Filters for filtering nucleated cells and filtration methods using them

Abstract

This invention provides a filter for filtering nucleated cells, which can improve the recovery rate of nucleated cells. The filter of this invention is a filter for filtering nucleated cells, which uses at least one of a metal and a metal oxide as its main component and forms a plurality of through-holes, wherein the plurality of through-holes does not include squares, 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.

Description

[0001] This application is a divisional application of the application filed on April 19, 2018, with application number 201810357055.6 and entitled "Filter for filtering nucleated cells and filtration method using the same". Technical Field

[0002] This invention relates to filters for filtering nucleated cells and filtration methods using the same. Background Technology

[0003] Patent Document 1 discloses a method for concentrating monocytes and platelets from a liquid containing red blood cells, nucleated cells, and platelets using a cell-capturing filter material. The cell-capturing filter material of Patent Document 1 captures nucleated cells and platelets while allowing unwanted cells such as red blood cells to pass through.

[0004] Existing technical documents

[0005] Patent documents

[0006] Patent Document 1: Japanese Patent Application Publication No. 2009-284860 Summary of the Invention

[0007] The problem the invention aims to solve

[0008] However, the cell-capturing filter material in Patent Document 1 still has room for improvement in terms of increasing the recovery rate of nucleated cells.

[0009] The purpose of this invention is to provide a filter for filtering nucleated cells and a filtration method using the same, wherein the filter can improve the recovery rate of nucleated cells.

[0010] Methods for solving problems

[0011] One aspect of the filter of the present invention is a filter for filtering nucleated cells.

[0012] It uses at least one of metals and metal oxides as its main components.

[0013] It also forms multiple through holes (excluding the square ones),

[0014] The major axis of the inscribed ellipse of the aforementioned through hole is smaller than the size of the nucleus of the aforementioned nucleated cell, and the inscribed ellipse of the aforementioned through hole is an ellipse that is tangent to all the sides that define the opening of the aforementioned through hole.

[0015] One aspect of the filtering method of the present invention is a method for filtering nucleated cells, which includes the following steps:

[0016] The step of preparing a filter, wherein the filter is composed of at least one of metal and metal oxide as the main component and is formed with a plurality of through holes (excluding squares), wherein the major axis of the inscribed ellipse of the through holes is smaller than the size of the nucleus of the nucleated cell, and the inscribed ellipse of the through holes is an ellipse that is tangent to all sides that define the opening of the through holes.

[0017] The step of passing the liquid containing the nucleated cells described above through the filter described above.

[0018] The effects of the invention

[0019] According to the present invention, a filter for filtering nucleated cells and a filtration method using the same are provided, the filter being capable of improving the recovery rate of nucleated cells. Attached Figure Description

[0020] Figure 1 This is a schematic diagram of the filter structure according to Embodiment 1 of the present invention.

[0021] Figure 2 This is an enlarged perspective view of a portion of the filter according to Embodiment 1 of the present invention.

[0022] Figure 3 Viewed from the thickness direction Figure 2 A diagram illustrating a portion of the filter.

[0023] Figure 4 This is a schematic diagram of the filter in Embodiment 1 of the present invention when a supporting substrate is provided.

[0024] Figure 5 This is a schematic diagram of a filter according to a modified embodiment 1 of the present invention.

[0025] Figure 6 This is an enlarged cross-sectional view of a part of a filter in another variation of Embodiment 1 of the present invention.

[0026] Figure 7 This is an enlarged cross-sectional view of a part of a filter in another variation of Embodiment 1 of the present invention.

[0027] Figure 8 This is an enlarged cross-sectional view of a part of a filter in another variation of Embodiment 1 of the present invention. Detailed Implementation

[0028] (The process of realizing this invention)

[0029] In Patent Document 1, a cell-capturing filter material made of nonwoven fabric is used to capture nucleated cells and platelets while allowing unwanted cells such as red blood cells to pass through, thereby separating monocytes from the blood. However, the monocyte recovery rate of the cell-capturing filter in Patent Document 1 is only about 74%, leaving room for improvement in increasing the recovery rate of the target cells.

[0030] The inventors conducted in-depth research and found that by using a filter with at least one of metals and metal oxides as the main component to filter liquids containing nucleated cells, the recovery rate of nucleated cells as the target of capture can be improved, thus completing the present invention.

[0031] One aspect of the filter of the present invention is a filter for filtering nucleated cells.

[0032] It uses at least one of metals and metal oxides as its main components.

[0033] It also forms multiple through holes (excluding the square ones),

[0034] The major axis of the inscribed ellipse of the aforementioned through hole is smaller than the size of the nucleus of the aforementioned nucleated cell, and the inscribed ellipse of the aforementioned through hole is an ellipse that is tangent to all the sides that define the opening of the aforementioned through hole.

[0035] This configuration can improve the recovery rate of nucleated cells.

[0036] In the above-mentioned filter, the shape of the through hole can be polygonal.

[0037] This configuration can improve the recovery rate of nucleated cells.

[0038] In the above-mentioned filter, the shape of the through hole can be rectangular.

[0039] This configuration allows for a reduction in filtration time while increasing the recovery rate of nucleated cells.

[0040] The above filters may further include:

[0041] A filter section having the aforementioned plurality of through holes, wherein the plurality of through holes penetrate a first main surface and a second main surface opposite to each other; and

[0042] A frame portion arranged to surround the outer periphery of the aforementioned filter section.

[0043] Within the aforementioned filter section, the membrane thickness of the filter section in the central region away from the aforementioned frame section is less than the membrane thickness of the filter section in the edge region closer to the aforementioned frame section than the central region.

[0044] With this configuration, a concave surface can be formed on a portion of at least one of the first and second main surfaces of the filter section. When cells are captured using the side with the concave surface, fluid volume easily forms on the concave surface due to the surface tension of the fluid, thus preventing the captured cells from drying out. Therefore, the processability of the cells captured by the filter section is improved.

[0045] In the above-mentioned filter, at least any of the above-mentioned metals and metal oxides may have an immersion potential higher than 0.03V in phosphate-buffered saline, with the immersion potential referenced by a reference electrode formed of silver chloride immersed in a saturated potassium chloride solution.

[0046] This configuration prevents the metal or metal oxides that serve as filter components from dissolving into the liquid containing nucleated cells.

[0047] In the above-mentioned filter, at least one of the above-mentioned metal and the above-mentioned metal oxide may contain at least one selected from gold, silver, copper, platinum, nickel, palladium, their alloys and their oxides.

[0048] This configuration can further improve the recovery rate of nucleated cells.

[0049] One aspect of the filtering method of the present invention is a method for filtering nucleated cells, which includes the following steps:

[0050] The step of preparing a filter, wherein the filter is composed of at least one of metal and metal oxide as the main component and is formed with a plurality of through holes (excluding squares), wherein the major axis of the inscribed ellipse of the through holes is smaller than the size of the nucleus of the nucleated cell, and the inscribed ellipse of the through holes is an ellipse that is tangent to all sides that define the opening of the through holes.

[0051] The step of passing the liquid containing the nucleated cells described above through the filter described above.

[0052] This configuration can improve the recovery rate of nucleated cells.

[0053] In the above filtering method, the shape of the through hole can be polygonal.

[0054] This configuration can improve the recovery rate of nucleated cells.

[0055] In the above filtration method, the shape of the through hole can be rectangular.

[0056] This configuration allows for a reduction in filtration time while further improving the recovery rate of nucleated cells.

[0057] In the above filtering method, the filter may have the following features:

[0058] A filter section having the aforementioned plurality of through holes, wherein the plurality of through holes penetrate a first main surface and a second main surface opposite to each other; and

[0059] A frame portion arranged to surround the outer periphery of the aforementioned filter section.

[0060] Within the aforementioned filter section, the membrane thickness of the filter section in the central region away from the aforementioned frame section is less than the membrane thickness of the filter section in the edge region closer to the aforementioned frame section than the central region.

[0061] With this configuration, cells can be captured using a concave surface formed on a portion of at least one of the first and second main surfaces of the filter section. Since fluid volume easily accumulates on the concave surface due to the surface tension of the fluid, drying of the captured cells can be suppressed. Therefore, the processability of cells captured by the filter section can be improved.

[0062] In the above filtration method, the immersion potential of at least one of the metals and the metal oxides in phosphate-buffered saline can be higher than 0.03V, and the immersion potential is referenced to a reference electrode formed of silver chloride immersed in a saturated potassium chloride solution.

[0063] This configuration prevents the metal or metal oxides that serve as filter components from dissolving into the liquid containing nucleated cells.

[0064] In the above filtration method, at least one of the metal and the metal oxide may contain at least one selected from gold, silver, copper, platinum, nickel, palladium, their alloys and their oxides.

[0065] This configuration can further improve the recovery rate of nucleated cells.

[0066] In the above filtration method, the step of passing the liquid containing the nucleated cells through the filter may include the step of separating live cells from dead cells.

[0067] This structure allows for the separation of living cells from dead cells.

[0068] One embodiment of the kit of the present invention comprises a filter for filtering nucleated cells and is used to perform the above-described filtration method.

[0069] The filter described above uses at least one of metal and metal oxide as its main component and forms a plurality of through holes (excluding square holes). The major axis of the inscribed ellipse of the through hole is smaller than the size of the nucleus of the nucleated cell. The inscribed ellipse of the through hole is an ellipse that is tangent to all sides that define the opening of the through hole.

[0070] This configuration can improve the recovery rate of nucleated cells.

[0071] Hereinafter, with reference to Embodiment 1 of the present invention, the accompanying drawings will be used. Figure 1 The accompanying explanation is provided. Furthermore, for ease of explanation, the elements are exaggerated in each diagram.

[0072] (Implementation Method 1)

[0073] [Filter Composition]

[0074] Figure 1 This is a schematic diagram of the structure of the filter 10 according to Embodiment 1 of the present invention. Figure 2 This is an enlarged perspective view of a portion of the filter 10 in Embodiment 1 of the present invention. Figure 1 and Figure 2 The X, Y, and Z directions in the diagram represent the longitudinal, transverse, and thickness directions of filter 10, respectively. For example... Figure 1 As shown, the filter 10 includes a filter section 11 and a frame section 15 disposed around the outer periphery of the filter section 11. Figure 2 As shown, the filter 10 has a first main surface PS1 and a second main surface PS2 that are opposite to each other. The filter section 11 includes a filter base section 14 having a plurality of through holes 12 that pass through the first main surface PS1 and the second main surface PS2.

[0075] The filter 10 filters nucleated cells by passing a liquid (cell suspension) containing nucleated cells through the filter section 11.

[0076] In this manual, "nucleated cell" refers to a cell in which the nucleolus is separated from the cytoplasm by the nuclear membrane.

[0077] <Material>

[0078] The filter substrate 14, which forms the base portion of the filter 10, is mainly composed of metals and / or metal oxides. The filter substrate 14 can be, for example, gold, silver, copper, platinum, nickel, palladium, their alloys, and their oxides.

[0079] The outermost layer of filter 10 can be made of a metal and / or metal oxide that is difficult to dissolve into the cell suspension. For example, when the outermost layer of filter 10 is covered with a metal with an immersion potential higher than 0.03V in phosphate-buffered saline, the leaching of the materials constituting filter 10 into the cell suspension can be suppressed, the immersion potential being referenced to a reference electrode formed of silver chloride immersed in a saturated potassium chloride solution. This reduces stress on the cells. Alternatively, the outermost layer of filter 10 can also be made of a hydrophilic material. For example, when treating aqueous cell suspensions, the treatment time can be shortened, thus reducing stress on the cells.

[0080] <Appearance>

[0081] The filter 10 has a shape that is, for example, circular, rectangular, or elliptical. In Embodiment 1, the filter 10 has a generally circular shape. By making the filter 10 generally circular, fluid can flow evenly across the main surface of the filter 10 (e.g., the first main surface PS1 of the filter section 11). It should be noted that, in this specification, "generally circular" means that the ratio of the length of the major axis to the length of the minor axis is 1.0 or more and 1.2 or less.

[0082] <Filtering Section>

[0083] The filter section 11 is a plate-shaped structure with multiple through holes 12. The shape of the filter section 11 is, for example, circular, rectangular, or elliptical. In Embodiment 1, the filter section 11 is approximately circular. By making the filter section 11 approximately circular, the fluid can flow uniformly over the first main surface PS1 of the filter section 11.

[0084] Figure 3 This is a schematic diagram showing a portion of the filter section 11 viewed from the thickness direction (Z direction). For example... Figure 3 As shown, a plurality of through holes 12 are periodically arranged on the first main surface PS1 and the second main surface PS2 of the filter section 11. Specifically, the plurality of through holes 12 are arranged in a matrix at equal intervals in the filter section 11.

[0085] In embodiment 1, the through hole 12 has a rectangular shape when viewed from the first main surface PS1 side of the filter section 11, i.e., in the Z direction. For example... Figure 3 As shown, the through hole 12 is a rectangle with the longer side d1 in the X direction and the shorter side d2 in the Y direction. It should be noted that the through hole 12, viewed from the Z direction, has a shape other than a square. The through hole 12, viewed from the Z direction, can be, for example, a rhombus or a polygon. It should be noted that the polygon can include regular polygons, excluding squares.

[0086] In Embodiment 1, the long side d1 of the through hole 12 is, for example, formed to be more than 1.2 times and less than 1.8 times the short side d2.

[0087] In Embodiment 1, the through-hole 12, projected onto the surface perpendicular to the first main surface PS1 of the filter section 11, has a rectangular shape (cross-sectional shape). Specifically, the cross-sectional shape of the through-hole 12 is a rectangle in which one side of the filter 10 in the radial direction is longer than the other side in the thickness direction. It should be noted that the cross-sectional shape of the through-hole 12 is not limited to a rectangle; it can be, for example, a parallelogram or a trapezoidal cone, a symmetrical shape, or an asymmetrical shape.

[0088] In embodiment 1, the plurality of through holes 12 are arranged along two directions parallel to the sides of the rectangle when viewed from the first main surface PS1 side (Z direction) of the filter section 11, i.e. Figure 3 The X and Y directions are arranged at equal intervals. By arranging multiple through holes 12 in a rectangular grid, the opening ratio is increased, reducing the flow resistance of fluid through the filter 10. This configuration shortens processing time and reduces stress on the cells. Furthermore, the increased symmetry of the arrangement of the multiple through holes 12 facilitates observation of the filter.

[0089] It should be noted that the arrangement of the multiple through holes 12 is not limited to a rectangular grid arrangement; it can be, for example, a quasi-periodic arrangement or a periodic arrangement. As an example of a periodic arrangement, as long as it is a square arrangement, it can be a rectangular arrangement with unequal intervals in two arrangement directions, or a triangular grid arrangement or an equilateral triangular grid arrangement, etc. It should be noted that as long as multiple through holes 12 are provided in the filter section 11, their arrangement is not limited.

[0090] It should be noted that the corners connecting the edges of the opening of the through-hole 12 can be rounded (R-shaped). That is, the corners of the through-hole 12 can be formed into a circle. With this configuration, cell damage can be suppressed at the corners of the through-hole 12, further ensuring cell viability.

[0091] The spacing of the through-holes 12 is appropriately designed according to the type (size, morphology, properties, elasticity) or quantity of cells to be separated. Here, the spacing of the through-holes 12 refers to: Figure 3 As shown, when viewing the through-hole 12 from the first main surface PS1 side of the filter section 11, the distances b1 and b2 between the center of any through-hole 12 and the center of an adjacent through-hole 12 are as follows: Specifically, interval b1 refers to the distance between the center of the through-hole 12 in the short side direction (X direction) and the center of an adjacent through-hole 12. Interval b2 refers to the distance between the center of the through-hole 12 in the long side direction (Y direction) and the center of an adjacent through-hole 12. It should be noted that in Embodiment 1, the center of any through-hole 12 refers to the intersection of the diagonals of the rectangular through-hole 12.

[0092] In the case of a periodically arranged structure, the spacing b1 of the through holes 12 is, for example, greater than 1 times and less than 10 times the long side d1 of the through holes 12, preferably less than 3 times the long side d1 of the through holes 12. The spacing b2 of the through holes 12 is, for example, greater than 1 times and less than 10 times the short side d2 of the through holes 12, preferably less than 3 times the short side d2 of the through holes 12. Alternatively, the opening ratio of the filter section 11 is, for example, 10% or more, and preferably 25% or more. With such a configuration, the flow resistance of fluid through the filter section 11 can be reduced. Therefore, the processing time can be shortened and the stress on the cells can be reduced. It should be noted that the opening ratio is calculated by (the area occupied by the through holes 12) / (the projected area of ​​the first main surface PS1 assuming that the through holes 12 are not open).

[0093] The design incorporates an inscribed ellipse of the through-hole 12 with a major axis smaller than the size of the nucleus of a nucleated cell. In this specification, the "inscribed ellipse of the through-hole 12" refers to an ellipse with both a major and a minor axis, specifically the ellipse with the largest major axis drawn within the through-hole 12 when viewed from the first principal surface PS1 of the filter section 11. In other words, the "inscribed ellipse of the through-hole 12" refers to the ellipse with the largest major axis among those tangent to the inner wall of the filter base 14 constituting the through-hole 12. Furthermore, the major and minor axes of the "inscribed ellipse of the through-hole 12" can be of different lengths or the same length. In this specification, the "inscribed ellipse of the through-hole 12" may include a perfect circle.

[0094] Furthermore, in this specification, "the size of the nucleus of a nucleated cell" refers to the average length of the nucleus of multiple nucleated cells when the nucleated cells are placed in a liquid and observed under a microscope, and the longest line segment connecting any two points on the periphery of the nucleus of the nucleated cell is taken as the length of the nucleus of the nucleated cell.

[0095] The dimensions of the multiple through holes 12 are designed to be approximately the same. When the multiple through holes 12 of the same shape are arranged periodically, the standard deviation of the dimensions of the multiple through holes 12 is preferably small.

[0096] The filter section 11 has a uniform film thickness from its center outwards. In other words, when cut along the Z direction, the filter section 11 has a flat cross-sectional shape. The thickness of the filter section 11 is preferably greater than 0.1 times and less than 100 times the shortest side (e.g., short side d2) among the sides defining the through-hole 12. More preferably, the thickness of the filter section 11 is greater than 0.5 times and less than 10 times the short side d2 of the through-hole 12. With this configuration, the resistance of the filter 10 to the fluid can be reduced, and the processing time can be shortened. As a result, the stress on the cells can be reduced.

[0097] The arithmetic mean roughness of the surface (first main surface PS1) of the filter section 11 is preferably smaller than the size of the nucleus of a nucleated cell. This configuration reduces cell adhesion to the surface (first main surface PS1) of the filter section 11, thereby improving cell recovery. It should be noted that the arithmetic mean roughness is measured using a DEKTAK150 stylus-type surface shape measuring machine (registered trademark) manufactured by ULVAC, Inc., and the average of five measurements taken at different points on the surface of the filter section 11 is used as the arithmetic mean roughness of the filter section 11.

[0098] In the filter section 11, the first main surface PS1, which comes into contact with the liquid containing nucleated cells, can be formed smoothly. Specifically, the first main surface PS1 of the filter section 11 can also be formed from a uniform plane without any bumps or depressions. In other words, the openings of the plurality of through holes 12 on the first main surface PS1 of the filter section 11 can be formed on the same plane. Furthermore, the portions of the filter section 11 where the through holes 12 are not formed, namely the filter base portion 14, are connected and integrally formed. With this configuration, the adhesion of cells to the surface of the filter section 11 (the first main surface PS1) is reduced, and the captured nucleated cells can be easily recovered.

[0099] Regarding the through hole 12 of the filter section 11, the opening on the first main surface PS1 side and the opening on the second main surface PS2 side are connected by a continuous wall. Specifically, the through hole 12 is configured such 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 section 11 is viewed from the first main surface PS1 side, the through hole 12 is configured such that the opening on the first main surface PS1 side and the opening on the second main surface PS2 side coincide. In Embodiment 1, the through hole 12 is configured such that its inner wall is perpendicular to the first main surface PS1 and the second main surface PS2.

[0100] <Frame>

[0101] The frame portion 15 is disposed on the outer periphery of the filter portion 11 and has a smaller number of through holes 12 per unit area compared to the filter portion 11. The number of through holes 12 in the frame portion 15 is less than 1% of the number of through holes 12 in the filter portion 11. The thickness of the frame portion 15 can be greater than the thickness of the filter portion 11. With this configuration, the mechanical strength of the filter 10 can be improved.

[0102] When the filter 10 is connected to and used with the device, the frame portion 15 can also function as a connecting part for connecting the filter 10 and the device. In addition, the frame portion 15 can also display information about the filter (such as the size of the through hole 12).

[0103] 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 viewed from the first main surface PS1 side of the filter 10, the center of the frame portion 15 is aligned with the center of the filter portion 11. That is, the frame portion 15 and the filter are formed on concentric circles.

[0104] From the viewpoint of ease of handling and ease of integration with the system, the filter 10 can be fixed to a clamp and used. The clamp can be made of, for example, a material capable of gamma sterilization. The clamp can be formed from materials including polyethylene, polyethylene terephthalate, polyurethane, polystyrene, silicone rubber, ABS resin, polyamide, polyamide-imide, polysulfone, natural rubber, latex, urethane rubber, silicone rubber, ethylene-vinyl acetate, polyesters, epoxy resins, phenols, silica, alumina, gold, platinum, nickel, stainless steel, titanium, etc. By constructing the clamp from such materials, stress on the cells can be reduced.

[0105] [Filtering Method]

[0106] The filtration method using filter 10 will be explained.

[0107] First, filter 10 is prepared. In this step, filter 10 with a through-hole size 12 selected according to the size of the nucleus of the nucleated cell is prepared. Specifically, filter 10 with through-holes 12 smaller than the size of the nucleus of the nucleated cell to be filtered is prepared.

[0108] For example, in the process of preparing filter 10, the size of the nuclei of multiple nucleated cells can be confirmed by using a micrometer or a blood cell counter, and a filter 10 with a through-hole 12 smaller than the size of the nuclei of the nucleated cells can also be selected. Alternatively, by taking photographs of multiple nucleated cells and measuring the size of the nuclei of multiple nucleated cells, a filter 10 with a through-hole 12 smaller than the size of the nuclei of the nucleated cells can also be selected. It should be noted that the selection of filter 10 is not limited to these methods.

[0109] The filter 10 is installed in the device. Specifically, the filter 10 is installed in the device by clamping the frame portion 15 of the filter 10.

[0110] Next, the cell suspension is passed through filter 10. In this specification, "cell suspension" refers to a fluid containing nucleated cells. In most cases, the fluid containing nucleated cells is a liquid. Liquids include, for example, culture solutions containing amino acids, proteins, serum, phosphate-buffered saline, or water. In addition to cells and fluid, the cell suspension may also contain non-biological materials such as resin particles, parts of tissue such as bone or flesh fragments, dead cells, etc.

[0111] In this way, by passing the liquid containing nucleated cells through the filter section 11, the nucleated cells are separated from the liquid. In Embodiment 1, the filter section 11 is designed such that the major axis of the inscribed ellipse of the through hole 12 is 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 section 11 without passing through the through hole 12.

[0112] As a method for passing the cell suspension through the filter 10, there are methods such as: passing the cell suspension through the filter 11 from approximately vertically above the first main surface PS1 relative to the filter section 11 by gravity. Other methods include: contacting the cell suspension with the first main surface PS1 of the filter section 11 and applying pressure to the cell suspension to pass it through (squeezing); or contacting the cell suspension with the first main surface PS1 of the filter section 11 and drawing it through from the second main surface PS2 to pass it through (suction), etc. It should be noted that in the process of passing liquid containing nucleated cells through the filter section 11, it is preferable to avoid applying stress to the cells as much as possible. For example, if pressure is applied, it is preferable to set a pressure that does not deform the nucleated cells. More preferably, the liquid is passed through the filter section 11 by its own weight without applying pressure. Alternatively, it is preferable to shorten the processing time and reduce the time stress acts on the nucleated cells by increasing the opening ratio of the filter section 11.

[0113] Furthermore, the cell suspension can be passed through filter 10 in a state where nucleated cells are suspended in the liquid. The nucleated cells suspended in the liquid are approximately spherical, thus improving the recovery rate of the nucleated cells to be captured. That is, it is possible to improve the size accuracy of the nucleated cells to be captured.

[0114] Furthermore, by passing the cells through the filter 10 multiple times, the size accuracy of the nucleated cells being captured can be improved.

[0115] In the filtration method using filter 10, a filter container can be used for filtration, for example. The filter container is, for example, a cylindrical container with an outer diameter of 14 mm, an inner diameter of 6 mm, and a height of 55 mm, on which filter 10 can be installed. It should be noted that the filter container is not limited to this; containers of various shapes and sizes can be used.

[0116] [Filter Manufacturing Method]

[0117] A representative manufacturing method for filter 10 will be described. Filter 10 is manufactured through the following steps.

[0118] <Formation of the power supply membrane>

[0119] A Cu power supply film is formed on the upper surface of a silicon substrate using a sputtering apparatus. This power supply film becomes a power source when the filter substrate 14 of the filter 10 (described later) is formed. At this time, an intermediate layer such as Ti can be formed to ensure the adhesion between the silicon substrate and the power supply film.

[0120] The conditions for forming a Cu power supply film are shown below.

[0121] Sputtering gas: Argon

[0122] Vacuum level of the sputtering unit: 5.0 × 10⁻⁶ -4 Pa

[0123] Power applied: DC 500W

[0124] Sputtering time: 27 minutes during Cu film formation

[0125] Ti film formation time / 3 minutes 5 seconds

[0126] <Formation of the resist image>

[0127] A resist image is formed on a power supply film formed on the upper surface of a silicon substrate.

[0128] A resist film of a specified thickness is formed on a power supply film formed on the upper surface of a silicon substrate using a spin coater or similar equipment. Then, the resist is exposed through a photomask with a specified pattern and developed to form a resist image.

[0129] The coating conditions for the resist film are shown below.

[0130] Corrosion resist: Phenolic varnish resin + organic solvent

[0131] Spin coater speed: 1130 rpm

[0132] Resist film thickness: 2μm

[0133] After the above-mentioned resist is applied to the upper surface of the silicon substrate using a spin coater, the solvent is evaporated at 130°C under a nitrogen atmosphere and then cooled to form a resist film.

[0134] Irradiation for 0.25 seconds included a wavelength of 365nm and an energy density of 2500J / m². 2 The light is used to expose the photoresist.

[0135] The exposed portion is brought into contact with an alkaline solution for development.

[0136] <Formation of the filter substrate>

[0137] A filter substrate 14 is formed at the opening of the resist image. Using a pre-formed power supply film as the power source, an electroplating method is used to form a filter substrate 14 containing a nickel-plated film.

[0138] The formation conditions of the filter substrate 14 are as follows.

[0139] Pretreatment: The surface of the power supply membrane is activated by immersion in dilute sulfuric acid for 60 seconds.

[0140] Plating bath: Nickel sulfamate plating bath, temperature = 55℃, pH = 4.0

[0141] Plating speed: 0.5 μm / min

[0142] Electroplating: Electroplating is performed while shaking.

[0143] <Resist Dissolution and Stripping>

[0144] The resist film is dissolved and stripped off by applying ultrasonic waves to the filter substrate 14 in an acetone solution for 15 minutes.

[0145] <Forming of Support Substrate>

[0146] When using filter 10 for filtration, a support substrate can be provided for filter 10 as needed.

[0147] Figure 4 The general structure of the filter 10, on which the support substrate 13 is mounted, is shown. (As shown) Figure 4 As shown, a support substrate 13 can be provided on the second main surface PS2 side of the filter 10. The support substrate 13 is provided with a plurality of square openings 13a. The thickness of the support substrate 13 is, for example, 14 μm, the spacing A of one side of the openings 13a, i.e., the horizontal strips, is 260 μm, and the width B of the horizontal strips is 14 μm.

[0148] In this way, by providing the support substrate 13, it is possible to prevent the filter 10 from breaking during filtration. The support substrate is manufactured through the following process.

[0149] After a photosensitive resist is coated again on the upper surface of the silicon substrate on which the filter 10 is formed to form a resist film, the resist is exposed through a photomask and then developed to form a resist image. At this time, the exposure and development processes are performed such that the resist image spans multiple substrates of the filter 10. It should be noted that the portion of the resist image that spans the substrate of the filter 10 becomes the opening of the filter 10 after the filter 10 is completed. In other words, the number of substrates of the filter 10 that the resist image spans is appropriately determined according to the required aperture ratio of the filter 10.

[0150] A filter substrate 14 is formed at the opening of the resist image. Using a pre-formed power supply film as the power source, a support substrate containing a nickel-plated film is formed by electroplating. It should be noted that the width of the support substrate is appropriately determined according to the required strength of the filter 10.

[0151] The resist film is dissolved by applying ultrasound to the support substrate in an acetone solution for 15 minutes, thereby stripping off the resist.

[0152] <Removal of the power supply membrane>

[0153] The power supply membrane is removed, and the filter substrate 14 and the support substrate are separated from the silicon substrate, thereby completing the filter 10 for filtering nucleated cells.

[0154] The removal of the power supply membrane was carried out by immersing it in an aqueous solution prepared by mixing 60% hydrogen peroxide aqueous solution with acetic acid and pure water in a ratio of 1:1:20 at 25°C for 48 hours.

[0155] [Effect]

[0156] The filter 10 according to Embodiment 1 can achieve the following effects.

[0157] The filter 10 uses at least one of metal and metal oxide as its main component. Furthermore, the filter 10 includes a filter section 11 with a plurality of through holes 12. With this configuration, the through holes 12 of the filter section 11 are less prone to deformation, enabling it to capture nucleated cells and improve the recovery rate of nucleated cells.

[0158] The design incorporates an inscribed ellipse with a major axis smaller than the size of the nucleus of a nucleated cell. This configuration further improves the recovery rate of nucleated cells.

[0159] The through-hole 12 is rectangular in shape. This configuration reduces filtration time compared to a square-shaped through-hole. Specifically, the cytoplasm of nucleated cells is more deformable than the nucleus. In the case of a square-shaped through-hole, if nucleated cells are captured by the filter section, the cytoplasm deforms, potentially clogging the through-hole. On the other hand, with the rectangular through-hole 12, even if the cytoplasm deforms and partially blocks the through-hole 12 when capturing nucleated cells, liquid can still pass through the rest of the through-hole 12. Therefore, the rectangular through-hole 12 allows liquid to pass through more easily than a square through-hole, reducing filtration time.

[0160] The first main surface PS1 of the filter section 11 is formed smoothly. With this configuration, nucleated cells captured by the first main surface PS1 can be easily separated from the filter section 11, thus simplifying recycling.

[0161] Regarding the shape of the nucleus of a nucleated cell, in addition to a perfect circle, there are various other shapes such as ellipses. In the filter 10, the major axis of the inscribed ellipse of the through-hole 12 is smaller than the size of the nucleus of the nucleated cell, and the inscribed ellipse of the through-hole 12 is an ellipse tangent to all the sides that define the opening of the through-hole 12. With this configuration, nuclei of various shapes other than perfect circles can be reliably captured, thereby improving the recovery rate.

[0162] Furthermore, in the filtration method using filter 10, by passing the liquid containing nucleated cells through filter 10, the nucleated cells that are the target of capture can be reliably captured, thus improving the recovery rate.

[0163] The size of the nucleus in nucleated cells varies depending on the type, culture conditions, and passage number. For example, even the same cell type can have different nuclei depending on culture conditions such as temperature, time, or environment. Therefore, even the same nucleated cell type can have diverse nuclei, and when the filter 10 is prepared without regard to nucleus size, nucleated cells may sometimes pass through the perforation pores 12. In the filtration method using the filter 10, the size of the perforation pores 12 is selected according to the size of the nucleated cell nucleus when preparing the filter 10. That is, in the filtration method using the filter 10, a filter 10 with perforation pores 12 smaller than the size of the nucleus of the nucleated cell being filtered is selected for filtration. Therefore, in the filtration method using the filter 10, nucleated cells can be reliably captured onto the filter 10, improving the recovery rate.

[0164] It should be noted that in Embodiment 1, an example with approximately the same size for the through holes 12 was described, but this is not a limitation. For example, the sizes of the through holes 12 can be different from each other. In this case, the maximum size of the through hole 12 can be designed to be smaller than the size of the nucleus of a nucleated cell.

[0165] In Embodiment 1, the method for manufacturing the filter 10 has been described with reference to an example including the step of forming a substrate portion, but it is not limited thereto. For example, the method for manufacturing the filter 10 may not include the step of forming a substrate portion.

[0166] In Embodiment 1, an example of filtering nucleated cells by passing a liquid containing nucleated cells through filter 10 was described, but it is not limited to this. For example, filter 10 can also be used to separate live cells from dead cells from a liquid containing live cells and dead cells.

[0167] As a method for separating live cells from dead cells, for example, the filter 10 can be used to capture live cells onto the first main surface PS1 of the filter section 11, while allowing dead cells to pass through. Alternatively, the filter 10 can also be used to capture dead cells onto the first main surface PS1 of the filter section 11, while allowing live cells to pass through.

[0168] In Embodiment 1, the filter 10 and the filtration method have been described, but are not limited thereto. For example, it may also be used in the form of a kit containing the filter 10 for filtering nucleated cells and for implementing the filtration method.

[0169] In Embodiment 1, an example with a uniform film thickness for the filter section 11 was described, but it is not limited to this. For example, the filter section 11 may be formed such that the film thickness on the central side is smaller than that on the edge side, and thus does not have a uniform film thickness.

[0170] Figure 5 The filter 10A is shown as a variation of embodiment 1. Figure 5 As shown, within the filter section 11a of filter 10A, the membrane thickness T1 of the central region R11, which is farther from the frame 15, is less than the membrane thickness T3 of the edge region R13, which is closer to the frame 15 than the central region R11. Furthermore, for filter 10A, within the filter section 11a, when the membrane thickness of the intermediate region R12, located between the central region R11 and the edge region R13, is denoted as T2, the membrane thickness of each region satisfies the relationship T1 < T2 < T3. That is, within the filter section 11a of filter 10, the membrane thickness is set such that it increases from the center of the filter section 11a toward the radial direction (outward in the radial direction). Furthermore, the membrane thickness can be increased continuously or in stages.

[0171] In addition, such as Figure 5As shown, the second main surface PS2 of the filter 10A is formed as a flat surface, and the surface corresponding to the filter portion 11a in the first main surface PS1 is formed as a concave surface, such that the edge side region is higher than the central side region. The frame portion 15 of the filter 10A is formed with a membrane thickness T0 that is approximately constant, and the membrane thickness T0 reaches or exceeds the membrane thickness T3 of the edge side region R13.

[0172] The filter 10A is formed as follows: for example, the diameter is 6 mm (the outer shape of the frame 15), the width of the frame 15 is 1 mm, and the spacing between adjacent through holes 12a is 1 μm or more and 500 μm or less. Furthermore, the filter section 11a is formed as follows: the membrane thickness T3 in the edge side region R13 near the frame 15 is 1.1 μm, and the membrane thickness T1 in the central side region R11 is 0.8 μm.

[0173] According to filter 10A, with respect to filter section 11a, the first main surface PS1 is formed as a concave surface such that the membrane thickness T1 of the central side region R11 is less than the membrane thickness T3 of the edge side region R13. Therefore, when cells are captured using the concave first main surface PS1, fluid volume easily forms on the concave surface due to the surface tension of the fluid. For example, by processing cells captured in the culture medium and retained on the concave surface, analysis and other processing can be performed while inhibiting cell drying. Thus, the processability of cells captured by filter section 11a can be improved.

[0174] Furthermore, within the filter section 11a, by continuously or progressively increasing the membrane thickness of the filter section 11a from the central side region R11 toward the edge side region R13, a smooth concave surface can be formed in the filter section 11a. As a result, fluid volume can be easily formed in the concave surface due to surface tension, further improving the processing capability of cells captured by the filter section 11a.

[0175] Furthermore, the second main surface PS2 of the filter section 11a is a flat surface, while the first main surface PS1 is concave. Therefore, for example, if it is desirable to utilize the fluid retention achieved by the concave surface, cells can be captured on the concave side of the first main surface PS1. On the other hand, if it is desirable to improve fluid removal without forming fluid retention, cells can be captured on the flat side of the second main surface PS2. By using the first main surface PS1 and the second main surface PS2 differently according to the purpose, the processing capacity of the cell filter can be improved.

[0176] It should be noted that in the description of filter 10A, the case in which a concave surface is formed within the filter section 11a such that the membrane thickness continuously increases or increases in stages from the central side region R11 towards the edge side region R13 is used as an example. However, filter 10A is not limited to this case. For example, there may be a portion within the filter section 11a where the membrane thickness increases from the central side region R11 towards the edge side region R13. Even in such a case, as long as the average membrane thickness of the central side region R11 is less than the average membrane thickness of the edge side region R13, the filter 10A can still function effectively.

[0177] In Embodiment 1, an example of a rectangular shape for the through hole 12 was described, but it is not limited to this. For example, the shape of the through hole 12 may be a polygon excluding squares.

[0178] Figure 6 An enlarged cross-sectional view of a portion of a filter 10B having a through-hole 12b formed in a regular hexagonal shape is shown. Figure 6 As shown, in the filter section 11b of filter 10B, a plurality of through holes 12b formed in the shape of regular hexagons are arranged in an equilateral triangular lattice (honeycomb structure).

[0179] Figure 7 An enlarged cross-sectional view of a portion of a filter 10C having a through-hole 12c formed in a regular octagonal shape is shown. Figure 7 As shown, in the filter section 11c of filter 10C, a plurality of through holes 12c formed in the shape of regular octagons form a square grid.

[0180] like Figure 6 and Figure 7 As shown, in the through holes 12b and 12c of regular polygonal shape, the angle between adjacent sides defining the opening of the through holes 12b and 12c can be made obtuse. That is, the corners of the through holes 12a and 12b are gentler compared to those of square through holes. Therefore, the through holes 12b and 12c have the advantage of being less likely to damage cells compared to square through holes. Furthermore, the through holes 12b and 12c of regular polygonal shape are easier to process than those of square through holes, thus improving processing accuracy. That is, the through holes 12b and 12c of regular polygonal shape can reduce the coefficient of variation compared to those of square through holes.

[0181] It should be noted that the through hole 12 can be formed into a regular polygon shape other than a regular hexagon or a regular octagon.

[0182] Furthermore, the through hole 12 can be a polygon with sides of varying lengths.

[0183] Figure 8An enlarged cross-sectional view of a portion of a filter 10D having through holes 12d formed in a polygonal shape is shown. Figure 8 As shown, in filter 10D, the through-hole 12d is formed as a transverse hexagonal shape extending along the Y direction. Specifically, the through-hole 12d is formed as a transverse hexagonal shape with two parallel sides defining the opening extending along the Y direction.

[0184] With this configuration, the filter 10D can achieve the same effect as the through holes 12b and 12c with regular polygonal shapes, and also the same effect as the through hole 12 with rectangular shapes.

[0185] It should be noted that the through-hole 12d of the filter 10D is not limited to a hexagonal shape, but can also be a polygonal shape such as an octagon. Furthermore, the extending direction of the through-hole 12d of the filter 10D is not limited. For example, the through-hole 12d can be formed as a longitudinal polygonal shape extending along the X direction.

[0186] Example

[0187] The performance of the filter 10 described in Embodiment 1 was evaluated using Examples 1-5 and Comparative Examples 1-3.

[0188] (1) Regarding the filters of Examples 1-5 and Comparative Examples 1-3

[0189] The filters of Examples 1-5 and Comparative Examples 1-3 were manufactured according to the specifications shown in Table 1.

[0190] [Table 1]

[0191]

[0192] It should be noted that in the arrangement intervals of Table 1, the longer side is equivalent to... Figure 3 The length of symbol b1 in the diagram, the shorter side is equivalent to Figure 3 The length of b2 in the equation.

[0193] The filters in Examples 1-5 and Comparative Examples 1-3 are circular, with an outer diameter of 7.8 mm, a filter section 11 diameter of 6 mm, and a frame thickness of 2 μm. The material is nickel (Ni).

[0194] Examples 1 and 2 use a filter 10 with a rectangular through-hole 12. Example 3 uses a filter 10B with a regular hexagonal through-hole 12b. Example 4 uses a filter 10C with a regular octagonal through-hole 12c. Example 5 uses a filter 10A with a rectangular through-hole 12a. Furthermore, in Example 5, the membrane thickness of the filter portion 11a in the central region away from the frame 15 is less than the membrane thickness of the filter portion 11a in the edge region closer to the frame 15 than the central region.

[0195] Comparative Example 1 uses a filter 10 with a rectangular through-hole 12. Comparative Example 2 uses a filter 10B with a regular hexagonal through-hole 12b. Comparative Example 3 uses a filter 10C with a regular octagonal through-hole 12c.

[0196] The filters of Examples 1-5 and Comparative Examples 1-3 were respectively installed in a filtration device, and cell suspensions were filtered to evaluate their performance. It should be noted that, as a pretreatment, each filter was immersed in an ethanol solution for 1 minute, and then immersed in pure water for 1 minute, thereby improving its hydrophilicity.

[0197] (2) About cell suspensions

[0198] HL-60, a leukemia cell line, was cultured for 5 days in 100 mm petri dishes in RPMI 1620 medium (containing L-glutamine) containing 10 vol% fetal bovine serum and 1 vol% penicillin-streptomycin.

[0199] A portion of the culture medium was transferred from a 100 mm petri dish to a 15 mL centrifuge tube using pipetting. The tube containing the culture medium was then centrifuged at 1000 rpm for 3 minutes, and the supernatant was removed. Next, phosphate-buffered saline was added to create a cell suspension. It should be noted that the amount of phosphate-buffered saline added was adjusted to achieve a cell concentration of 102. 5 per mL.

[0200] Cells were stained by mixing 30 μL of cell suspension with 15 μL of the fluorescent reagent DAPI in a microtube. The stained cell suspension (cell staining solution) was then incubated at 37°C for 20 minutes under dark conditions. Subsequently, 10 μL of the cell staining solution was added to a glass slide, covered with a coverslip, and fluorescence was observed using a fluorescence microscope with an excitation wavelength of 345 nm and a bandpass filter centered at 455 nm. The size of the nuclei that developed a blue-violet color was measured, and the result showed that the nucleus size of HL-60 was 3.66 μm. It should be noted that the nucleus size of HL-60 was calculated as an average value based on the size measurements of 100 HL-60 nuclei.

[0201] For a portion of the culture medium in a 100 mm petri dish, after dispersing the cells by pipetting, 10 μL was taken out using a micropipette. The average cell concentration, viability, and cell size were measured using a cell counter (Thermo Fisher Scientific, Countess II FL). The result showed a cell concentration of 5 × 10⁻⁶ cells / mL. 5 The average size of viable cells (HL-60) was 13.4 μm, and the survival rate was 90%. Specifically, the cell suspension was stained blue by mixing the cell suspension with 0.4% trypan blue solution at a 1:1 volume ratio. 10 μL of the cell suspension and trypan blue solution mixture was added to a cell counting slide (Thermo Fisher Scientific, Countess Cell Counting Chamber Slide), and cell morphology was observed. In cell counting, the stained cell membrane was used as a marker for image analysis to determine the cell count (concentration), average cell size, and survival rate.

[0202] The culture medium in the 100 mm petri dish was further mixed with RPMI 1620 medium at any ratio to prepare the following HL-60 cell suspension.

[0203] Concentration of viable cells (HL-60)... 3.06 × 10⁻⁶ 5 cells / mL

[0204] Liquid volume…1mL

[0205] It should be noted that when the cell viability was measured using the above method for culture medium in a 100mm petri dish placed in a clean bench at room temperature for 4 hours, the viability decreased to 81%. This means that prolonged placement of cells at room temperature led to a decrease in cell activity.

[0206] (3) Regarding filtering methods

[0207] Regarding the filters of Examples 1-5 and Comparative Examples 1-3 installed in the filtration apparatus, cell suspension is filtered by adding cell suspension dropwise to the first main surface PS1 of the filter section 11 using a pipette and aspirating the cell suspension from the second main surface PS2 side. As operating conditions, aspiration is performed at a pressure of 0.5 kPa. Aspiration is stopped after visually confirming that approximately 0.8 ml of liquid has passed through the filter (through liquid). Evaluation is performed by measuring the time from the start of aspiration to the stop of aspiration (filtration time), the volume of through liquid, and the number of cells contained in the through liquid.

[0208] (4) Regarding the evaluation results

[0209] Table 2 shows the evaluation results.

[0210] [Table 2]

[0211]

[0212] In Examples 1-5, the cell (HL-60) recovery rate was 100%. In contrast, in Comparative Examples 1-3, the cell recovery rates were 29.3%, 35.7%, and 40.9%, respectively.

[0213] As shown in Table 1, in Comparative Examples 1-3, the major axes of the inscribed ellipse of the through-hole 12 are 9.0 μm, 6.6 μm, and 6.5 μm, respectively, which are larger than the size of the cell (HL-60) nucleus (3.66 μm). Therefore, it can be considered that the cell passed through the through-hole 12.

[0214] On the other hand, in Examples 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, it can be considered that cells can be reliably captured in Examples 1-5.

[0215] It should be noted that in this specification, "recovery rate" refers to the ratio of the number of active cells introduced to the number of cells captured on the first main surface PS1 of the filter, and is calculated as (number of active cells introduced - number of active cells contained in the fluid) / (number of active cells introduced).

[0216] It should be noted that the number of viable cells in the fluid was measured as follows: After the cells in the fluid were dispersed by pipetting, ten 10 μL samples of the fluid were taken using a micropipette, and the number of cells in the samples was measured using a cell counter (ThermoFisher, Countess II FL).

[0217] In this way, 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 deformable, while the cell nucleus is not easily deformable.

[0218] As in Comparative Examples 1-3, when the major axis of the inscribed ellipse of the through-hole 12 is larger than the size of the cell nucleus, even if the major axis of the inscribed ellipse of the through-hole 12 is smaller than the size of the cell as a whole, the cell may sometimes pass through the filter 10 due to deformation of the cytoplasm.

[0219] On the other hand, as in Examples 1-5, when the major axis of the inscribed ellipse of the through hole 12 is designed to be smaller than the cell nucleus, the cell nucleus is less deformable than the cytoplasm, and therefore, it is easier to capture the cell on the filter 10 compared to Comparative Examples 1-3.

[0220] Furthermore, the fact that filter 10 is made of metal also helps to improve cell recovery. By using a filter 10 formed of metal, the deformation of the through-holes 12 of filter 10 is less compared to resin-based filters such as membranes. Therefore, filter 10 is more likely to capture cells.

[0221] It should be noted that when filtering nucleated cells, it is self-evident that it is preferable to inhibit impurities from mixing into the cell suspension, that is, to inhibit the metal constituting filter 10 from dissolving into the cell suspension.

[0222] In Example 5, cells captured on the first main surface PS1 of the filter section 11a were observed after filtration. The results showed that, similar to the cells before filtration, the cells were round in shape and their activity was maintained. This is because fluid accumulates in the central side region R11 of the filter section 11a, and the cells are captured on the first main surface PS1 of the filter section 11a while immersed in the fluid.

[0223] For nickel, the immersion potential, an indicator of metal ionization tendency, was measured. The immersion potential was measured for 3 minutes in phosphate-buffered saline solution. The results showed that the immersion potential of nickel shifted within a range above 0.03 V, with a reference electrode formed of silver chloride immersed in a saturated potassium chloride solution as a reference. In other words, it can be said that, under the same conditions, any metal and / or metal oxide exhibiting an immersion potential at least above 0.03 V can be filtered without impairing cell activity.

[0224] Furthermore, Examples 1, 2, and 5 have shorter filtration times compared to Examples 3 and 4. This is because forming the through-hole 12 into a rectangular shape can suppress clogging caused by cytoplasmic deformation of the cells. Specifically, when the through-hole 12 is rectangular, even if part of the through-hole 12 is blocked due to cytoplasmic deformation, the liquid can easily pass through the other parts of the through-hole 12. Therefore, it can be considered that Examples 1, 2, and 5 have shorter filtration times compared to Examples 3 and 4.

[0225] It should be noted that, in the embodiments, the cell suspension contains 10 5 The example described is the filtration of cell suspensions with a high concentration of nucleated cells per mL or more to capture nucleated cells. However, even when filtering cell suspensions with a very low concentration of nucleated cells per mL, the filter 10 can capture nucleated cells that are to be recovered.

[0226] The preferred embodiments of the present invention have been fully described with reference to the accompanying drawings; however, various modifications and variations will be apparent to those skilled in the art. It should be understood that such modifications and variations are included in the present invention as long as they do not depart from the scope of the invention as defined in the appended claims.

[0227] Industrial availability

[0228] The filter of the present invention can improve the recovery rate of nucleated cells, and is therefore useful for the purpose of separating nucleated cells from cell suspensions. Attached Figure Description

[0230] Filters 10, 10A, 10B, 10C, and 10D

[0231] Filter sections 11, 11a, 11b, 11c, 11d

[0232] Through holes 12, 12a, 12b, 12c, 12d

[0233] 13 Support substrate

[0234] 13a Opening

[0235] 14 Filter base

[0236] 15. Frame

[0237] PS1 First Main Page

[0238] PS2 Second Main Page

[0239] R11 Central Side Area

[0240] R12 Middle Area

[0241] R13 Edge Side Region

[0242] T0, T1, T2, T3 film thickness

Claims

1. A filter, which is a filter for filtering nucleated cells, and has at least one of metal and metal oxide as the main component, and forms a plurality of through-holes, and the through-holes do not include squares, the arithmetic mean roughness of the surface of the filter is smaller than the size of the nucleus of the nucleated cell, the filter includes: a filtering portion having the plurality of through-holes, and the plurality of through-holes penetrate through a first main surface and a second main surface facing each other; and a frame portion disposed so as to surround the outer periphery of the filtering portion, a central side region far from the frame portion, an intermediate region located between the central side region and the frame portion, and an edge side region close to the frame portion are provided, and has a stepped film thickness distribution satisfying the relationship of T1 < T2 < T3 when the film thickness of the central side region is denoted as T1, the film thickness of the intermediate region is denoted as T2, and the film thickness of the edge side region is denoted as T3.

2. The filter according to claim 1, wherein, The immersion potential of at least one of the metal and the metal oxide in phosphate buffered saline is higher than 0.03V, and the immersion potential is based on a reference electrode formed of silver chloride immersed in a saturated potassium chloride solution.

3. The filter according to claim 1 or 2, wherein, At least one of the metal and the metal oxide contains at least one selected from gold, silver, copper, platinum, nickel, palladium, their alloys, and their oxides.

4. A filtering method, which is a method for filtering nucleated cells, and includes the following steps: a step of preparing a filter, the filter has at least one of metal and metal oxide as the main component, and forms a plurality of through-holes, the through-holes do not include squares, and the arithmetic mean roughness of the surface of the filter is smaller than the size of the nucleus of the nucleated cell; a step of passing a liquid containing the nucleated cells through the filter, in, the filter includes: a filtering portion having the plurality of through-holes, and the plurality of through-holes penetrate through a first main surface and a second main surface facing each other; and a frame portion disposed so as to surround the outer periphery of the filtering portion, the filter is provided with a central side region far from the frame portion, an intermediate region located between the central side region and the frame portion, and an edge side region close to the frame portion, and the filter has a stepped film thickness distribution satisfying the relationship of T1 < T2 < T3 when the film thickness of the central side region is denoted as T1, the film thickness of the intermediate region is denoted as T2, and the film thickness of the edge side region is denoted as T3.

5. The filtering method according to claim 4, wherein, The immersion potential of at least one of the metal and the metal oxide in phosphate buffered saline is higher than 0.03V, and the immersion potential is based on a reference electrode formed of silver chloride immersed in a saturated potassium chloride solution.

6. The filtering method according to claim 4 or 5, wherein, At least one of the metal and the metal oxide contains at least one selected from gold, silver, copper, platinum, nickel, palladium, their alloys, and their oxides.

7. The filtering method according to claim 4 or 5, wherein, The step of passing a liquid containing the nucleated cells through the filter includes a step of separating live cells from dead cells.

8. A kit, which is a kit containing a filter for filtering nucleated cells and for implementing the method according to any one of claims 4 to 7, The filter has at least one of metal and metal oxide as the main component, and has a plurality of through holes, and the through holes do not include squares, and the arithmetic mean roughness of the surface of the filter is smaller than the size of the nucleus of a nucleated cell. The filter includes: a filtering portion having the plurality of through holes, the plurality of through holes penetrating through a first main surface and a second main surface facing each other; and a frame portion disposed so as to surround the outer periphery of the filtering portion, The filter is provided with a central side region away from the frame portion, an intermediate region between the central side region and the frame portion, and an edge side region close to the frame portion. The filter has a stepped film thickness distribution that satisfies the relationship of T1 < T2 < T3 when the film thickness of the central side region is denoted as T1, the film thickness of the intermediate region is denoted as T2, and the film thickness of the edge side region is denoted as T3.

Images