substantially the same. When a plurality of through holes 12 having the same shape are periodically arranged, it is preferable that the standard deviation in the sizes of the plurality of through holes 12 is small.

The filter unit 11 has a uniform film thickness from the center of the filter unit 11 to the outside. In other words, when the filter portion 11 is cut in the Z direction, the filter portion 11 has a flat cross-sectional shape. The thickness of the filter portion 11 is preferably larger than 0.1 times and 100 times or less of the shortest side (for example, the short side d2) of the sides defining the through hole 12. More preferably, the thickness of the filter portion 11 is larger than 0.5 times the short side d2 of the through hole 12 and 10 times or less. With such a configuration, the resistance of the filter 10 to the fluid can be reduced, and the processing time can be shortened. As a result, stress on cells can be reduced.

The arithmetic mean roughness of the surface of the filter unit 11 (first main surface PS1) is preferably smaller than the size of the nuclei of nucleated cells. With such a configuration, the adhesion of cells to the surface of the filter unit 11 (first main surface PS1) can be reduced, and the cell recovery rate can be increased. The arithmetic mean roughness was measured by using a stylus type surface shape measuring machine DEKTAK150 (registered trademark) manufactured by ULVAC Co., Ltd., and the average value of the measured values at five points on the surface of the filter unit 11 was measured by the filter unit 11. Arithmetic mean roughness.

In the filter unit 11, the first main surface PS1 to which the liquid containing nucleated cells comes into contact may be formed smoothly. Specifically, the first main surface PS1 of the filter unit 11 may be formed on a uniform flat surface without unevenness. In other words, the openings of the plurality of through holes 12 on the first main surface PS1 of the filter portion 11 are formed on the same plane. Further, the filter base portion 14 which is a portion of the filter portion 11 in which the through hole 12 is not formed is connected and integrally formed. With such a configuration, the adhesion of cells to the surface of the filter unit 11 (first main surface PS1) is reduced, and the captured nucleated cells can be easily recovered.

The through hole 12 of the filter portion 11 communicates with the opening on the first main surface PS1 side and the opening on the second main surface PS2 side through a continuous wall surface. Specifically, the through hole 12 is provided so that the opening on the first main surface PS1 side can be projected onto the opening on the second main surface PS2 side. That is, when the filter portion 11 is viewed from the first main surface PS1 side, the through hole 12 is provided so that the opening on the first main surface PS1 side overlaps with the opening on the second main surface PS2 side. In the first embodiment, the through hole 12 is provided so that the inner wall thereof is perpendicular to the first main surface PS1 and the second main surface PS2.

<Frame part>
The frame portion 15 is provided on the outer periphery of the filter portion 11, and is a portion in which the number of through holes 12 per unit area is smaller than that of the filter portion 11. The number of through holes 12 in the frame portion 15 is 1% or less of the number of through holes 12 in the filter portion 11. The thickness of the frame portion 15 may be thicker than the thickness of the filter portion 11. With such a configuration, the mechanical strength of the filter 10 can be increased.

When the filter 10 is connected to the device for use, the frame portion 15 may function as a connecting portion for connecting the filter 10 and the device. Further, the frame portion 15 may display filter information (such as the dimensions of the through hole 12).

The frame portion 15 is formed in a ring shape when viewed from the first main surface PS1 side of the filter portion 11. When the filter 10 is viewed from the first main surface PS1 side, the center of the frame portion 15 coincides with the center of the filter portion 11. That is, the frame portion 15 is formed on a concentric circle with the filter.

The filter 10 may be fixed to a jig and used from the viewpoint of ease of handling and ease of joining with the system. The jig can be made of, for example, a gamma sterilizable material. The jigs include, for example, polyethylene, polyethylene terephthalate, polyurethane, polystyrene, silicone rubber, ABS resin, polyamide, polyamideimide, polysulfone, natural rubber, latex, urethane rubber, silicone rubber, ethylene vinyl acetate, polyesters, epoxys, etc. It may be made of a material containing phenols, silica, alumina, gold, platinum, nickel, stainless steel, titanium, and the like. By constructing the jig with such a material, stress on cells can be reduced.

[Filtration method]
A filtration method using the filter 10 will be described.

First, the filter 10 is prepared. In this step, a filter 10 in which the size of the through hole 12 is selected according to the size of the nucleus of the nucleated cell is prepared. Specifically, a filter 10 having a through hole 12 smaller than the size of the nucleus of a nucleated cell to be filtered is prepared.

For example, in the step of preparing the filter 10, the size of the nuclei of a plurality of nucleated cells is confirmed by using a micrometer, a hemocytometer, or the like, so that the through holes 12 are smaller than the size of the nuclei of the nucleated cells. A filter 10 having a size may be selected. Alternatively, by taking a picture of a plurality of nucleated cells and measuring the size of the nuclei of the plurality of nucleated cells, a filter 10 having a through-hole 12 size smaller than the size of the nuclei of the nucleated cells can be obtained. You may choose. The selection of the filter 10 is not limited to these.

The filter 10 is attached to the device. Specifically, the filter 10 is attached to the device by sandwiching the frame portion 15 of the filter 10.

The cell suspension is then passed through the filter 10. As used herein, a "cell suspension" is a fluid containing nucleated cells. Often, the fluid containing nucleated cells is a liquid. The liquid is, for example, a culture solution containing amino acids, proteins, serum, etc., phosphate buffered saline, or water. In addition to cells and fluids, cell suspensions may contain non-living substances such as resin particles, parts of tissues such as bone fragments or meat fragments, dead cells, and the like.

In this way, the nucleated cells are separated from the liquid by passing the liquid containing the nucleated cells through the filter unit 11. In the first embodiment, the major axis of the inscribed ellipse of the through hole 12 of the filter portion 11 is designed to be smaller than the size of the nucleus of the nucleated cell. Therefore, the nucleated cells are captured on the first main surface PS1 of the filter unit 11 without passing through the through hole 12.

As a method of passing the cell suspension through the filter 10, for example, there is a method of passing the cell suspension through the first main surface PS1 of the filter unit 11 from substantially vertically above using gravity. In addition to this, a method (pressing) in which the cell suspension is brought into contact with the first main surface PS1 of the filter unit 11 and then passed through the cell suspension by applying pressure, or the first main surface PS1 of the filter unit 11 There is a method (suction) in which the cell suspension is brought into contact with the cell suspension and sucked from the second main surface PS2 to pass through the cell suspension. In the step of passing the liquid containing the nucleated cells through the filter unit 11, it is preferable that the cells are not stressed as much as possible. For example, when pressure is applied, it is preferable that the pressure is such that the nucleated cells are not deformed. More preferably, the liquid is passed through the filter unit 11 by the weight of the liquid without applying pressure. Alternatively, it is preferable to shorten the treatment time by increasing the aperture ratio of the filter unit 11 and reduce the time during which the nucleated cells are stressed.

Alternatively, the cell suspension may be passed through the filter 10 with the nucleated cells suspended in the liquid. Since the nucleated cells suspended in the liquid have a substantially spherical shape, the recovery rate of the nucleated cells to be captured can be improved. That is, the dimensional accuracy of the nucleated cells to be captured can be improved.

Further, by passing the filter 10 through the filter 10 a plurality of times, the dimensional accuracy of the nucleated cells to be captured can be improved.

In the filtration method using the filter 10, for example, filtration may be performed using a filtration container. The filtration container is, for example, a cylindrical container having an outer diameter of 14 mm, an inner diameter of 6 mm, and a height of 55 mm, and the filter 10 can be attached to the bottom thereof. The filtration container is not limited to this, and containers of various shapes and dimensions may be used.

[Manufacturing method of filter]
A typical manufacturing method of the filter 10 will be described. The filter 10 is manufactured by the following steps.

<Formation of feeding film>
A Cu feeding film is formed on the upper surface of the silicon substrate using a sputtering device. This feeding film serves as a power supply when forming the filter base portion 14 of the filter 10 described later. At this time, an intermediate layer such as Ti may be formed for the purpose of ensuring the adhesiveness between the silicon substrate and the feeding film.

The conditions for forming the Cu feeding film are as follows.
Sputtering gas: Argon gas Vacuum degree of sputtering equipment: 5.0 × 10 ^-4 Pa
Applied power: DC500W
Sputtering time: Cu film formation / 27 minutes
At the time of Ti film formation / 3 minutes 5 seconds

<Formation of resist image>
A resist image is formed on the feeding film formed on the upper surface of the silicon substrate.
A resist film having a predetermined film thickness is formed on the feeding film formed on the upper surface of the silicon substrate by using a spin coater or the like. Next, a resist image is formed by exposing the resist through a photomask on which a predetermined pattern is formed and developing it.

The conditions for applying the resist film are as follows.
Resistant: Novolac resin + organic solvent Spin coater rotation speed: 1130 rpm
Resist film thickness: 2 μm
A resist film is formed by applying the resist agent to the upper surface of a silicon substrate with a spin coater, volatilizing the solvent at 130 ° C. in a nitrogen atmosphere, and then cooling the mixture.

A light beam having an energy density of 2500 J / m ² containing a wavelength of 365 nm is irradiated for 0.25 seconds to expose the resist agent.

The exposed portion is brought into contact with an alkaline solution for development processing.

<Formation of filter substrate>
A filter base portion 14 is formed in the opening of the resist image. The filter base portion 14 made of a nickel plating film is formed by using an electrolytic plating method using the previously formed feeding film as a power supply.

The conditions for forming the filter base portion 14 are as follows.
Pretreatment: The surface of the feeding film is activated by immersing it in dilute sulfuric acid for 60 seconds.
Plating solution: Nickel sulfamate plating solution Liquid temperature 55 ° C pH = 4.0
Plating speed: 0.5 μm / min
Plating: Electroplating is performed while shaking.

<Dissolution and peeling of resist>
The resist film is dissolved and the resist is peeled off by applying ultrasonic waves to the filter substrate portion 14 in an acetone solution for 15 minutes.

<Formation of supporting base material>
When performing filtration using the filter 10, a support base material may be provided on the filter 10 if necessary.

FIG. 4 shows a schematic configuration of the filter 10 to which the support base material 13 is attached. As shown in FIG. 4, the support base material 13 may be provided on the second main surface PS2 side of the filter 10. The support base material 13 is provided with a plurality of square openings 13a. The thickness of the support base material 13 is, for example, 14 μm, one side of the opening 13a, that is, the distance A between the crosspieces is 260 μm, and the width B of the crosspieces is 14 μm.

By providing the support base material 13 in this way, it is possible to prevent the filter 10 from being damaged during filtration. The supporting base material is produced by the following steps.

A photosensitive resist is applied again to the upper surface of the silicon substrate on which the substrate of the filter 10 is formed to form a resist film, and then the resist is exposed through a photomask and developed to form a resist image. At this time, the exposure and development processing are performed so that the resist image straddles a plurality of substrates of the filter 10. The portion where the resist image straddles the substrate of the filter 10 becomes an opening of the filter 10 after the completion of the filter 10. That is, the number of substrates of the filter 10 that the resist image straddles is appropriately determined according to the aperture ratio required for the filter 10.

A filter base portion 14 is formed in the opening of the resist image. Using the previously formed feeding film as a power supply, an electrolytic plating method is used to form a supporting base material made of a nickel plating film. The width of the supporting base material is appropriately determined according to the strength required for the filter 10.

The resist is peeled off by dissolving the resist film by applying ultrasonic waves to the supporting substrate in an acetone solution for 15 minutes.

<Removal of feeding film>
By removing the feeding film and separating the filter base portion 14 and the supporting base material from the silicon substrate, the filter 10 for filtering nucleated cells is completed.

The feeding film is removed by immersing the feed film in an aqueous solution prepared by mixing 60% hydrogen peroxide solution, acetic acid and pure water at a mixing ratio of 1: 1: 20 in an environment of 25 ° C. for 48 hours.

[effect]
According to the filter 10 according to the first embodiment, the following effects can be obtained.

The filter 10 contains at least one of a metal and a metal oxide as a main component. Further, the filter 10 includes a filter portion 11 in which a plurality of through holes 12 are formed. With such a configuration, the through hole 12 of the filter portion 11 is not easily deformed, and the nucleated cells to be captured can be captured, and the recovery rate of the nucleated cells can be improved.

The major axis of the inscribed ellipse of the through hole 12 is designed to be smaller than the size of the nucleus of a nucleated cell. With such a configuration, the recovery rate of nucleated cells can be further improved.

The shape of the through hole 12 is formed in a rectangular shape. With such a configuration, the filtration time can be reduced as compared with the square-shaped through hole. Specifically, the cytoplasm of nucleated cells is more susceptible to deformation than the nucleus. In the case of a square-shaped through hole, when nucleated cells are captured by the filter portion, the cytoplasm is deformed, and the through hole may be blocked by the deformed cytoplasm. On the other hand, in the rectangular through hole 12, even if the cytoplasm is deformed and the through hole 12 is partially blocked when the nucleated cells are captured, the liquid can pass through from the other part of the through hole 12. it can. Therefore, in the rectangular through hole 12, the liquid can easily pass through and the filtration time can be reduced as compared with the square through hole.

The first main surface PS1 of the filter unit 11 is formed smoothly. With such a configuration, the nucleated cells captured by the first main surface PS1 can be easily separated from the filter unit 11, so that the cells can be easily collected.

As for the shape of the nucleus of a nucleated cell, there are various shapes such as an ellipse other than a perfect circle. In the filter 10, the major axis of the inscribed ellipse of the through hole 12 is formed smaller than the size of the nucleus of the nucleated cell, and the inscribed ellipse of the through hole 12 defines all the openings of the through hole 12. It is an ellipse that touches the side. With such a configuration, it is possible to reliably capture nuclei having various shapes other than the perfect circular nuclei, and it is possible to improve the recovery rate.

Further, in the filtration method using the filter 10, the nucleated cells to be captured can be reliably captured by passing the liquid containing the nucleated cells through the filter 10, so that the recovery rate can be improved. it can.

The size of the kernel of nucleated cells varies depending on the type, culture conditions, number of passages, and the like. For example, even in the same cell, the size of the nucleus differs depending on the culture conditions such as the culture temperature, time or environment. Therefore, even if the cells are the same nucleated cells, the size of the nuclei varies, and if the filter 10 is prepared without paying attention to the size of the nuclei, the nucleated cells pass through the through hole 12. There is. In the filtration method using the filter 10, the filter 10 in which the size of the through hole 12 is selected according to the size of the nucleus of the nucleated cell is prepared. That is, in the filtration method using the filter 10, a filter 10 having a through hole 12 smaller than the size of the nucleus of the nucleated cell to be filtered is selected, and the filter 10 to be used for filtration is prepared. Therefore, in the filtration method using the filter 10, nucleated cells can be reliably captured on the filter 10, and the recovery rate can be improved.

In the first embodiment, an example in which the dimensions of the through holes 12 are substantially the same has been described, but the present invention is not limited to this. For example, the dimensions of the through holes 12 may be different. In this case, the maximum size of the through hole 12 may be designed to be smaller than the size of the nucleus of the nucleated cell.

In the first embodiment, the method for manufacturing the filter 10 has described an example including a step of forming a backing layer substrate portion, but the method is not limited thereto. For example, the method for manufacturing the filter 10 does not have to include the step of forming the backing layer substrate portion.

In the first embodiment, an example in which the nucleated cells are filtered by passing a liquid containing the nucleated cells through the filter 10 has been described, but the present invention is not limited thereto. For example, a filter 10 may be used to separate live and dead cells from a liquid containing live and dead cells.

As a method for separating the living cells and the dead cells, for example, the living cells may be captured on the first main surface PS1 of the filter unit 11 by the filter 10 and passed through the dead cells. In addition, dead cells may be captured on the first main surface PS1 of the filter unit 11 by the filter 10 and passed through the living cells.

In the first embodiment, the filter 10 and the filtration method have been described, but the present invention is not limited thereto. For example, it may be used as a kit for carrying out a filtration method, which comprises a filter 10 for filtering nucleated cells.

In the first embodiment, an example in which the filter portion 11 has a uniform film thickness has been described, but the present invention is not limited to this. For example, the filter portion 11 does not have a uniform film thickness, but may be formed so that the film thickness on the central side is smaller than that on the peripheral side.

FIG. 5 shows a filter 10A of a modified example of the first embodiment. As shown in FIG. 5, in the filter portion 11a of the filter 10A, the film thickness T1 in the central region R11 away from the frame portion 15 is the film thickness in the peripheral region R13 closer to the frame portion 15 than the central region R11. It is formed smaller than T3. Further, in the filter 10A, when the film thickness in the intermediate region R12 located between the central region R11 and the peripheral region R13 in the filter portion 11a is T2, the film thickness in each region is T1 <T2. <Satisfies the relationship of T3. That is, the film thickness is set so that the film thickness increases in the filter portion 11a of the filter 10 from the center of the filter portion 11a toward the radial direction (outward in the radial direction). In addition, the film thickness may be increased continuously or stepwise.

Further, as shown in FIG. 5, the second main surface PS2 of the filter 10A is formed as a flat surface, and the surface of the first main surface PS1 corresponding to the filter portion 11a has a peripheral region rather than a central region. It is formed as a raised concave surface. The frame portion 15 of the filter 10A has a film thickness T0 formed to have a substantially constant thickness, and the film thickness T0 is equal to or greater than the film thickness T3 in the peripheral region R13.

The filter 10A is formed, for example, with a diameter of 6 mm (outer shape of the frame portion 15), a width dimension of the frame portion 15 of 1 mm, and an interval of adjacent through holes 12a of 1 μm or more and 500 μm or less. Further, in the filter portion 11a, the film thickness T3 in the peripheral region R13 near the frame portion 15 is formed to be 1.1 μm, and the film thickness T1 in the central region R11 is formed to be 0.8 μm.

According to the filter 10A, in the filter portion 11a, the first main surface PS1 is formed as a concave surface so that the film thickness T1 of the central region R11 is smaller than the film thickness T3 of the peripheral region R13. As a result, when cells are captured by the first main surface PS1 on which the concave surface is formed, a fluid pool is likely to be formed on the concave surface due to the surface tension of the fluid. For example, by handling the cells captured in a state where the culture solution is stored in a concave surface as a liquid pool, it is possible to perform processing such as analysis while suppressing the drying of the cells. Therefore, the handleability of the cells captured by the filter unit 11a can be improved.

Further, in the filter portion 11a, the film thickness of the filter portion 11a is increased continuously or stepwise from the central side region R11 to the peripheral side region R13, so that the filter portion 11a becomes smooth. Can form a concave surface. As a result, a fluid pool can be easily formed on the concave surface by the action of surface tension, and the handleability of the cells captured by the filter portion 11a can be further improved.

Further, the second main surface PS2 of the filter portion 11a is a flat surface, and the first main surface PS1 is a concave surface. Thereby, for example, when it is desired to utilize the fluid pool using the concave surface, the cells can be captured on the first main surface PS1 side which is the concave surface. On the other hand, when it is desired to improve the fluid drainage without forming a fluid pool, cells can be captured on the PS2 side of the second main surface, which is a flat surface. By properly using the first main surface PS1 and the second main surface PS2 according to the purpose in this way, the handleability of the cell filtration filter can be improved.

In the description of the filter 10A, a case where a concave surface is formed in the filter portion 11a so that the film thickness increases continuously or stepwise from the central region R11 to the peripheral region R13 is taken as an example. .. However, the filter 10A is not limited to such a case. For example, in the filter portion 11a, a portion where the film thickness increases from the central side region R11 to the peripheral side region R13 may be partially included. Even in such a case, if the average film thickness in the central region R11 is smaller than the average film thickness in the peripheral region R13, the effect of the filter 10A can be exhibited.

In the first embodiment, an example in which the shape of the through hole 12 is rectangular has been described, but the present invention is not limited to this. For example, the shape of the through hole 12 may be a polygon other than a square.

FIG. 6 shows an enlarged cross-sectional view of a part of the filter 10B having the through hole 12b formed in a regular hexagonal shape. As shown in FIG. 6, in the filter portion 11b of the filter 10B, a plurality of through holes 12b formed in a regular hexagonal shape are formed in a regular triangular lattice arrangement (honeycomb structure).

FIG. 7 shows an enlarged cross-sectional view of a part of the filter 10C having a through hole 12c formed in a regular octagonal shape. As shown in FIG. 7, in the filter portion 11c of the filter 10C, a plurality of through holes 12c formed in a regular octagonal shape are formed by a square lattice.

As shown in FIGS. 6 and 7, in the regular polygonal through holes 12b and 12c, the angle between the adjacent sides defining the openings of the through holes 12b and 12c can be obtuse. .. That is, the corners of the through holes 12a and 12b are gentler than the square-shaped through holes. Therefore, the through holes 12b and 12c have an advantage that they are less likely to damage cells as compared with the square-shaped through holes. Further, since the regular polygonal through holes 12b and 12c are easier to process than the square through holes, the processing accuracy can be improved. That is, in the regular polygonal through holes 12b and 12c, the coefficient of variation can be made smaller than that in the square through holes.

The through hole 12 may be formed in a regular polygonal shape other than a regular hexagon and a regular octagon.

Further, the shape of the through hole 12 may be a polygon including sides having different lengths. FIG. 8 shows an enlarged cross-sectional view of a part of the filter 10D having the through holes 12d formed in a polygonal shape. As shown in FIG. 8, in the filter 10D, the through hole 12d is formed in a horizontally long hexagonal shape extending in the Y direction. Specifically, the through hole 12d is formed in a horizontally long hexagonal shape in which two parallel sides defining the opening are extended in the Y direction.

With such a configuration, the filter 10D can have the same effect as the regular polygonal through holes 12b and 12c, and can also have the same effect as the rectangular through hole 12.

The filter 10D is not limited to the hexagonal through hole 12d, and may be a polygonal through hole such as an octagon. Further, the extending direction of the through hole 12d of the filter 10D is not limited. For example, the through hole 12d may be formed in a vertically long polygonal shape extending in the X direction.

The performance evaluation of the filter 10 according to the first embodiment was carried out using Examples 1-5 and Comparative Example 1-3.

(1) Filters of Examples 1-5 and Comparative Example 1-3 The filters of Examples 1-5 and Comparative Example 1-3 were prepared according to the specifications shown in Table 1.

In addition, in the arrangement interval of Table 1, the long side corresponds to the length of reference numeral b1 in FIG. 3, and the short side corresponds to the length of b2 in FIG.

The filters of Examples 1-5 and Comparative Example 1-3 are circular, have an outer diameter of 7.8 mm, a filter portion 11 diameter of 6 mm, and a frame portion thickness of 2 μm. The material is nickel (Ni).

In Examples 1 and 2, a filter 10 having a rectangular through hole 12 was used. In Example 3, a filter 10B having a regular hexagonal through hole 12b was used. In Example 4, a filter 10C having a regular octagonal through hole 12c was used. In Example 5, a filter 10A having a rectangular through hole 12a was used. Further, in the fifth embodiment, the film thickness of the filter portion 11a in the central region away from the frame portion 15 is smaller than the film thickness of the filter portion 11a in the peripheral region closer to the frame portion 15 than the central region.

In Comparative Example 1, a filter 10 having a rectangular through hole 12 was used. In Comparative Example 2, a filter 10B having a regular hexagonal through hole 12b was used. In Comparative Example 3, a filter 10C having a regular octagonal through hole 12c was used.

The filters of Examples 1-5 and Comparative Example 1-3 were respectively installed in the filtration device, and the performance was evaluated by filtering the cell suspension. As a pretreatment, each filter was immersed in an ethanol solution for 1 minute and then immersed in pure water for 1 minute to increase hydrophilicity.

(2) Cell suspension Using a 100 mm dish, HL-60, a leukemia cell line, was used in RPMI1620 medium (containing L-glutamine) containing 10 vol% fetal bovine serum and 1 vol% penicillin-streptomycin. Was cultured for 5 days.

A portion of the culture broth from the 100 mm dish was transferred by pipetting to a 15 mL centrifuge tube. Next, the centrifuge tube containing the culture solution was centrifuged at a rotation speed of 1000 rpm for 3 minutes, and then the supernatant was removed. Phosphate buffered saline was then added to generate a cell suspension. Incidentally, by adjusting the amount of phosphate buffered saline so that the cell concentration of the cell suspension at 10 ⁵ cells / mL.

30 μL of cell suspension and 15 μL of fluorescent reagent DAPI are mixed in a microtube to stain cells, and the stained cell suspension (cell stain) is incubated at 37 ° C. for 20 minutes under shading. did. Then, 10 μL of the cell staining solution was dropped onto the slide glass, the cover glass was overlapped, and fluorescence observation was performed with a fluorescence microscope through a bandpass filter centered on 455 nm using an excitation light source having a wavelength of 345 nm. When the size of the nuclei that developed bluish purple was measured, the size of the nuclei of HL-60 was 3.66 μm. The size of the HL-60 core is an average value calculated by measuring the size of 100 HL-60 cores.

After the cells were dispersed by pipetting to a part of the culture medium in a 100 mm dish, 10 μL was taken out using a micropipette, and a cell counter (Automato Fisher automatic cell counter, Countess® II FL) was taken out. ), The average values of cell concentration, viability, and cell size were measured. As a result, the cell concentration was 5 × 10 ⁵ cells / mL, the average size of active cells (HL-60) was 13.4 μm, and the survival rate was 90%. Specifically, the cell suspension and 0.4% trypan blue solution are mixed at a volume ratio of 1: 1 to stain the cell membrane in blue, and 10 μL of the mixed solution of the cell suspension and the trypan blue solution is prepared. The cells were dropped onto a cell counting slide (Countess (registered trademark) Cell Counting Chamber Slide manufactured by Thermo Fisher), and the morphology of the cells was observed. In the cell count, the number (concentration) of cells, the average value of cell size, and the survival rate were determined by image analysis using the stained cell membrane as a marker.

The following HL-60 cell suspension was prepared by further mixing RPMI1620 medium with the culture medium in a 100 mm dish at an arbitrary ratio.
Concentration of active cells (HL-60) ・ ・ ・ 3.06 × 10 ⁵ cells / mL
Liquid volume: 1 mL

As a result of measuring the cell viability of the culture solution in a 100 mm dish left in a clean bench at room temperature for 4 hours by the above-mentioned method, the viability was reduced to 81%. This means that the activity decreases when the cells are left at room temperature for a long time.

(3) Filtration method In the filters of Examples 1-5 and Comparative Example 1-3 attached to the filtration device, the cell suspension was dropped onto the first main surface PS1 of the filter unit 11 with a pipette, and the second The cell suspension was filtered by sucking the cell suspension from the PS2 side of the main surface. As an operating condition, suction was performed at a pressure of 0.5 kPa. As soon as it was visually confirmed that the amount of liquid (passing liquid) that had passed through the filter was about 0.8 ml, suction was stopped. The evaluation was performed by measuring the time from the start of suction to the end of suction (filtration time), the amount of the passing liquid, and the number of cells contained in the passing liquid.

(4) Evaluation results Table 2 shows the evaluation results.

In Examples 1-5, the recovery rate of cells (HL-60) was 100%. On the other hand, in Comparative Example 1-3, the cell recovery rates were 29.3%, 35.7% and 40.9%, respectively.

As shown in Table 1, in Comparative Example 1-3, the major axis of the inscribed ellipse of the through hole 12 is 9.0 μm, 6.6 μm, and 6.5 μm, respectively, and the size of the nucleus of the cell (HL-60). It is larger than 3.66 μm. Therefore, it is considered that the cells have passed through the through hole 12.

On the other hand, in Example 1-5, the major axis of the inscribed ellipse of the through hole 12 is 3.0 μm or more and 3.5 μm or less, which is smaller than the size of the cell nucleus. Therefore, in Example 1-5, it is considered that the cells could be reliably captured.

In addition, in this specification, "recovery rate" means the ratio of the number of the charged active cells to the cells captured on the first main surface PS1 of a filter, and (the number of input active cells-passage). It was calculated by (the number of active cells contained in the solution) / (the number of active cells input).

As for the number of active cells contained in the passing fluid, after dispersing the cells in the passing fluid by pipetting, 10 samples of 10 μL of the passing fluid were taken out using a micropipette, and a cell count meter (Automatic cell manufactured by Thermo Fisher) was used. The number of cells in the sample was measured with a counter, Countess (registered trademark) II FL).

As described above, by designing the major axis of the inscribed ellipse of the through hole 12 to be smaller than the size of the cell nucleus, the recovery rate can be improved. The reason for this is that the cytoplasm surrounding the cell nucleus is easily deformed, and the cell nucleus is not easily deformed.

When the major axis of the inscribed ellipse of the through hole 12 is larger than the size of the cell nucleus as in Comparative Example 1-3, the cytoplasm is poor even if the major axis of the inscribed ellipse of the through hole 12 is smaller than the size of the entire cell. The deformation may cause cells to pass through the filter 10.

On the other hand, when the major axis of the inscribed ellipse of the through hole 12 is designed to be smaller than the cell nucleus as in Example 1-5, the cell nucleus is less likely to be deformed than the cytoplasm, and therefore, as compared with Comparative Example 1-3, Easy to capture cells on filter 10.

In addition, the fact that the filter 10 is made of metal also contributes to the improvement of the cell recovery rate. By using the filter 10 made of metal, the amount of deformation of the through hole 12 itself of the filter 10 is smaller than that of a resin filter such as a membrane. Therefore, the filter 10 makes it easier to capture cells.

Needless to say, when filtering nucleated cells, it is preferable to suppress the contamination of the cell suspension with impurities, that is, the elution of the metal constituting the filter 10 into the cell suspension.

In Example 5, after filtration, the cells captured on the first main surface PS1 of the filter unit 11a were observed. As a result, it was found that the morphology of the cells was circular and the activity was maintained, similar to the cells before filtration. This is because a liquid pool was generated in the central region R11 of the filter unit 11a, and the cells were captured on the first main surface PS1 of the filter unit 11a in a state of being immersed in the liquid pool.

For nickel, the immersion potential, which is an index of metal ionization tendency, was measured. When the reference electrode-referenced immersion potential of silver chloride immersed in a saturated potassium chloride solution in phosphate buffered saline was measured for 3 minutes, the immersion potential of nickel changed in the noble range from 0.03 V. That is, it can be said that a metal and / or a metal oxide showing a noble immersion potential of at least 0.03 V when measured under the same conditions can be filtered without impairing the activity of cells.

Further, in Examples 1, 2 and 5, the filtration time is shorter than that in Examples 3 and 4. This is because the through hole 12 is formed in a rectangular shape, so that clogging due to deformation of the cytoplasm of the cell can be suppressed. Specifically, when the shape of the through hole 12 is rectangular, even if a part of the through hole 12 is blocked by the deformation of the cytoplasm, the liquid easily passes from the other part of the through hole 12. Therefore, it is considered that the filtration time in Examples 1, 2 and 5 was shorter than that in Examples 3 and 4.

In the embodiment, the nucleated cells has been described an example of capturing 10 ⁵ cells / relatively high concentrations of cell suspension present over mL was filtered nucleated cells in the cell suspension, The filter 10 can capture the nucleated cells to be collected even when the cell suspension is filtered with an extremely low concentration of several nucleated cells / mL. ..

Although the present invention has been fully described in connection with preferred embodiments with reference to the accompanying drawings, various modifications and modifications are obvious to those skilled in the art. It should be understood that such modifications and modifications are included therein, as long as they do not deviate from the scope of the invention according to the appended claims.

Since the filter of the present invention can improve the recovery rate of nucleated cells, it is useful for the purpose of separating nucleated cells from a cell suspension.

10, 10A, 10B, 10C, 10D Filter 11, 11a, 11b, 11c, 11d Filter part 12, 12a, 12b, 12c, 12d Through hole 13 Support base material 13a Opening 14 Filter base part 15 Frame part PS1 1st main Surface PS2 Second main surface R11 Central area R12 Intermediate area R13 Peripheral area T0, T1, T2, T3 Film thickness

Claims (12)

A method for recovering nucleated cells
At least one of metals and metal oxides as a main component are formed a plurality of through holes (excluding a square) is the arithmetic average roughness of the surface of the filter, rather smaller than the size of the nucleus of nucleated cells, Prepare a filter in which the major axis of the inscribed ellipse of the through hole is smaller than the size of the nucleus of the nucleated cell, and the inscribed ellipse of the through hole is an ellipse tangent to all sides defining the opening of the through hole. Steps to do,
A step of passing a liquid containing the nucleated cells through the filter,
Including
The filter is
A filter unit having the plurality of through holes penetrating the first main surface and the second main surface facing each other,
A frame portion arranged so as to surround the outer circumference of the filter portion and
With
A recovery method in which the film thickness of the filter is continuously or gradually increased from the center of the filter portion toward the frame portion in the filter portion. The recovery method according to claim 1, wherein the shape of the through hole is polygonal. The recovery method according to claim 2, wherein the shape of the through hole is rectangular. Claimed that at least one of the metal and the metal oxide has a reference electrode-referenced immersion potential of silver chloride immersed in a saturated potassium chloride solution in phosphate buffered physiological saline, noble than 0.03 V. Item 8. The collection method according to any one of Items 1 to 3. 1. At least one of the metal and the metal oxide contains at least one selected from the group consisting of gold, silver, copper, platinum, nickel, palladium, alloys thereof and oxides thereof. The collection method according to any one of 4 to 4. The recovery method according to any one of claims 1 to 5, wherein the step of passing the liquid containing nucleated cells through the filter includes a step of separating live cells and dead cells. A method of capturing nucleated cells
The main component is at least one of metal and metal oxide, and a plurality of through holes (excluding squares) are formed, and the arithmetic mean roughness of the surface of the filter is smaller than the size of the nucleus of nucleated cells and penetrates. Prepare a filter in which the major axis of the inscribed ellipse of the hole is smaller than the size of the nucleus of the nucleated cell, and the inscribed ellipse of the through hole is an ellipse tangent to all sides defining the opening of the through hole. Step,
A step of passing a liquid containing the nucleated cells through the filter,
Including
The filter
A filter unit having the plurality of through holes penetrating the first main surface and the second main surface facing each other,
A frame portion arranged so as to surround the outer circumference of the filter portion and
With
A capturing method in which the film thickness of the filter is continuously or gradually increased from the center of the filter portion toward the frame portion in the filter portion. The capture method according to claim 7, wherein the shape of the through hole is polygonal. The capture method according to claim 8, wherein the shape of the through hole is rectangular. Claimed that at least one of the metal and the metal oxide has a reference electrode reference immersion potential of silver chloride immersed in a saturated potassium chloride solution in phosphate buffered physiological saline, noble than 0.03 V. Item 8. The capture method according to any one of Items 7 to 9. 7. At least one of the metal and the metal oxide contains at least one selected from the group consisting of gold, silver, copper, platinum, nickel, palladium, alloys thereof and oxides thereof. The capture method according to any one of 10 to 10. The capture method according to any one of claims 7 to 11, wherein the step of passing the liquid containing the nucleated cells through the filter includes a step of separating live cells and dead cells.

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