Cell harvesting device
The cell retrieval device addresses scaffold and cell damage issues by employing a flexible tip with recesses and a gentle operation method, enhancing recovery efficiency.
Patent Information
- Authority / Receiving Office
- JP · JP
- Patent Type
- Patents
- Current Assignee / Owner
- TOYODA GOSEI CO LTD
- Filing Date
- 2023-03-27
- Publication Date
- 2026-05-25
Smart Images
Figure 0007864292000003 
Figure 0007864292000004 
Figure 0007864292000005
Abstract
Description
Technical Field
[0001] The present invention relates to a cell recovery tool for recovering cells cultured on a scaffold.
Background Art
[0002] Cell culture is carried out in various fields such as drug discovery and regenerative medicine. To culture scaffold-dependent cells, it is common to attach cells to a scaffold, add a nutrient medium to culture the cells, and then detach and recover the cultured cells from the scaffold using a cell recovery tool. The surrounding of the scaffold is often surrounded by a wall.
[0003] As a cell recovery tool, a scraping tool called a cell scraper as described in Patent Document 1 is generally used. The cell scraper is composed of a scraping part consisting of a blade made of a rubber-like elastic material and its support, and a handle for holding it. Then, an operator holds the handle and performs an operation of moving the blade as long as possible from one side of the scaffold to the other side, and the blade scrapes and recovers the cells from the scaffold.
[0004] However, the scraping with this cell scraper has the following problems. (A) When the scaffold is weak like a membrane, excessive force may be applied from the blade and the scaffold may be damaged, and contamination (mixing of damaged substances) may occur. (B) Also, although the blade is made of an elastic material, it is considerably hard compared to cells. Therefore, during the above operation, the cells may be damaged by physical stimulation, and the cell recovery rate may decrease. (C) Since the operation method is as described above (move the blade as long as possible), when the surrounding of the scaffold is surrounded by a wall, the blade hits the wall and its movement is obstructed, making it difficult to scrape cells from a wide range of the scaffold, and the cell recovery rate deteriorates.
[0005] Other cell harvesting devices include one disclosed in Patent Document 2, which features a harvesting section made of a monofiber deformed fiber formed by the bonding of multiple synthetic resin ribbons along their long sides. Patent Document 3 also discloses a transport device in which a sheet-like graft is pierced by multiple piercing points protruding from the side of a support. However, these have not yet been widely used. [Prior art documents] [Patent Documents]
[0006] [Patent Document 1] CD-ROM of Utility Model Application No. 4-10788 (Utility Model Publication No. 5-80300) [Patent Document 2] Japanese Patent Publication No. 2021-132574 [Patent Document 3] Japanese Patent Publication No. 2013-198442 [Overview of the project] [Problems that the invention aims to solve]
[0007] Therefore, the object of the present invention is to provide a cell retrieval device that can retrieve cells without damaging the scaffold. [Means for solving the problem]
[0008] [1] In a cell retrieval device for retrieving cultured cells from a scaffold, A tip surface that is pressed against the scaffolding in a facing position, and a tip portion having a plurality of dispersed recesses or a network of connected recesses that are recessed from the tip surface, A flexible portion provided on the rear side of the aforementioned tip, having a thickness of 2 mm or more and a 25% compressive stress of 0.5 to 300 kPa, It includes a pinch portion that can be pinched with the fingers, which is provided on the rear side of the flexible portion. A cell retrieval device characterized in that the area of the tip surface is 0.8 times or less the area of the scaffold. Here, "area of the tip surface" refers to the area within the contour of the tip surface, including the area of any recesses.
[0009] [Effect] Pinch the pinch with your fingers, lightly press the tip against the scaffolding in a position facing it, and then rotate or slide the tip in short bursts along the surface of the scaffolding. This operation causes cells to adhere to the tip surface, where the surface area is increased by the recesses. Cells are also wiped off and adhere around the tip. In this way, cells can be attached to a wide area of the scaffolding. (a) During this operation, the flexible portion of the aforementioned thickness and compressive stress compresses appropriately to act as a cushion, so that the damage to the cells caused by physical stimulation is small and the cell recovery rate does not decrease. (i) Furthermore, even if the scaffolding is as weak as a membrane, the load on the scaffolding is mitigated by the cushion, making it less likely to be damaged. (c) As described above, the operation method is to lightly press the tip against the scaffolding and then rotate and slide it briefly. Therefore, even if the scaffolding is surrounded by a wall, the movement of the tip and flexible part is hardly obstructed by the wall, allowing cells to be wiped from a wide area of the scaffolding, resulting in a better cell recovery rate. These effects can be obtained.
[0010] If the thickness of the flexible section is less than 2 mm, the compression allowance of the flexible section will be insufficient, increasing the load on the scaffolding. A thickness of 3 mm or more is preferable for the flexible section. There is no specific upper limit for the thickness of the flexible section, but if we had to specify one, it would be 15 mm. A 25% compressive stress of 0.5 to 300 kPa in the flexible section ensures sufficient force to swab cells while minimizing damage to the scaffold. If the 25% compressive stress of the flexible section is less than 0.5 kPa, the force to swab cells weakens, and if it exceeds 300 kPa, the load on the scaffold becomes too great, potentially damaging the membrane. A 25% compressive stress of 0.5 to 200 kPa in the flexible section is desirable as it further reduces the risk of scaffold damage. Because the surface area of the tip is 0.8 times or less the surface area of the scaffolding, the tip can be easily rotated and slid to move it in the direction of the scaffolding surface, as described above.
[0011] Once cells adhere to the tip, lift the gripping part to peel the cells from the scaffold and collect them.
[0012] [2] The cell recovery tool according to [1], wherein the tip surface is a flat surface. Since the scaffold is generally a flat surface, having a flat tip surface makes it easier for cells to adhere.
[0013] [3] The cell recovery tool according to [1] or [2], wherein the shape of the tip surface is a sided shape having at least one side that is a straight line or a curve concave inward. When the tip is rotated, this side intersects the rotation locus, making it easy to wipe off the cells.
[0014] [4] The cell recovery tool according to any one of [1] to [3], wherein the area of the tip surface is 0.35 times or more the area of the scaffold. When this magnification is 0.35 times or more, the amount of cells adhering increases. [[ID=*23]]
[0015] [[ID=*24]] [5] The cell recovery tool according to any one of [1] to [4], wherein the recess is a recess into which a virtual sphere having a diameter of any of 30 to 500 μm can be inserted. Here, the virtual sphere is a virtual sphere for specifying the size of the recess and is not something to be inserted during use. Also, insertion means being inserted almost exactly so as to be sandwiched from at least two sides of the recess. If the recess is one into which a virtual sphere with a diameter of 30 μm or more can be inserted, the cell surface can be locked in the recess and it is easy to adhere. If the recess is one into which a virtual sphere with a diameter of 500 μm or less can be inserted, it is less likely that the cells will completely enter the recess.
[0016] [6] The cell recovery tool according to any one of [1] to [5], wherein the flexible part is made of a polymer foam. Polymer foam is easy to maintain a constant quality, less likely to cause contamination, and can be made inexpensive.
[0017] [7] The cell recovery tool according to [6], wherein the tip portion is integral with the flexible portion, and the tip surface is the cut end surface of the flexible portion. This enables the tip portion and the flexible portion to be integrated.
[0018] [8] The cell recovery tool according to any one of [1] to [7], wherein the gripping portion is composed of a rod-shaped body. This makes it easy to rotate the gripping portion around the central axis.
[0019] [9] The cell recovery tool according to [8], wherein the central axis of the gripping portion is perpendicular to the tip surface or has an inclination within 15° from the perpendicular. This makes it easy to perform both rotating the gripping portion around the central axis and moving it in a direction intersecting the central axis.
[0020]
[10] The cell recovery tool according to [9], wherein the cross-sectional shape of the gripping portion is a circle, an ellipse, or a polygon with 5 or more sides. This makes it easy to grip the gripping portion with a finger and rotate it around the central axis.
Effect of the Invention
[0021] [[ID=ed=26]] [[ID=2c=27]]According to the cell recovery tool of the present invention, cells can be recovered without damaging the scaffold.
Brief Description of the Drawings
[0022] [Figure 1] FIG. 1 is an exploded perspective view showing the cell recovery tool of the embodiment and the cell incubator using the same. [Figure 2] FIG. 2 is a cross-sectional view showing the incubator during cell culture. [Figure 3] FIG. 3 shows the cell recovery tool, (a) is a perspective view, (b) is an enlarged view of the tip surface, and (c) is an enlarged cross-sectional view. [Figure 4]Figure 4 shows how to use the cell retrieval device, where (a) is a cross-sectional view when the tip surface is in contact with the scaffold, (b) is a plan view of the same, (c) is a cross-sectional view when the tip is rotated, (d) is a plan view of the same, (e) is a cross-sectional view when the cell retrieval device is lifted, and (f) is a plan view of the same. [Figure 5] Figure 5 shows a photograph taken from the bottom of a scaffold after culturing Caco-2 cells on the top surface of the scaffold, which were then nuclear-stained. [Figure 6] Figure 6 is a photograph taken from the underside of the scaffold after the Caco-2 cells were swiped off using the cell harvesting device of this example. [Figure 7] Figure 7 illustrates the shapes (a) to (e) of the tip surfaces of the cell harvesting device in Example 2. [Figure 8] Figure 8(a) is an enlarged view of the tip surface of Example 3, (b) is an enlarged cross-sectional view thereof, (c) is an enlarged view of the tip surface of Example 4, (d) is an enlarged cross-sectional view thereof, (e) is an enlarged view of the tip surface of Example 5, (f) is an enlarged cross-sectional view thereof, (g) is an enlarged view of the tip surface of Example 6, and (h) is an enlarged cross-sectional view thereof. [Modes for carrying out the invention]
[0023] 1. Scaffolding The form of the scaffolding is not particularly limited, but examples include membranes, plates, fibers, etc. While not particularly limited, scaffolding materials include polycarbonate, polyethylene terephthalate, polyimide, polyurethane, and collagen vitrigel.
[0024] 2. Enclosure The area around the scaffolding is not particularly limited, but the cell retrieval tool of the present invention is especially useful when it is surrounded by a wall. This is because the operation method of the cell retrieval tool of the present invention is as described above (lightly pressing the tip against the scaffolding in a position facing it, then rotating and sliding it), making it easier to retrieve cells even when the surrounding wall is small, unlike conventional cell scrapers. The enclosure wall is not particularly limited, but examples include the cylindrical enclosure wall of an insert (inserted into a well plate), the side wall of a petri dish, and so on.
[0025] 3.Tip As mentioned above, the tip surface is preferably a flat surface, but it may also be a curved surface that curves gently towards the tip (preferably with a radius of curvature of 20 mm or more) (a partially cylindrical surface or a partially spherical surface), or the central part of the tip surface may be a flat surface and only the end part may be curved.
[0026] Examples of the aforementioned "bordered shape having at least one side that is a straight line or an inwardly curved side" include polygons, partial circles, and the like. (a) The polygon may be a general polygon that does not have any vertices with interior angles exceeding 180°, or a concave polygon that has such vertices. • For general polygons, those with 3 to 6 sides are preferred, with quadrilaterals being the most preferred. If the polygon has 7 or more sides (i.e., the closer it is to a circle), the smaller the angle at which the sides intersect the rotational trajectory, and the easier it is to wipe away cells. • In the case of concave polygons, the angles at which the sides intersect the rotational trajectory tend to be small, so polygons with 4 to 12 sides are preferable. (i) A partial circle is a shape obtained by cutting off a part of a circle along the aforementioned edge, and examples include a semicircle and a sector.
[0027] The tip portion having the aforementioned "recess" (preferably "a recess into which a virtual sphere having a diameter of 30 to 500 μm can be fitted") is not particularly limited, but the following are examples: (a) Cross-section of polymer foam: The following explanation regarding the flexible part can be used for polymer foam. (i) Polymer sheet body with multiple recesses dispersed therein: See Figures 8(a) and 8(b) described later. (c) Polymer sheet body with a network of interconnected recesses: See Figures 8(c) and 8(d) described later. (e) Woven fabric with recessed areas in the weave: See Figures 8(e) and 8(f) described later. (e) Surface of the cotton-like material where the gaps between fibers form depressions: See Figures 8(g) and 8(h) described later.
[0028] 4. Flexibility The "flexible part having the same cross-sectional shape as the tip, a thickness of 2 mm or more, and a 25% compressive stress of 0.5 to 300 kPa (preferably 0.5 to 200 kPa)" is not particularly limited, but the following are examples. (a) Polymer foam: Polymers include resins, rubbers, and elastomers. The cut surface of the polymer foam has depressions due to cut cells, so the cut end can be the tip and the cut surface can be the tip surface. The foam may be closed-cell or open-cell, but open-cell foam is more likely to satisfy the compressive stress. The resin is not particularly limited, but examples include polyurethane (PUR), polyethylene (PE), ethylene-vinyl acetate copolymer resin (EVA), and polypropylene. The rubber used is not particularly limited, but examples include ethylene-propylene-diene copolymer rubber (EPDM), natural rubber (NR), chloroprene rubber (CR), nitrile rubber (NBR), styrene-butadiene rubber (SBR), silicone rubber, and urethane rubber. While not particularly limited, examples of elastomers include dynamically crosslinked thermoplastic elastomers (TPV), olefin-based elastomers (TPO), polyester-based thermoplastic elastomers (TPEE, TPC), and thermoplastic polyurethane elastomers (TPU). (i) Fiber-based cotton-like material: This is made by forming fibers into a cotton-like material. If the leading surface of this fiber-based cotton-like material has the aforementioned recess, the leading surface can be considered the tip.
[0029] The cross-sectional shape of the flexible portion is not particularly limited, but it is preferable that it be the same as the shape of the tip surface at least in the portion connected to the tip, and more preferably that it be the same as the shape of the tip surface throughout. This is to ensure uniform support for the entire tip.
[0030] 5. Pinching part As mentioned above, the shape of the knob is preferably rod-shaped, but it may also be plate-shaped or the like. The rod-shaped object is not limited to one whose diameter is constant along its length, but may also have a diameter that changes. The means of joining the gripping part and the flexible part are not particularly limited, but examples include bonding, welding, clamping, gripping, etc. Alternatively, the tip of the knob may be joined to the flexible part. [Examples]
[0031] Next, embodiments of the present invention will be described with reference to the drawings. Note that the structure, materials, shape, and dimensions of each part of the embodiments are illustrative and can be modified as appropriate without departing from the spirit of the invention.
[0032] First, let's briefly describe an example of a cell culture vessel. The culture vessel shown in Figures 1 and 2 consists of a well plate 10, inserts 13 to be inserted into each well 11, and culture medium 18 injected into each well 11 and insert 13. The well plate 10 has multiple (e.g., 6, 12, 24, 96, etc.) cylindrical wells 11 attached to a plate 12. The insert 13 consists of a cylindrical enclosure 14 that is slightly smaller than the well 11, a porous membrane that serves as a scaffold 15 fixed to the bottom surface of the enclosure 14, a flange 16 above the enclosure 14, and multiple stays 17 connecting the enclosure 14 and the flange 16. When the enclosure 14 and scaffold 15 of the insert 13 are inserted into the well 11, the flange 16 locks into the upper end of the well 11, and the scaffold 15 and the plate 12 are separated.
[0033] When cells C are attached to the upper surface of the scaffold 15 (cells C can also be attached to the lower surface of the scaffold 15), and the nutrient medium 18 is added, the cells C are cultured so that they spread out on the upper surface of the scaffold 15, as shown in Figure 2. The insert 13 used in the example has a scaffold area of 33.2 mm² (the area enclosed by the surrounding wall 14). 2 It is (6.5 mm in diameter).
[0034] [Example 1] The cell harvesting device of Example 1 shown in Figures 1, 3, 4, and 5(a) is The tip portion 1 has a tip surface 2 that is pressed against the scaffolding 15 in a position facing it, and a plurality of dispersed recesses 3 that are recessed from the tip surface 2, A flexible part 4 is provided on the rear side of the tip 1, having a thickness of 2 mm or more and a 25% compressive stress of 0.5 to 300 kPa (preferably 0.5 to 200 kPa), It includes a pinch portion 5 that can be pinched with fingers, which is provided on the rear side of the flexible portion 4.
[0035] In this embodiment, the tip portion 1 and the flexible portion 4 are made of a polymer foam and together form a rectangular parallelepiped. The cut surface of the polymer foam has recesses 3 due to cut air bubbles, so the cut end is designated as the tip portion 1 and the cut surface as the tip surface.
[0036] The tip surface 2 is a flat surface. The shape of the tip surface 2 (the shape when viewed from the opposite side) is a square. The cross-sectional shape of the flexible part 4 is the same and constant as this. The side length of the square is 3.4 to 5.2 mm, and therefore the area of the tip surface 2 is 0.35 to 0.8 times the area of the scaffolding 15.
[0037] As described above, recess 3 is caused by a broken bubble and is a recess into which a virtual sphere with a diameter of 30 to 500 μm can be fitted. The bubbles are either closed or open, and Figures 3(b) and 3(c) illustrate open bubbles. In this example, when the tip portion 1 and the flexible portion 4 are a single unit, their boundary is around the bottom of the recess 3 that opens onto the tip surface.
[0038] The knob portion 5 consists of a rod-shaped body with a circular cross-section. The central axis of the knob portion 5 is perpendicular to the tip surface 2. The tip of the knob portion 5 enters the flexible portion 4 and is joined to it with adhesive.
[0039] To retrieve cells cultured on the scaffold 15 using the cell retrieval device of this embodiment configured as described above, the following procedure is followed. As shown in Figures 4(a) and 4(b), pinch the handle 5 with your fingers and lightly press the tip surface 2 against the scaffolding 15 in a position facing it. As shown in Figures 4(c) and 4(d), the gripping part 5 is rotated with the fingers, and the tip part 1 is rotated or slid briefly to move it in the direction of the scaffold 15 surface. Through this operation, cells C adhere to the tip surface 2, where the surface area is increased by the recess 3. Cells C are also wiped off and adhere to the area around the tip part 1 and the flexible part 4. In this way, cells C can be attached to a wide area of the scaffold 15. As shown in Figures 4(e) and 4(f), the pinching part 5 is lifted to detach and collect the cells C from the scaffold 15.
[0040] Figure 5 is a photograph taken from the bottom surface of the scaffold after culturing Caco-2 cells on the top surface of the scaffold, staining the nuclei with DAPI, and before swabbing the cells with the cell retrieval tool of this example. Figure 6 is a photograph taken from the bottom surface of the scaffold after swabbing the cells with the cell retrieval tool of this example, and it can be observed that the cells wrapped around a portion of the membrane after swabbing.
[0041] As described above, the cell retrieval device of this embodiment provides the aforementioned effects and allows for cell retrieval without damaging the scaffold.
[0042] Next, we will explain the considerations that led to this embodiment. First, we obtained commercially available polymer foams as shown in Table 1 below. The grades listed in the Grade column, "E-7010, E-8000, N-148, N-149, C-4205, C-4215, C-4505, C-4600, E-4070, E-4188, E-4288, E-4390, E-4408, NBR-4112E, TT-4102, TT-4103, A-050F, A-8, HD-80, A-080, RP-300S, RP-300FRE, RP-300S, RP-300FRND, A-082," are product names of Inoac Corporation. "Value Sponge No. 845521" is a product name of Rosie Rosa Co., Ltd. "PORON LE-20, PORON TR-24, PORON L-24, and PORON TM-2" are product names of Rogers Inoac Corporation.
[0043] [Table 1]
[0044] These polymer foams were cut into rectangular parallelepipeds with a tip surface of 4.5 mm x 4.5 mm and a thickness (height) of 3 mm, and the gripping part 5 was attached as in Example 1 above to create a cell retrieval tool. Then, a cell retrieval experiment was performed using cells cultured on a scaffold as in Example 1 above. Table 1 shows the results of cell swabbing. The meaning of the symbols is as follows. ○: Cells were successfully swabbed away. ×: The scaffolding (porous membrane) was damaged or the scaffolding detached due to air pressure. These results showed that materials with a 25% compressive stress of 300 kPa or less could be wiped away, while those exceeding 300 kPa could not. Furthermore, the material type did not affect whether or not wiping was possible.
[0045] Next, using the "EPDM-based E-7010" shown in Table 1, the shape of the tip surface was changed from a square to a circle, and the side length or diameter was changed for each to create a rectangular parallelepiped (however, the thickness (height) was kept constant at 3 mm). Then, as in Example 1 above, the gripping part 5 was attached to create a cell harvesting tool.
[0046] [Table 2]
[0047] Then, as in Example 1 above, an experiment was conducted to recover the cells cultured on the scaffold. Table 1 shows the results of cell swab removal. The meaning of the symbols is as follows. ○: Wiped evenly across the entire surface (95% or more) △: The edges were not wiped clean (approximately 10% of the surface remained unwiped). ×: The scaffolding (porous membrane) was damaged or the scaffolding detached due to air pressure. This result shows that if the area ratio is 0.8 or less, a certain degree of wiping is possible.
[0048] [Example 2] The cell harvesting device of Example 2 shown in Figure 7 differs from Example 1 only in that the shape of the tip surface 2 and the cross-sectional shape of the flexible part, which is not shown in the figure, have been changed; otherwise, it is the same as Example 1. Specifically, (a) is a square from Example 1, (b) is a square with curved sides that are concave inward, (c) is a concave octagon, (d) is a partially circular shape obtained by cutting off a part of a circle along one side, and (e) is a sector shape obtained by cutting off a part of a circle along two sides.
[0049] [Example 3] The cell harvesting device of Example 3 shown in Figures 8(a) and 8(b) differs from Example 1 in that it has a polymer sheet body with multiple recesses 3 dispersed on its tip surface 2 as the tip portion 1, and the flexible portion 4 of Example 1 is attached to this tip portion 1; otherwise, it is the same as Example 1.
[0050] [Example 4] The cell harvesting device of Example 4 shown in Figures 8(c) and 8(d) differs from Example 1 in that it has a polymer sheet body with network-like recesses 3 formed on its tip surface 2 as the tip portion 1, and the flexible portion 4 of Example 1 is attached to this tip portion 1; otherwise, it is the same as Example 1.
[0051] [Example 5] The cell harvesting device of Example 5, shown in Figures 8(e) and 8(f), differs from Example 1 in that its tip surface 2 is made of thread and its weave is recessed 3, forming the tip 1, to which the flexible part 4 of Example 1 is attached; otherwise, it is the same as Example 1.
[0052] [Example 6] The cell harvesting device of Example 6 shown in Figures 8(g) and 8(h) differs from Example 1 in that the tip surface 2 is made of fibers and the gaps between them form recesses 3, with the surface portion of the cotton-like material being designated as the tip portion 1, and the other portion of the cotton-like material that is integrally continuous with the surface portion being designated as the flexible portion 4. Otherwise, it is the same as Example 1.
[0053] These examples 2-6 also demonstrate that cells can be recovered with a high recovery rate without damaging the scaffold.
[0054] It should be noted that the present invention is not limited to the embodiments described above, and can be appropriately modified and implemented without departing from the spirit of the invention. [Explanation of symbols]
[0055] 1 Tip 2 Tip surface 3. recess 4. Flexible parts 5. The pinching part 10-well plate 11 wells 12 plates 13 Inserts 14 Enclosure 15 Scaffolding 16 flange 17 Stay 18 Culture medium C cell
Claims
1. In a cell retrieval device for collecting cells cultured on a scaffold, The tip portion has a tip surface that is pressed against the scaffolding in a facing position, and the tip surface has a plurality of dispersed recesses or a network of connected recesses, A flexible portion is provided on the rear side of the aforementioned tip, having a thickness of 2 mm or more and a 25% compressive stress of 0.5 to 300 kPa, A pinch that can be grasped with fingers is provided on the rear side of the flexible part. It includes, The area of the tip surface is 0.8 times or less the area of the scaffolding. A cell harvesting device characterized by the following features.
2. The cell harvesting device according to claim 1, wherein the tip surface is a flat surface.
3. The cell harvesting device according to claim 1, wherein the shape of the tip surface is a bevel shape having at least one side that is a straight line or an inwardly concave curve.
4. The cell retrieval device according to claim 1, wherein the area of the tip surface is 0.35 times or more the area of the scaffold.
5. The cell retrieval device according to claim 1, wherein the recess is a recess into which a virtual sphere having a diameter of 30 to 500 μm can be fitted.
6. The cell recovery device according to claim 1, wherein the flexible part is made of a polymer foam.
7. The cell retrieval device according to claim 6, wherein the tip portion is integral with the flexible portion, and the tip surface is the cut end surface of the flexible portion.
8. The cell harvesting device according to claim 1, wherein the gripping portion is a rod-shaped body.
9. The cell harvesting device according to claim 8, wherein the central axis of the pinching portion is perpendicular to the tip portion or inclined at an angle of 15° or less from the perpendicular.
10. The cell harvesting device according to claim 9, wherein the cross-sectional shape of the pinching portion is a circle, an ellipse, or a polygon with five or more sides.