Filter, cell membrane disruption device, and cell membrane disruption method

Abstract

A filter according to the present disclosure disrupts a cell membrane, the filter being equipped with a filter base part that has a first main surface having a projection part and a second main surface on the opposite side from the first main surface, and that defines a plurality of through-holes that allow the first main surface and the second main surface to communicate. The projection part is positioned between adjacent through-holes, and projects in a first direction away from the filter base part. In a cross-section cut in a second direction in which the through-holes are adjacent to each other, the projection part is positioned inside the end part of the filter base part positioned between the through-holes adjacent to each other, and the thickness of the projection part in the second direction decreases toward the top part of the projection part.

Description

Filter, cell membrane destruction device, and cell membrane destruction method

[0001] The present disclosure relates to a filter, a cell membrane disruption device, and a cell membrane disruption method.

[0002] For example, Patent Document 1 discloses a method for extracting intracellular substances, which comprises the steps of suspending cells in a solution prepared using benzalkonium chloride as a surfactant at a concentration ranging from 0.0005% to 0.01%, and mixing the cells with a liquid having an osmotic pressure different from that inside the cells.

[0003] Furthermore, Patent Document 2 discloses a method for forming a high-resistance (gigaohm) electric seal between a region of the cell membrane of a cell attached to a patch clamp electrode and the tip of the electrode, and then destroying the cell membrane at the sealed portion. The method described in Patent Document 1 includes the steps of (a) contacting a test cell with the tip of a patch clamp pipette, and (b) applying a negative pressure controlled by a pressure calibrator to the cell via the patch clamp pipette.

[0004] JP 2009-42 A JP 2004-294211 A

[0005] In recent years, there has been a demand for efficient destruction of cell membranes.

[0006] An object of the present disclosure is to provide a filter capable of efficiently destroying cell membranes, a cell membrane destruction device, and a cell membrane destruction method.

[0007] A filter according to one embodiment of the present invention is a filter for disrupting cell membranes, comprising a filter base having a first main surface with protrusions and a second main surface opposite the first main surface, the filter base defining a plurality of through holes connecting the first main surface and the second main surface, wherein the protrusions are located between adjacent through holes and protrude in a first direction away from the filter base, and in a cross section cut in a second direction in which the through holes are adjacent to each other, the protrusions are located more inward than the ends of the filter base located between the adjacent through holes, and the thickness of the protrusions in the second direction decreases toward the tops of the protrusions.

[0008] A cell membrane disruption device according to one aspect of the present invention includes the filter according to the above aspect.

[0009] A method for disrupting cell membranes according to one aspect of the present invention includes the steps of: preparing a filter having a filter base portion having a first main surface with protrusions and a second main surface opposite the first main surface, the filter base portion defining a plurality of through holes connecting the first main surface and the second main surface; and introducing cells containing cell membranes into the first main surface side of the filter base portion of the filter.

[0010] According to the present disclosure, it is possible to provide a filter capable of efficiently destroying cell membranes, a cell membrane destruction device, and a cell membrane destruction method.

[0011] FIG. 1 is a schematic plan view of an example of a filter according to a first embodiment of the present disclosure, as seen from the first main surface side. FIG. 2 is an enlarged plan view of a portion of the filter section. FIG. 3 is an enlarged perspective view of a portion of the filter section. FIG. 4 is a schematic cross-sectional view of a portion of the filter section. FIG. 5 is a schematic view of an example of a manufacturing process for a filter. FIG. 6 is a schematic view of an example of a manufacturing process for a filter. FIG. 7 is a schematic view of an example of a manufacturing process for a filter. FIG. 8 is a schematic view of an example of a manufacturing process for a filter. FIG. 9 is a schematic view of an example of a cell membrane disruption device according to the first embodiment of the present disclosure. FIG. 10 is a flowchart of an example of a cell membrane disruption method according to the first embodiment of the present disclosure. FIG. 11 is a schematic view of an example of a cell membrane disruption device according to a second embodiment of the present disclosure. FIG. 12 is a flowchart of an example of a cell membrane disruption method according to the second embodiment of the present disclosure. Fig. 1 is a schematic diagram showing a schematic cross section of a part of a filter of Modification 1. Fig. 2 is a schematic diagram showing a schematic cross section of a part of a filter of Modification 2. Fig. 3 is a flowchart of an example of a method for disrupting cell membranes of Modification 3. Fig. 4 is a schematic diagram showing a schematic cross section of a part of a filter of Modification 4.

[0012] Hereinafter, a first embodiment of the present invention will be described with reference to the accompanying drawings. In each drawing, elements are exaggerated for ease of explanation.

[0013] 1 is a schematic plan view of an example of a filter 1 according to a first embodiment of the present disclosure, viewed from a first main surface PS1 side. In the figure, the X, Y, and Z directions indicate the vertical, horizontal, and thickness directions of the filter 1, respectively.

[0014] As shown in Figure 1, the filter 1 breaks down the cell membrane by filtering cells containing the cell membrane, and extracts the cell contents by breaking down the cell membrane.

[0015] "Cells" refer to cells having a cell membrane, and include, for example, induced pluripotent stem cells (iPS cells), ES cells, stem cells, mesenchymal stem cells, mononuclear cells, single cells, cell clumps, floating cells, adhesive cells, nerve cells, leukocytes, cells for regenerative medicine, autologous cells, cancer cells, circulating cancer cells (CTCs), HL-60, HELA, eubacteria, fungi, archaea, and fungi such as yeast. Alternatively, "cells" include substances having a membrane containing lipids and proteins.

[0016] "Cellular contents" refers to materials present within the cell membrane, including, for example, the cytosol, nucleus, mitochondria, plastids, endoplasmic reticulum, Golgi apparatus, lysosomes, peroxisomes, endosomes, genetic elements including RNA, proteins, etc.

[0017] The material constituting the filter 1 may be, for example, primarily composed of at least one of metal, oxide, polymer, and ceramic. In the case of metal and metal oxide, the material constituting the filter 1 may be, for example, gold, silver, copper, platinum, nickel, palladium, titanium, aluminum, alloys thereof, and oxides thereof. In the case of polymer, the material constituting the filter 1 may be, for example, polyethylene or polypropylene. In particular, by using nickel-palladium alloy, titanium, aluminum, oxides thereof, or polyethylene, the amount of elution is reduced, thereby reducing the impact on cells and their contents. Furthermore, the reduced amount of elution reduces noise during the recovery and analysis of cell contents.

[0018] The filter 1 comprises a filter portion 10 and a frame portion 20 provided on the outer periphery of the filter portion 10. The filter portion 10 is provided with a plurality of through-holes 11 and breaks cell membranes to extract cellular contents. The frame portion 20 is provided on the outer periphery of the filter portion 10 and is used to attach the filter 1 to a container or device. Alternatively, the frame portion 20 is held with tweezers.

[0019] In the first embodiment, the filter section 10 and the frame section 20 are integrally formed.

[0020] <Filter section> Fig. 2 is an enlarged plan view of a portion of the filter section 10. Fig. 3 is an enlarged perspective view of a portion of the filter section 10. Fig. 4 is a schematic cross-sectional view of a portion of the filter section 10.

[0021] 2 to 4, the filter section 10 includes a filter base section 12 that defines a plurality of through holes 11. The filter base section 12 is also provided with protrusions 13.

[0022] The filter base 12 has a first main surface PS1 having protrusions 13, and a second main surface PS2 opposite the first main surface PS1. For example, the first main surface PS1 includes an uneven surface, and the uneven surface is formed by the protrusions 13. The second main surface PS2 includes a flat surface. The filter base 12 also defines a plurality of through holes 11 that communicate between the first main surface PS1 and the second main surface PS2.

[0023] The plurality of through holes 11 have a substantially circular shape when viewed from the first main surface PS1 side of the filter base portion 12, i.e., from the Z direction. The term "substantially circular shape" includes a perfect circle shape and an ellipse shape with an error of 10% or less between the length of the major axis and the length of the minor axis.

[0024] For example, the diameter of the through-holes 11 is appropriately designed depending on the type (size, morphology, properties, elasticity) or quantity of cells to be disrupted. The diameter of the through-holes 11 is, for example, 5 nm or more and 200 μm or less. Target cells ranging from small fungi with a size of approximately 5 nm to cell clumps (aggregates of cells) with a total size of approximately 200 μm can be processed. Furthermore, the diameter of the through-holes 11 is preferably 5 nm or more and 40 μm or less. Isolated cells can be efficiently processed. Furthermore, the diameter is more preferably 5 nm or more and 5 μm or less. Cell contents such as cell nuclei can easily pass through the through-holes 11, while undisrupted cells cannot pass through the through-holes 11, allowing efficient collection of cell contents from the cells. Furthermore, preferably, the through-holes 11 penetrate between both main surfaces but do not branch. Cell contents passing through the through-holes 11 do not remain within the through-holes, thereby increasing the recovery rate of cell contents. The diameter of the through hole 11 can be measured using an SEM and an image analysis application (Hakuto Co., Ltd., product name: Image-Pro (registered trademark)). For example, the outline of the hole is identified by analyzing an image of the hole observed with an SEM using an image analysis application (Hakuto Co., Ltd., product name: Image-Pro (registered trademark)). The outline of the hole may be identified automatically by the image analysis application, or may be identified manually by the user operating an input interface. Next, the center of gravity of the planar shape defined by the outline of the hole is calculated, and the long and short sides of the planar shape are calculated based on the center of gravity. The average value of the long and short sides of the planar shape is calculated as the diameter of the through hole 11.

[0025] For example, the interval between adjacent through holes 11 is more than 1 time and not more than 10 times the diameter of the through holes 11. Preferably, the interval between adjacent through holes 11 is not more than 3 times the diameter of the through holes 11. For example, the interval between adjacent through holes 11 is 10 nm or more and 600 μm or less. More preferably, the interval between adjacent through holes 11 is 10 nm or more and 120 μm or less. Even more preferably, the interval between adjacent through holes 11 is 10 nm or more and 15 μm or less. By adjusting the interval between adjacent through holes 11, the destruction area ratio can be adjusted.

[0026] The shape of the plurality of through-holes 11 is not limited to a substantially circular shape when viewed from the first main surface PS1 side. For example, the shape of the plurality of through-holes 11 may include a triangle, a rectangle, or a polygon when viewed from the first main surface PS1 side. The size and shape of the plurality of through-holes 11 are designed appropriately depending on the size, form, properties, elasticity, or amount of the cells or the substances within the cells.

[0027] The sizes and intervals of the multiple through holes 11 may be different or the same.

[0028] For example, the aperture ratio of the filter portion 10 is 10% or more, and preferably 25% or more. The aperture ratio is calculated by (area occupied by the through holes 11) / (projected area of ​​the first main surface PS1 when the through holes 11 are not formed).

[0029] Protrusions 13 are provided on the first main surface PS1 side of the filter base 12. The protrusions 13 are located between adjacent through holes 11. The protrusions 13 protrude in a first direction D1 in which the filter base 12 moves away from each other. The "first direction D1" may be parallel to the thickness direction (Z direction) of the filter 1, or may be inclined relative to the thickness direction.

[0030] The thickness of the protrusion 13 decreases toward the top 14 of the protrusion 13. In other words, the thickness of the protrusion 13 decreases in the first direction D1 as it moves away from the filter base 12. The thickness of the protrusion 13 is the dimension of the protrusion 13 in the second direction D2 in which the through holes 11 are adjacent to each other.

[0031] When viewed from the first main surface PS1 side of the filter base portion 12, the protrusion portion 13 surrounds one or more through holes 11. For example, the protrusion portion 13 has a cylindrical shape surrounding one or more through holes 11. For example, the protrusion portion 13 surrounds one to three through holes 11. Preferably, the protrusion portion 13 surrounds one through hole 11.

[0032] As shown in Figure 2, when viewed from the first main surface side of the filter base 12, the tops 14 of the protrusions 13 are curved and surround one or more through-holes 11. The tops 14 of the protrusions 13 are the tips of the protrusions 13 in the first direction D1. When viewed from the first main surface side of the filter base 12, the area of ​​a region R1 surrounded by the tops 14 of the protrusions 13 is larger than the opening area of ​​one or more through-holes 11. For example, the area of ​​the region R1 is greater than 1 time and not more than 36 / π times the opening area of ​​one or more through-holes 11. By having the tops 14 curved, the destruction area of ​​the cell membrane can be made larger than the area of ​​the protrusions 13, thereby delaying the repair of the cell membrane.

[0033] 4 , in a cross section of the filter 1 cut in the second direction D2, the protrusions 13 are located inside end portions 15 in the second direction D2 of the filter base portion 12 that are located between adjacent through holes 11. The protrusions 13 protrude in the first direction D1 inside end portions 15 in the second direction D2 of the filter base portion 12. Specifically, the apexes 14 of the protrusions 13 are located inside end portions 15 in the second direction D2 of the filter base portion 12.

[0034] The protrusions 13 have outer surfaces 16 that curve from a central region in the second direction D2 of the filter base 12 located between adjacent through holes 11 toward the end portions 15. The central region refers to a region extending from the center of the filter base 12 in the second direction D2 to within 40% of the dimension of the filter base 12 in the second direction D2. For example, the outer surface 16 is curved in a rounded shape. In a cross section of the filter 1 cut in the second direction D2, the radius of curvature of the outer surface 16 is appropriately designed depending on the thickness of the filter 1 (the distance between the first main surface PS1 and the second main surface PS2) or the diameter of the through holes 11. For example, the radius of curvature of the outer surface 16 is 1 nm or more and 200 μm or less.

[0035] 3 and 4 , the heights of the protrusions 13 vary. For example, the height of the protrusions 13 varies continuously. The height of the protrusions 13 refers to the distance from the second main surface PS2 of the filter base 12 to the tops 14 of the protrusions 13. For example, the height of the protrusions 13 is the distance from the tops 14 of the protrusions 13 to the second main surface PS2 of the filter base 12 in a direction perpendicular to the second main surface PS2.

[0036] For example, the height of protrusions 13 is 5 nm to 100 μm, which allows protrusions 13 to destroy the cell membrane and penetrate deeper into the cell. Preferably, the height of protrusions 13 is 5 nm to 1 μm, which allows the thickness of filter 1 to be thinned and reduces pressure loss to the fluid in filter 1. More preferably, the ratio of the diameter of protrusions 13 to the diameter of through-holes 11 is 1:1 to 0.2.

[0037] As shown in FIG. 4, attention will be focused on the first protrusion 13A provided on the first filter base 12A and the second protrusion 13B provided on the second filter base 12B.

[0038] The first filter base 12A and the second filter base 12B are parts of the filter base 12. The first filter base 12A is located between adjacent through holes 11. The second filter base 12B is a part different from the first filter base 12A and is located between adjacent through holes 11.

[0039] 4, the first filter base 12A and the second filter base 12B are adjacent to each other. However, the first filter base 12A and the second filter base 12B do not have to be adjacent to each other. The first filter base 12A and the second filter base 12B only need to be provided at different positions on the filter base 12.

[0040] The first protrusion 13A and the second protrusion 13B are part of the protrusion 13. The first protrusion 13A protrudes from the first main surface PS1 of the first filter base 12A. The second protrusion 13B protrudes from the first main surface PS1 of the second filter base 12B.

[0041] The height H1 of the first protrusion 13A is different from the height H2 of the second protrusion 13B. In the example shown in Fig. 4, the height H1 of the first protrusion 13A is greater than the height H2 of the second protrusion 13B.

[0042] As shown in Fig. 3, the protrusion 13 has a pointed apex 14a. The pointed apex 14a has a shape in which the tip of the protrusion 13 is tapered. The pointed apex 14a is provided on at least a portion of the apex 14 of the protrusion 13. In the apex 14 of the protrusion 13, the apex 14b other than the pointed apex 14a may be pointed or may not be pointed. The other apex 14b may be rounded.

[0043] When viewed from the first main surface PS1 side of the filter base portion 12, a region R2 is defined by connecting three imaginary straight lines L1, L2, and L3 that are tangent to the outer peripheries of two adjacent through holes 11A, 11B, and 11C, and the region R2 includes the three through holes 11A, 11B, and 11C. The pointed apex 14a is located within the region R2.

[0044] For example, as shown in Figure 2, when viewed from the first main surface PS1 side of the filter base portion 12, a first imaginary line L1, a second imaginary line L2 and a third imaginary line L3 are drawn that are tangent to the outer circumferences of the adjacent first through hole 11A, second through hole 11B and third through hole 11C.

[0045] The first imaginary line L1 is tangent to the outer periphery of the first through hole 11A and the outer periphery of the second through hole 11B. The second imaginary line L2 is tangent to the outer periphery of the second through hole 11B and the outer periphery of the third through hole 11C. The third imaginary line L3 is tangent to the outer periphery of the first through hole 11A and the outer periphery of the third through hole 11C.

[0046] When viewed from the first main surface PS1 side of the filter base portion 12, the region R2 is an area surrounded by the first imaginary line L1, the second imaginary line L2, and the third imaginary line L3 and has a triangular shape. The first through hole 11A, the second through hole 11B, and the third through hole 11C are located within the region R2.

[0047] For example, when viewed from the first main surface PS1 side of the filter base portion 12, the pointed apex 14a is located in the center of the region R2.

[0048] The height of the protrusion 13 at the pointed apex 14a is greater than the height of the protrusion 13 at the other apex 14b. For example, the height of the protrusion 13 at the pointed apex 14a is greater than one time and less than two times the height of the protrusion 13 at the other apex 14b.

[0049] 1 , the frame portion 20 is provided on the outer periphery of the filter portion 10, and is a portion having a smaller number of through holes 11 per unit area than the filter portion 10. The number of through holes 11 in the frame portion 20 is 25% or less of the number of through holes 11 in the filter portion 10.

[0050] The frame portion 20 is formed in a ring shape when viewed from the first main surface PS1 side of the filter portion 10. When the filter 10 is viewed from the first main surface PS1 side, the center of the frame portion 20 coincides with the center of the filter portion 10. In other words, the frame portion 20 is formed concentrically with the filter 1.

[0051] 5A to 5G are schematic diagrams showing an example of a manufacturing process for the filter 1.

[0052] 5A, electrolytic polishing is performed on the aluminum plate 50. As a result, the surface of the aluminum plate 50 becomes flat.

[0053] As shown in Fig. 5B, an anodization process is performed on the aluminum plate 50. For example, the aluminum plate 50 is subjected to an electrolytic process using an anode. As a result, a first oxide film 51 is formed on the surface layer of the aluminum plate 50. For example, the outer surface of the first oxide film 51 has an irregular shape.

[0054] 5C, the first oxide film 51 is removed from the aluminum plate 50. For example, the first oxide film 51 is removed from the aluminum plate 50 by performing an etching process. As a result, a plurality of dents 52 are formed in the portions of the aluminum plate 50 from which the first oxide film 51 has been removed. For example, the plurality of dents 52 have a round shape.

[0055] 5D , an anodization process is performed on the aluminum plate 50 having the plurality of recesses 52 formed therein. As a result, a second oxide film 53 is formed on the side of the aluminum plate 50 where the plurality of recesses 52 is provided. A plurality of holes 54 extending toward the bottoms of the plurality of recesses 52 are formed in the second oxide film 53.

[0056] 5E and 5F , the aluminum plate 50 is peeled off from the second oxide film 53. For example, the aluminum plate 50 is peeled off from the second oxide film 53 by anodic electrolytic stripping using the aluminum plate 50 as the anode and the cathode plate 55 as the cathode.

[0057] In addition, in stripping the aluminum plate 50, cathodic electrolytic stripping may be performed using the aluminum plate 50 as the cathode.

[0058] 5G, the bottom of the second oxide film 53 is etched to form a filter base 12 that opens a plurality of holes 54 and defines a plurality of through-holes 11. Furthermore, etching is performed to enlarge the plurality of holes 54, thereby forming through-holes 11 with a desired pore diameter.

[0059] In this manner, the filter 1 can be produced.

[0060] [Cell Membrane Disruption Device] The cell membrane disruption device will be described with reference to Fig. 6. Fig. 6 is a schematic diagram of an example of the cell membrane disruption device 2 according to the first embodiment of the present disclosure.

[0061] As shown in FIG. 6, the cell membrane disruption device 2 includes a filter 1 and a container 30 in which the filter 1 is attached.

[0062] The container 30 has an opening. The filter 1 is attached to the opening of the container 30. For example, the container 30 is a centrifuge tube, a test tube, a microtube, a flask, or a Petri dish.

[0063] [Cell Membrane Disruption Method] The cell membrane disruption method will be described with reference to Figure 7 and Figures 8A to 8C. Figure 7 is a flowchart of an example of the cell membrane disruption method according to the first embodiment of the present disclosure. Figures 8A to 8C are schematic diagrams showing an example of steps of the cell membrane disruption method according to the first embodiment of the present disclosure.

[0064] As shown in FIG. 7, in step ST10, a filter 1 is prepared.

[0065] In step ST20, cells containing cell membranes are introduced into the filter 1. For example, a cell suspension containing a plurality of cells is introduced into the first main surface PS1 side of the filter substrate 12 of the filter 1.

[0066] 8A , the cell 3 has a plurality of cellular contents 5 within the cell membrane 4. In step ST20, the cell 3 having the cell membrane 4 is introduced onto the first main surface PS1 side of the filter base portion 12 of the filter 1.

[0067] Returning to Figure 7, in step ST30, the cell membrane 4 is destroyed by the protrusions 13 of the filter 1. As shown in Figure 8B, the cells 3 introduced onto the first main surface PS1 side of the filter base 12 fall vertically downward due to gravity. The first main surface PS1 of the filter base 12 is provided with protrusions 13. As a result, the cell membrane 4 of the cells 3 is pierced by the tops 14 of the protrusions 13 and destroyed. This causes the intracellular contents 5 to be expelled from the cell membrane 4.

[0068] 8C , the plurality of through-holes 11 in the filter 1 are formed to a size that allows the passage of the intracellular contents 5. Therefore, the intracellular contents 5 discharged to the outside of the cell membrane 4 pass through the through-holes 11. Meanwhile, the cell membrane 4 is captured by the filter 1.

[0069] In this way, the filter 1 can be used to disrupt the cell membrane 4 of the cell 3 .

[0070] [Effects] The filter 1 according to the first embodiment can provide the following effects.

[0071] The filter 1 is a filter that disrupts cell membranes 4 and includes a filter base 12. The filter base 12 has a first main surface PS1 with protrusions 13 and a second main surface PS2 opposite the first main surface PS1, and defines a plurality of through-holes 11 that communicate with the first main surface PS1 and the second main surface PS2. The protrusions 13 are located between adjacent through-holes 11 and protrude in a first direction D1 away from the filter base 12. In a cross section cut in a second direction D2 in which the through-holes 11 are adjacent to each other, the protrusions 13 are located inside an end 15 of the filter base 12 that is located between adjacent through-holes 11. The thickness of the protrusions 13 in the second direction D2 decreases toward the tops 14 of the protrusions 13. This makes the circumference of the protrusions 13 approximately equal to the circumference of the through-holes 11, improving the disruption rate.

[0072] This configuration allows efficient destruction of the cell membrane 4. According to the filter 1, the cell membrane 4 can be destroyed by the protrusions 13 of the filter 1. This allows the cell membrane 4 to be destroyed in a relatively short time. Furthermore, the protrusions 13 can be used to puncture and destroy a single cell at multiple locations almost simultaneously. This allows the cell membrane to be destroyed in an even shorter time and the cell contents to be extracted from multiple locations. Furthermore, a relatively large number of cells 3 can be processed at once. Furthermore, by recovering the cell contents through the through-holes 11 after destroying the cell membrane 4, post-destruction processing is either unnecessary or simplified. Furthermore, by destroying most of the introduced cells in a short time, clogging of the filter 1 can be reduced, and pressure loss relative to the fluid in the filter 1 can be reduced. Furthermore, reduced pressure loss, etc., reduces heat generation, thereby reducing deactivation of the cell contents (e.g., cell contents containing proteins).

[0073] In the present disclosure, efficient destruction of cell membranes 4 may be determined based on the following: (a) destruction rate: The number of cells destroyed by this filter relative to the total number of cells input. (b) destruction rate: The number of cells destroyed by this filter per unit time. (c) destruction area rate: The ratio of the voids opened per cell by this filter to the surface area of ​​the cell. (d) destruction yield rate: The ratio of the number of cells destroyed by this filter to the activity of the biological material obtained by the destruction. Common means of expressing the activity of a biological material include mass, concentration, potential, and fluorescence spectrum. (e) destructible range: The range of cell diameters that can be destroyed by this filter.

[0074] In the second direction D2, the protrusions 13 have outer surfaces 16 that curve from the central region of the filter base 12 located between adjacent through-holes 11 toward the end 15. This configuration makes it easier to destroy the cell membrane 4. In addition, the intracellular contents 5 discharged from the cell membrane 4 can more easily flow into the through-holes 11, reducing pressure loss of the fluid in the filter 1.

[0075] The protrusion 13 has, on at least a part of the outer surface 16 of the protrusion 13, a flat inclined surface 16a that is inclined toward the through-hole 11. With this configuration, the intracellular contents 5 discharged outside the cell membrane 4 can more easily flow into the through-hole 11.

[0076] The filter base 12 includes a first filter base 12A located between adjacent through holes 11 and a second filter base 12B, a different portion from the first filter base 12A and located between adjacent through holes 11. The protrusions 13 include a first protrusion 13A protruding from a first main surface PS1 of the first filter base 12A and a second protrusion 13B protruding from a second main surface PS2 of the second filter base 12B. If the distance from the second main surface PS2 of the filter base 12 to the top 14 of the protrusion 13 is defined as the height of the protrusion 13, the height H1 of the first protrusion 13A and the height H2 of the second protrusion 13B are different. This configuration enables more efficient destruction of cell membranes 4. For example, if the size of the cells 3 introduced into the filter 1 varies, larger cells will be more likely to be destroyed by the taller protrusions 13, and smaller cells will be more likely to be destroyed by the shorter protrusions 13. This increases the surface area of ​​the filter 1 used for destruction, shortening the processing time and increasing the destruction speed. It also allows more cells to be processed at once. Furthermore, the destruction area can be expanded, allowing cells 3 with cell membranes 4 of various types and sizes to be destroyed.

[0077] When viewed from the first main surface PS1 side of the filter base portion 12, the protrusions 13 surround one or more through-holes 11. With this configuration, the cell membranes of the cells are destroyed before they reach the through-holes 11, so that the cell membranes 4 can be destroyed more efficiently.

[0078] The height of the protrusions 13 changes continuously. This configuration makes it easier to destroy the cell membrane 4, and allows for more efficient destruction of the cell membrane 4. After the tops 14 of the protrusions 13 puncture the cell membrane, the holes created in the cell membrane are gradually widened, allowing the cell membrane to be destroyed with little force and making it easier to extract the cell contents. In addition, the pressure loss of the filter 1 relative to the fluid can be reduced.

[0079] When viewed from the first main surface PS1 side of the filter base portion 12, the area of ​​the region R1 surrounded by the tops 14 of the protrusions 13 is larger than the opening area of ​​one or more through-holes 11. This configuration makes it possible to more efficiently destroy the cell membranes 4. Furthermore, with this configuration, when viewed from the first main surface PS1 side, the protrusions 13 do not block the through-holes 11, so no pressure loss occurs in the filter.

[0080] The protrusion 13 has a sharp tip 14a. This configuration makes it easier to destroy the cell membrane 4, and the cell membrane 4 can be destroyed more efficiently.

[0081] When viewed from the first main surface PS1 side of the filter base portion 12, the sharp apex 14a is defined by connecting three imaginary lines L1, L2, and L3 that tangent to the outer circumferences of two adjacent through holes 11A, 11B, and 11C. When a region R2 is drawn that includes the three through holes 11A, 11B, and 11C, the sharp apex 14a is located within region R2. This configuration makes it easier to destroy the cell membrane 4, and more efficiently destroys the cell membrane 4. Furthermore, because the distance to the through holes 11A, 11B, and 11C through which the cellular contents pass is short, the cellular contents can pass through the through holes 11A, 11B, and 11C more easily, thereby increasing the recovery rate of the cellular contents.

[0082] The height of the protrusion 13 at the sharp tip 14a is greater than the height of the protrusion 13 at the other tip 14b. This configuration makes it easier to destroy the cell membrane 4, and allows for more efficient destruction of the cell membrane 4. This is because, after puncturing with the tip 14 of the protrusion 13, the hole created in the cell membrane is gradually widened, allowing the cell membrane to be destroyed with little force and making it easier to extract the cell contents. In addition, pressure loss of the filter 1 relative to the fluid can be reduced.

[0083] The interval between adjacent through holes 11 in the filter 1 is more than 1 time and not more than 10 times the diameter of the through holes 11. Preferably, the interval between adjacent through holes 11 is not more than 3 times the diameter of the through holes 11. For example, the interval between adjacent through holes 11 is 10 nm or more and 600 μm or less. More preferably, the interval between adjacent through holes 11 is 10 nm or more and 120 μm or less. Even more preferably, the interval between adjacent through holes 11 is 10 nm or more and 15 μm or less. With this configuration, the aperture ratio can be increased by reducing the interval between the through holes 11, thereby improving liquid permeability.

[0084] When viewed from the first main surface PS1 side of the filter base portion 12, the protrusion portion 13 surrounds one or more through holes 11. For example, the protrusion portion 13 has a cylindrical shape surrounding one or more through holes 11. For example, the protrusion portion 13 surrounds one to three through holes 11. Preferably, the protrusion portion 13 surrounds one through hole 11. With such a configuration, the strength of the filter base portion 12 in the tensile direction can be increased.

[0085] The cell membrane 4 disruption device 2 includes the filter 1 of the above-described embodiment. Therefore, the disruption device 2 also has the same effect as the filter 1.

[0086] The method for destroying cell membranes 4 includes step ST10 of preparing a filter 1, step ST20 of introducing cells 3 containing cell membranes 4 onto the first main surface PS1 side of the filter base portion 12 of the filter 1, and step ST30 of destroying the cell membranes 4 with the protrusions 13 of the filter 1. This configuration allows the cell membranes 4 to be destroyed efficiently. Therefore, the cell membranes 4 can be destroyed in a relatively short time. Furthermore, a relatively large number of cells can be processed at one time. Furthermore, processing after the destruction of the cell membranes 4 is either unnecessary or simplified.

[0087] In the present embodiment, an example in which the filter 1 includes the frame portion 20 has been described, but the present invention is not limited to this. For example, the filter 1 does not need to include the frame portion 20. The entire filter 1 may be composed of the filter portion 10.

[0088] In the present embodiment, an example has been described in which the protrusions 13 are provided on the first main surface PS1 of the filter base 12. However, the present invention is not limited to this. For example, the protrusions 13 may be provided on the second main surface PS2 of the filter base 12.

[0089] In this embodiment, an example has been described in which only the protrusions 13 are used to destroy the cell membrane 4, but the apex 14b formed by providing the protrusions 13 may also be used to destroy the cell membrane 4. With this configuration, the diameter of the hole formed by puncturing becomes larger, and the time required for repairing the cell membrane can be extended.

[0090] In the present embodiment, an example has been described in which the through holes 11 are substantially circular and arranged at different intervals, but this is not limiting. For example, the through holes 11 may have a square shape. In this case, the through holes 11 may be arranged along two arrangement directions parallel to each side of the square when viewed from the first main surface PS1 side (Z direction) of the filter 1. In this way, by arranging the through holes 11 in a square lattice arrangement, it is possible to increase the aperture ratio and improve the liquid permeability of the filter 1.

[0091] The arrangement of the through holes 11 is not limited to a square lattice arrangement, and may be, for example, a quasi-periodic arrangement or a periodic arrangement. Examples of the periodic arrangement include a rectangular arrangement in which the intervals in the two arrangement directions are not equal, a triangular lattice arrangement, or a regular triangular lattice arrangement, as long as the arrangement is a square arrangement.

[0092] (Embodiment 2) A cell membrane disruption device and a cell membrane disruption method according to embodiment 2 of the present disclosure will be described. Note that in embodiment 2, components that are the same as or equivalent to those in embodiment 1 will be denoted by the same reference numerals, and descriptions that overlap with embodiment 1 will be omitted.

[0093] FIG. 9 is a schematic diagram of an example of a cell membrane 4 disruption device 2A according to the second embodiment of the present disclosure.

[0094] The second embodiment differs from the first embodiment in that the destruction device 2A includes a suction device 40 .

[0095] As shown in FIG. 9, the destruction device 2A includes a suction device 40.

[0096] The suction device 40 sucks the cells 3 including the cell membranes 4 located on the first main surface PS1 side of the filter base portion 12 to the second main surface PS2 side through the plurality of through-holes 11.

[0097] The suction device 40 is connected to the container 30 downstream of the filter 1. The suction device 40 suctions gas from within the container 30. By suctioning the gas from within the container 30, the suction device 40 suctions cells 3 in a direction from the first main surface PS1 toward the second main surface PS2 of the filter base portion 12 through the multiple through-holes 11 of the filter 1. For example, the suction device 40 is a syringe.

[0098] FIG. 10 is a flowchart illustrating an example of a method for disrupting the cell membrane 4 according to the second embodiment of the present disclosure.

[0099] In this embodiment, step ST30 of destroying the cell membrane 4 includes step ST31 of sucking the cell 3.

[0100] In step ST31, the suction device 40 sucks the cells 3 located on the first main surface PS1 side of the filter base part 12 through the plurality of through-holes 11 to the second main surface PS2 side.

[0101] In this embodiment, the suction device 40 suctions gas from the container 30 downstream of the filter 1. This causes the cells 3 to be sucked in the direction from the first main surface PS1 to the second main surface PS2 of the filter base portion 12.

[0102] [Effects] According to the cell membrane 4 disruption device 2A according to the second embodiment, the following effects can be achieved.

[0103] The disruption device 2A includes a suction device 40 that sucks cells 3, including cell membranes 4, located on the first main surface PS1 side of the filter base portion 12 toward the second main surface PS2 side through the multiple through-holes 11. This configuration makes it easier to disrupt the cell membranes 4. For example, by sucking the cells 3, the cell membranes 4 are pulled in the suction direction, making them more likely to be pierced by the protrusions 13. This allows the cell membranes 4 to be disrupted more efficiently.

[0104] The disruption device 2A is located downstream of the filter 1 and sucks gas from the container 30 to which the filter 1 is attached. With this configuration, the cell membranes 4 can be disrupted more efficiently.

[0105] In the method for destroying the cell membrane 4, step ST30 of destroying the cell membrane 4 includes step ST31 of sucking the cells 3 located on the first main surface PS1 side of the filter base part 12 to the second main surface PS2 side through the plurality of through-holes 11. With this configuration, the cell membrane 4 is more easily destroyed, and the cell membrane 4 can be destroyed more efficiently.

[0106] In the method for disrupting cell membranes 4, the preparation step ST10 includes preparing a filter 1 attached to a container 30. The suction step ST31 includes suctioning gas from within the container 30 downstream of the filter 1. This configuration allows the cell membranes 4 to be disrupted more efficiently.

[0107] In the present embodiment, the suction device 40 is a syringe, but the present invention is not limited to this. The suction device 40 may be any device capable of suction. For example, the suction device 40 may be a blower, an aspirator, or a pump.

[0108] The following describes modified examples.

[0109] [Variation 1] Fig. 11 is a schematic diagram showing a cross section of a portion of a filter of Variation 1. As shown in Fig. 11, the apex 14A of the protrusion 13A in Variation 1 need only have a shape in which the thickness of the protrusion 13 decreases toward the apex 14A, and does not have to be pointed. For example, the apex 14A of the protrusion 13A may be substantially trapezoidal or semicircular. Even with this configuration, the cell membrane 4 can be efficiently destroyed.

[0110] [Modification 2] Fig. 12 is a schematic diagram showing a cross section of a portion of a filter of Modification 2. As shown in Fig. 12, the protrusions 13B in Modification 2 may have flat inclined surfaces 16a that slope toward the through-holes 11 on at least a portion of the outer surface 16 of the protrusions 13B. This may form a taper on the protrusions 13B. With this configuration, the intracellular contents 5 discharged outside the cell membrane 4 gradually move toward the through-holes 11, thereby suppressing damage to the intracellular contents 5.

[0111] 13 is a flowchart of an example of a method for disrupting a cell membrane 4 according to Modification 3. As shown in FIG. 13, the method for disrupting a cell membrane 4 according to Modification 3 includes step ST40 of recovering the intracellular content 5 that has passed through the filter 1. For example, the intracellular content 5 that has passed through the filter 1 can be recovered using a container 30 disposed below the filter 1. This configuration allows the intracellular content 5 within the cell membrane 4 to be easily recovered.

[0112] The collected cell contents 5 can be used for analysis or measurement, such as genetic analysis, cancer testing, and gene concentration measurement. Genetic analysis may involve decoding gene sequences using a sequencer. Cancer testing may involve exosome analysis. Gene concentration measurement may involve obtaining information used for calibrating real-time PCR.

[0113] The method for disrupting the cell membrane 4 in variant 3 may include a step of analyzing the recovered cell contents 5.

[0114] The method for disrupting the cell membrane 4 may include a step of collecting the cell membrane 4 remaining on the first main surface PS1 of the filter 1. The method for disrupting the cell membrane 4 may also include a step of analyzing the cell membrane 4 remaining on the first main surface PS1 of the filter 1.

[0115] Furthermore, the cell membrane 4 disruption device 2 may include, for example, a vibration device that vibrates the filter 1. Vibrating the filter 1 may reduce friction at the contact point between the cell membrane 4 and the sharp apex 14a, thereby disrupting the cell membrane 4 passing through the through-hole 11.

[0116] The vibration source of the vibration device provided in the destruction device 2 may include vibrations due to rotation, impact, or ultrasonic waves. The frequency of the vibration source can be selected from a range of 1 kHz to 100 kHz, which can enhance the destruction effect of the sharp apex 14a.

[0117] Furthermore, the vibration device provided in the disruption device 2 may be provided at a location within the device away from the filter 1, as long as the vibration is transmitted to the filter 1. For example, if no vibration-isolating material is provided between the filter 1 and the culture vessel to block vibration of the filter 1, the vibration device may be configured to vibrate the culture vessel.

[0118] [Modification 4] Fig. 14 is a schematic diagram showing a cross section of a portion of a filter of Modification 4. As shown in Fig. 14, in Modification 4, the protrusion 13 has a side protrusion 17 that protrudes from the outer surface 16. The side protrusion 17 may protrude in a direction different from the protrusion direction of the protrusion 13. For example, the side protrusion 17 may protrude in a direction intersecting the protrusion direction of the protrusion 13.

[0119] The side protrusions 17 may be sharp or rounded.

[0120] Side protrusions 17 can capture cells whose cell contents have decreased due to the cell membrane being destroyed by protrusions 13, as well as destroyed cell membranes, and prevent them from re-floating. Furthermore, because the cell membranes destroyed by filter 1 are held by side protrusions 17, information about the cell membranes can be obtained by directly analyzing the cell membranes held by filter 1 with a microscope or the like.

[0121] In Example 1, an experiment was carried out to disrupt the cell membrane 4 using the filter 1 of the first embodiment. 100 ml of a suspension of cells (HL-60) and culture medium in a culture vessel was prepared, 10 μl of the cell suspension was taken out with a micropipette, and the cell concentration was measured using a ThermoFisher Countess (AMQAF2000) and found to be 6×10 ５ The average cell size was 14 μm. 1 ml of the culture vessel was extracted with a micropipette and used as a sample (total number of cells: 6 × 10 ５ pieces).

[0122] The filter 1 was made of aluminum oxide. When viewed from the main surface direction, the filter 1 had a substantially circular outer shape. The diameter of the filter 1 was measured at eight locations with a vernier caliper, and the average diameter was 13.2 mm. The thickness of the filter 1 was measured at eight locations with a vernier caliper, and the average thickness was 1.9 mm. The vicinity of the through holes 11 from the main surface direction of the filter 1 was observed with a scanning electron microscope (SEM). The hole sizes of the eight through holes 11 were measured with the SEM, and the average hole size was 728 nm. When the vicinity of the through holes 11 was observed with the SEM from a direction tilted 30 degrees from the first main surface PS1, protrusions 13 were found to surround the through holes 11, and multiple pointed shapes were observed at the tops 14 of the protrusions 13.

[0123] Filter 1 was placed in the inlet of a newly prepared 15 ml centrifuge tube, and 1 ml of sample was dropped onto the first main surface PS1 of filter 1. The sample was held on the first main surface PS1 of filter 1. Next, a syringe connected to the 15 ml centrifuge tube was used to create negative pressure inside the 15 ml centrifuge tube. As a result, liquid passed from the first main surface PS1 of filter 1 to the second main surface PS2 through the multiple through-holes 11 into the 15 ml centrifuge tube. The syringe operation took 2 seconds, and liquid passage was confirmed within approximately 2 seconds.

[0124] The filter 1 was removed, and the first main surface PS1 of the filter 1 was observed under a microscope. As a result, a plurality of unbroken cells 3, parts of broken cell membranes 4, and spherical cell nuclei were observed, and it was found that the cell membranes 4 of many cells 3 had been broken by the filter 1. The number of unbroken cells was counted under the microscope, and it was found that 100 μm ２ This corresponds to an area of ​​the first main surface PS1 of the filter 1 of 0.81 × 10 ５ That is, the cell membranes 4 of 86.5% of the cells 3 contained in the sample were destroyed.

[0125] A mixed solution of 25 nm fluorescent dye Mito Tracker Red FM and 1.5% DMSO was added to the liquid in a 15 ml centrifuge tube, and after leaving it at 37°C for 30 minutes, it was observed under a fluorescence microscope. As a result, multiple red spherical objects were observed, indicating that mitochondria, one of the intracellular contents 5, had been extracted. Furthermore, the size of the mitochondria was smaller than the pore size of filter 1 (average 728 nm). In other words, intracellular contents 5 had been extracted.

[0126] Example 2 In Example 2, an experiment was carried out in which the cell membrane 4 was destroyed using the filter 1 of the first embodiment, and the cell contents 5 were extracted and analyzed. The procedure will be described below.

[0127] (1) The E. coli culture solution was stirred for 5 seconds using a vortex mixer to adjust the culture solution to a uniform dispersion state. (2) 1 ml of the E. coli culture solution stirred in (1) was dispensed into a centrifuge tube using a micropipette. (3) The E. coli culture solution was introduced into the filter 1 of embodiment 1. At this time, the tip of the micropipette was moved to drop the liquid so that the entire main surface of the filter 1 was evenly wetted with the liquid. (4) The filter 1 was left to stand for 3 minutes until the liquid on the first main surface PS1 had completely passed through. (5) The filter 1 was removed from the container 30. (6) The liquid containing the cellular contents 5 and the cell membranes 4 that had passed through the filter 1 were analyzed.

[0128] In Example 2, when the liquid containing the intracellular contents 5 was observed under a microscope, the nuclei were not aggregated but were present in an isolated state. Because the intracellular contents 5 passed through the multiple through-holes 11 of the filter 1 in a divided manner, the intracellular contents 5 were less likely to aggregate.

[0129] In Example 2, when the liquid containing the intracellular contents 5 was observed under a fluorescence microscope, intracellular organelles were extracted that retained the organelle contact sites that had been fluorescently modified in advance. The intracellular contents 5 within the cell membrane 4 could be extracted without any change in chemical composition.

[0130] In Example 2, the first main surface PS1 of the filter 1 removed from the container 30 was observed under a microscope, subjected to X-ray structural analysis, and subjected to infrared spectroscopy, and membrane fragments that retained the mosaic structure or molecular arrangement of the cell membrane 4 were confirmed. The cell membrane 4 was able to be retained on the first main surface PS1 of the filter 1.

[0131] In Example 2, the first main surface PS1 of the filter 1 removed from the container 30 was subjected to X-ray structural analysis, and the three-dimensional structure of the cell membrane protein was analyzed. The cell membrane 4 was able to be retained on the first main surface PS1 of the filter 1.

[0132] In Example 2, lipid rafts were confirmed by X-ray structural analysis of the first main surface PS1 of the filter 1 removed from the container 30. Because the cell membrane 4 can be held on the first main surface PS1 of the filter 1 while protecting the intracellular environment (such as pH or the charge state used for signal transduction), analysis was possible without destroying the lipid rafts.

[0133] Example 3 In Example 3, an experiment was conducted to destroy the cell membrane 4 using a destruction device 2 equipped with a vibration device that vibrates the filter 1. The procedure will be described below.

[0134] (1) A 100 ml suspension of suspended cells (HL-60) and medium was prepared, and the cell count was measured using a ThermoFisher Countess (AMQAF2000). (2) (1) was poured onto filter 1, which was being vibrated by a vibrator. (3) The number of cells in the cell contents 5 that had passed through filter 1 was measured using a ThermoFisher Countess (AMQAF2000). (4) The cell disruption efficiency was calculated from the number of cells before and after passing through filter 1.

[0135] In Example 3, the total cell count and disruption efficiency were compared with those in Example 2, which does not disrupt cells using vibration. While the efficiency was 60-80% (standard error rate 14.8%) in Example 2, it was improved to 65-95% (standard error rate 5.5%) in Example 3.

[0136] In Example 3, a container 31 with a filter 1 at the bottom was prepared, and when culture medium and adherent cells (VERO) were added and cultured, the adherent cells were able to be retained on the first main surface PS1 of the filter 1. When the container 31 was vibrated, the contents of the destroyed adherent cells flowed out onto the second main surface PS2 of the filter 1, and the membrane proteins that make up the cell membrane could be observed from the cell membrane 4 remaining on PS1 without destroying the arrangement of the cells.

[0137] It should be noted that, in this specification, terms such as "first," "second," etc. are used for descriptive purposes only and should not be understood as expressing or implying relative importance or ranking of technical features. Features qualified as "first" and "second" expressly or imply the inclusion of one or more of such features.

[0138] Although the present invention has been fully described in connection with the preferred embodiments with reference to the accompanying drawings, various changes and modifications will be apparent to those skilled in the art, and it is to be understood that such changes and modifications are included within the scope of the present invention as defined by the appended claims unless they depart therefrom.

[0139] (Summary of embodiments) (1) The filter of the present disclosure is a filter that destroys cell membranes, and includes a filter base portion having a first main surface with protrusions and a second main surface opposite the first main surface, and defining a plurality of through-holes that communicate with the first main surface and the second main surface, wherein the protrusions are located between adjacent through-holes and protrude in a first direction away from the filter base portion, and in a cross section cut in a second direction in which the through-holes are adjacent to each other, the protrusions are located more inward than the ends of the filter base portion that are located between the adjacent through-holes, and the thickness of the protrusions in the second direction decreases toward the tops of the protrusions.

[0140] (2) In the filter of (1), in the second direction, the protrusion may have an outer surface that curves from a central region of the filter base portion located between adjacent through holes toward the end portion.

[0141] (3) In the filter of (2), the protrusion may have a flat inclined surface inclined toward the through hole on at least a part of the outer surface of the protrusion.

[0142] (4) In any one of the filters (1) to (3), the filter base may include a first filter base located between adjacent through holes and a second filter base that is a different part from the first filter base and is located between adjacent through holes, and the protrusions may include a first protrusion protruding from the first main surface side of the first filter base and a second protrusion protruding from the second main surface of the second filter base, and when the distance from the second main surface of the filter base to the top of the protrusion is defined as the height of the protrusion, the height of the first protrusion and the height of the second protrusion may be different.

[0143] (5) In the filter of any one of (1) to (4), the protrusion may surround one or more through holes when viewed from the first main surface side of the filter base.

[0144] (6) In any one of the filters (1) to (5), when the distance from the second main surface of the filter base to the top of the protrusion is defined as the height of the protrusion, the height of the protrusion may change continuously.

[0145] (7) In the filter of (5), the area of ​​the region surrounded by the tops of the protrusions when viewed from the first main surface side of the filter base may be larger than the opening area of ​​the one or more through holes.

[0146] (8) In the filter of any one of (1) to (7), the protrusion may have a pointed top.

[0147] (9) In the filter of (8), when viewed from the first main surface side of the filter base portion, the area is defined by connecting three imaginary straight lines tangent to the outer circumferences of two adjacent through holes among three adjacent through holes, and when an area including the three through holes is drawn, the pointed apex may be located within the area.

[0148] (10) In the filter of (8), when the distance from the second main surface of the filter base to the top of the protrusion is defined as the height of the protrusion, the height of the protrusion at the pointed top may be greater than the height of the protrusion at other tops.

[0149] (11) In the filter of (2), the protrusion may have a side protrusion protruding from the outer surface.

[0150] (12) The cell membrane disruption device of the present disclosure includes any one of the filters (1) to (11).

[0151] (13) The cell membrane destruction device of (12) may further include a suction device that sucks cells containing the cell membrane located on the first main surface side of the filter base portion toward the second main surface side through the multiple through holes.

[0152] (14) The cell membrane disruption device of (13) may further include a container to which the filter is attached, and the suction device may suck gas from the container downstream of the filter.

[0153] (15) The cell membrane disruption device according to any one of (12) to (14) may further include a vibration device that vibrates the filter.

[0154] (16) The method for disrupting cell membranes disclosed herein includes the steps of: preparing a filter having a first main surface with protrusions and a second main surface opposite the first main surface, the filter base defining a plurality of through holes connecting the first main surface and the second main surface; introducing cells containing cell membranes into the first main surface side of the filter base of the filter; and disrupting the cell membranes with the protrusions of the filter.

[0155] (17) The cell membrane disruption device of (16) may further include a step of recovering the cell contents that have passed through the filter.

[0156] (18) In the cell membrane destruction method of (16) or (17), the step of destroying the cell membrane may further include sucking the cells located on the first main surface side of the filter base portion toward the second main surface side through the multiple through holes.

[0157] (19) In the cell membrane disruption method of (18), the preparing step may include preparing the filter attached to a container, and the suctioning may include suctioning gas in the container downstream of the filter.

[0158] The filters of the present disclosure are useful for disrupting cell membranes or extracting cellular contents within the cell membrane.

[0159] DESCRIPTION OF SYMBOLS 1 Filter 2, 2A Breaking device 3 Cell 4 Cell membrane 5 Cell contents 10 Filter part 11 Through-hole 12 Filter base part 13 Projection part 14 Top part 14a Pointed top part 14b Other top part 15 End part 16 Outer surface 16a Inclined surface 17 Side projection part 20 Frame part 30 Container 40 Suction device 50 Aluminum plate 51 First oxide film 52 Depression 53 Second oxide film 54 Hole 55 Cathode plate

Claims

1. A filter for disrupting cell membranes, comprising: a filter base portion having a first main surface having protrusions, and a second main surface opposite to the first main surface, and defining a plurality of through holes connecting the first main surface and the second main surface, wherein when viewed from the first main surface side, the protrusions are located between adjacent through holes and protrude in a first direction away from the filter base portion, and in a cross section cut in a second direction in which the through holes are adjacent to each other, the protrusions are located more inward than ends of the filter base portion located between the adjacent through holes, and the thickness of the protrusions in the second direction decreases toward the apex of the protrusions.

2. The filter according to claim 1, wherein in the second direction, the protrusions have outer surfaces that curve from a central region of the filter base portion located between adjacent through holes toward the end portions.

3. The filter according to claim 2, wherein the protrusion has, on at least a portion of the outer surface of the protrusion, a flat inclined surface that is inclined toward the through hole.

4. A filter as claimed in any one of claims 1 to 3, wherein the filter base portion includes a first filter base portion located between adjacent through holes, and a second filter base portion which is a portion different from the first filter base portion and is located between adjacent through holes, and the protrusion portion includes a first protrusion portion protruding from the first main surface of the first filter base portion, and a second protrusion portion protruding from the first main surface of the second filter base portion, and when the distance from the second main surface of the filter base portion to the top of the protrusion portion is defined as the height of the protrusion portion, the height of the first protrusion portion and the height of the second protrusion portion are different.

5. The filter according to any one of claims 1 to 4, wherein the protrusion surrounds one or a plurality of through holes when viewed from the first main surface side of the filter base.

6. The filter according to any one of claims 1 to 5, wherein, when the distance from the second main surface of the filter base to the top of the protrusion is defined as the height of the protrusion, the height of the protrusion changes continuously.

7. The filter according to claim 5, wherein an area of ​​a region surrounded by the tops of said protrusions when viewed from the first main surface side of said filter base is larger than an opening area of ​​said one or more through holes.

8. A filter according to any one of claims 1 to 7, wherein the protrusions have pointed tips.

9. A filter as described in claim 8, wherein, when viewed from the first main surface side of the filter base portion, a region is defined by connecting three imaginary straight lines tangent to the outer peripheries of two adjacent through holes among three adjacent through holes, and when a region including the three through holes is drawn, the pointed apex is located within the region.

10. The filter according to claim 8, wherein, when the distance from the second main surface of the filter base to the apex of the protrusion is defined as the height of the protrusion, the height of the protrusion at the pointed apex is greater than the height of the protrusion at the other apex.

11. The filter of claim 2, wherein the projections include side projections projecting from the outer surface.

12. A device for disrupting a cell membrane, comprising a filter according to any one of claims 1 to 11.

13. The cell membrane disruption device according to claim 12, further comprising a suction device that sucks cells containing the cell membrane located on the first main surface side of the filter base portion through the multiple through holes to the second main surface side.

14. The cell membrane disruption device according to claim 13, further comprising a container in which the filter is attached, and the suction device suctions gas from within the container downstream of the filter.

15. The destruction device according to any one of claims 12 to 14, further comprising a vibration device for vibrating the filter.

16. A method for disrupting a cell membrane, comprising the steps of: preparing a filter having a filter base portion having a first main surface with protrusions and a second main surface opposite the first main surface, the filter base portion defining a plurality of through holes connecting the first main surface and the second main surface; introducing cells including a cell membrane onto the first main surface side of the filter base portion of the filter; and disrupting the cell membrane with the protrusions of the filter.

17. The method for disrupting cell membranes according to claim 16, further comprising the step of recovering the cellular contents that pass through the filter.

18. A method for disrupting a cell membrane as described in claim 16 or 17, wherein the step of disrupting the cell membrane further includes sucking the cells located on the first main surface side of the filter base portion to the second main surface side through the multiple through holes.

19. The method for disrupting a cell membrane according to claim 18, wherein the preparing step comprises preparing the filter attached to a container, and the aspirating comprises aspirating gas from within the container downstream of the filter.

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