Cell culture vessel and method for manufacturing the same

Abstract

To provide a cell culture vessel in which a spheroid can be easily formed, and a spheroid can be easily observed.SOLUTION: A cell culture vessel to be used for cell culture and observation has: a transparent bottom part 10 in which a plurality of recesses 11 are formed; and a frame body which is provided on the bottom part 10 and has a side wall part, and forms a plurality of through-holes at positions corresponding to the plurality of recesses 11 when seen from above, in which a plurality of wells are formed by each recess 11 and the through-holes, where an inner surface 11a of the recess 11 is a cell low adhesive surface, the maximum height roughness Rz of the inner surface 11a of the recess 11 is 0.170 μm or less, a thickness ratio tA / tB when an average thickness of a bottom of the recess 11 is tA(mm) and an average thickness of the recess at a position on a circular region of 50 to 60% the radius of an opening the recess 11 from the bottom seen from above the recess 11 is tB(mm) is 0.80 to 1.20 or more, and a region inside the circular region when seen from above in the recess 11 is a culture part.SELECTED DRAWING: Figure 2

Description

[Technical field]

[0001] The present invention relates to a cell culture vessel and a method for producing the same. [Background technology]

[0002] Cell masses (spheroids) obtained by 3D culture are expected to have biological activity closer to that in the body than conventional 2D culture. Patent Document 1 discloses a cell culture vessel for three-dimensional culture having multiple wells, which is formed by combining an opaque perforated plate portion obtained by resin injection molding or thermoforming with a transparent, rounded well bottom plate portion obtained by resin injection molding or thermoforming. Patent Document 2 discloses a cell culture vessel for three-dimensional culture, which has a well consisting of a side wall that forms an opening and a bottom that covers the lower end of the opening, with multiple recesses formed on the upper surface of the bottom that constitutes the well, and a surface layer that suppresses cell adhesion formed on the inner surface of the recesses. [Prior art documents] [Patent documents]

[0003] [Patent Document 1] Special Publication No. 2016-520307 [Patent Document 2] International Publication No. 2019 / 151114 Summary of the Invention [Problem to be solved by the invention]

[0004] However, in conventional cell culture vessels such as those disclosed in Patent Documents 1 and 2, problems may arise such as insufficient formation of spheroids depending on the well, or the formed spheroids may be difficult to observe.

[0005] An object of the present invention is to provide a cell culture vessel in which spheroids can be easily formed and in which the spheroids can be easily observed. [Means for solving the problem]

[0006] As a result of the inventors' investigation, it was found that in a conventional cell culture vessel having a bottom obtained by injection molding or thermoforming of a resin as in Patent Document 1, the cutting marks of the mold are transferred to form unevenness on the surface of the bottom recess, which may impede the smooth movement of cells and reduce the formation of spheroids and the observability of spheroids. In addition, in a cell culture vessel having a bottom with multiple recesses on the upper surface as in Patent Document 2, the thickness of the recesses varies depending on the location, which may deteriorate the observation image. Further investigation was carried out to determine the maximum height roughness Rz of the inner surface of the recess and the thickness ratio t A / t B The present inventors have found that controlling the amount of spheroids in a specific range improves the formability and observability of the spheroids, and have completed the present invention.

[0007] That is, the present invention includes the following aspects. [1] A cell culture vessel for use in culturing and observing cells, a transparent bottom portion on which a plurality of recesses are formed, and a frame body having a side wall portion provided on the bottom portion and having a plurality of through holes formed at positions corresponding to the plurality of recesses when viewed from above; a plurality of wells are formed by the respective recesses and the through holes; The recess has at least a rounded bottom, the inner surface of the recess is a low cell-adhesive surface; The average thickness of the bottom of the recess is t A (mm), and the average thickness of the recess at a position on a circular region that is 50 to 60% of the radius of the opening of the recess from the bottom when viewed from above is t B (mm), the thickness ratio t A / t B is 0.80 to 1.20, A region inside the annular region when viewed from above in the recess is a culture region, A cell culture vessel, wherein the maximum height roughness Rz of the surface of the culture portion is 0.170 μm or less. [2] The cell culture vessel according to [1], wherein the surface roughness Ra of the culture portion is 0.024 μm or less. [3] The cell culture vessel according to [1] or [2], wherein the maximum ten-point height roughness Rzjis of the surface of the culture portion is 0.150 μm or less. [4] The cell culture vessel according to any one of [1] to [3], wherein the S / N ratio when the spheroids in the culture section are stained with a fluorescent staining reagent and subjected to fluorescence measurement is 11.5 or more. [5] The cell culture vessel according to any one of [1] to [4], wherein the bottom is made of glass. [6] The cell culture vessel according to any one of [1] to [5], wherein the side wall portion of the frame contains a coloring agent. [7] A cell culture vessel for use in culturing and observing cells, a transparent bottom portion on which a plurality of recesses are formed, and a frame body having a side wall portion provided on the bottom portion and having a plurality of through holes formed at positions corresponding to the plurality of recesses when viewed from above; a plurality of wells are formed by the respective recesses and the through holes; The recess has at least a rounded bottom, The average thickness of the bottom of the recess is t A (mm), and the average thickness of the recess at a position on a circular region that is 50 to 60% of the radius of the opening of the recess from the bottom when viewed from above is t B (mm), the thickness ratio t A / t B is 0.80 to 1.20, A region inside the annular region when viewed from above in the recess is a culture region, A cell culture vessel, wherein the maximum height roughness Rz of the surface of the culture portion is 0.170 μm or less. [8] The cell culture vessel according to [7], wherein the surface roughness Ra of the culture portion is 0.024 μm or less. [9] The cell culture vessel according to [7] or [8], wherein the maximum ten-point height roughness Rzjis of the surface of the culture portion is 0.150 μm or less.

[10] The cell culture vessel according to any one of [7] to [9], wherein the bottom is made of glass.

[11] The cell culture vessel according to any one of [7] to

[10] , wherein the side wall portion of the frame contains a coloring agent.

[12] A transparent bottom portion having a plurality of recesses formed therein, and a frame body having a side wall portion provided on the bottom portion and having a plurality of through holes formed at positions corresponding to the plurality of recesses when viewed from above, A method for manufacturing a cell culture vessel for use in culturing and observing cells, in which a plurality of wells are formed by each of the recesses and the through-holes, comprising: placing a plate-like member on a die having a plurality of holes, and drawing a vacuum from the side opposite to the side on which the plate-like member is placed while heating the plate-like member, thereby partially deforming the plate-like member to form a plurality of recesses and obtain the bottom portion; and bonding the bottom and the frame.

[13] The method for producing a cell culture vessel described in

[12] , wherein the plate-like member is a glass sheet. Effect of the Invention

[0008] According to the present invention, there is provided a cell culture vessel in which spheroids can be easily formed and the spheroids can be easily observed. [Brief description of the drawings]

[0009] [Figure 1] 1 is a cross-sectional view that illustrates a schematic diagram of a cell culture vessel according to an embodiment. [Diagram 2] 2 is an enlarged cross-sectional view of a recess in the bottom of the cell culture vessel of FIG. 1. FIG. [Diagram 3] FIG. 4 is an enlarged cross-sectional view showing a bottom portion of a cell culture vessel according to another embodiment. [Figure 4] 1A to 1C are cross-sectional views that diagrammatically show a state in which the bottom part of the cell culture vessel of the embodiment is manufactured. [Diagram 5] 4 shows photographs of each well after culture in Experimental Example 1, observed with a fluorescence microscope. [Figure 6]13 shows photographs of each well after culture in Experimental Example 3, observed with a fluorescence microscope. [Figure 7] FIG. 1 shows the results of S / N ratios obtained by optical measurement of spheroids in wells in Experimental Examples 1 and 3. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0010] Hereinafter, an embodiment of the cell culture vessel of the present invention will be described with reference to the drawings. Note that the dimensions of the drawings shown in the following description are merely examples, and the present invention is not necessarily limited thereto, and can be appropriately modified and implemented within the scope of the present invention.

[0011] [Cell culture container] The cell culture vessel of the present invention is a cell culture vessel for use in culturing and observing cells. As shown in FIG. 1, a cell culture container 1 according to one embodiment comprises a transparent bottom 10 having a plurality of recesses 11 formed therein, and a frame 20 provided on the bottom 10 and having a side wall portion 22 that forms a plurality of through holes 21.

[0012] Each recess 11 formed in the bottom 10 and each through hole 21 formed in the frame 20 are located in corresponding positions when the cell culture container 1 is viewed from above, and multiple wells 30 are formed by each recess 11 and through hole 21. The shape of the cell culture vessel 1 as viewed from above is typically rectangular, but is not limited thereto, and may be square, circular, or the like.

[0013] The arrangement pattern of the wells 30 in the cell culture vessel 1, i.e., the arrangement pattern of the recesses 11 in the bottom 10, or the arrangement pattern of the through-holes 21 in the frame 20, is not particularly limited. For example, the arrangement pattern may be a square lattice pattern, a staggered pattern, or the like. In the case of a normal microplate, the number of wells 30 is standardized to 6, 12, 24, 48, 96, 384, 1536, etc., but is not particularly limited and can be appropriately adjusted depending on the size of the cell culture vessel 1 and the desired size of the wells 2. It is preferable to have about 2 to 1600 wells. The number of recesses 11 and the number of through-holes 21 are the same as the number of wells 30.

[0014] The bottom portion 10 is made of a plate-like member, and each of the recesses 11 is formed by curving the plate-like member so as to be partially recessed. The shape of the recess 11 may be any shape having at least a rounded bottom, and may be, for example, a substantially conical shape having a rounded bottom, a hemispherical shape, a mortar shape, a bell shape, etc. The shape of the recess 11 may also be a curved shape in which the curvature of the inner surface 11a increases from the bottom toward the opening. 3, the shape of the recess 11 in a cut surface passing through the bottom (deepest part) of the recess 11 and parallel to the thickness direction of the bottom may be an arc shape (a downwardly convex arc shape in FIG. 3) whose center of curvature is located inside the inner surface 11a of the recess 11, and which widens linearly from the bottom toward the opening edge. In this case, the opening edge may be rounded. The radius of curvature of the bottom of the recess 11 is preferably from more than 0 mm to 2.8 mm, more preferably from 0.5 mm to 2.5 mm, and even more preferably from 0.8 mm to 2.3 mm, so that cells tend to gather therein.

[0015] 3, the inclination angle θ0 of the straight portion above the arc portion on the inner surface 11a of the recess 11 is preferably 23° to 63°, more preferably 33° to 58°, and even more preferably 38° to 53°. If the inclination angle θ0 is within the above range, cells tend to gather. The inclination angle θ0 is the angle between the straight line portion of the inner surface 11a of the recess 11 and the direction parallel to the surface direction of the upper surface of the portion of the bottom 10 other than the recess 11 in the cross section of FIG.

[0016] The opening shape of the recess 11 is typically circular, but is not limited thereto, and may be elliptical, rectangular, honeycomb, donut-shaped, or the like. The diameter of the opening of the recess 11 is not particularly limited, and is preferably, for example, 2.0 to 36.0 mm. In the case of a 96-hole well, the diameter is about 6.0 to 8.0 mm. When the opening shape of the recess 11 is not circular, the diameter of the opening means the diameter of the smallest circle that can contain the opening. The depth of the bottom (deepest part) of the well 11 (the length from the opening surface of the well to the bottommost part of the culture surface) is not particularly limited and can be about 1 to 15 mm. In the case of the recess 11 illustrated in FIG. 3, in a cut surface parallel to the thickness direction of the bottom, the depth D from the opening surface of the recess 11 to the boundary point d between the arc portion and the straight portion of the inner surface 11a of the recess 11 is not particularly limited and can be, for example, 1 to 2 mm. The diameter of the opening of the recess 11 and the depth of the bottom are appropriately adjusted depending on the size of the cell culture vessel 1, the number of wells 30, the shape of the opening, and the like.

[0017] As shown in FIG. 2, the average thickness of the bottom of the recess 11 (point a in FIG. 2, the deepest part) is t A The average thickness of the recess 11 at a position (point b in FIG. 2) on the annular region C that is 50 to 60% of the radius r of the opening of the recess from the bottom (point a in FIG. 2, the deepest part) when viewed from above, that is, the position (point b in FIG. 2) on the annular region C surrounded by a circle of radius 0.5r and a circle of radius 0.6r centered on the bottom (point a), is t B (mm), where the average thickness t A is the average value of the bottom thicknesses measured for two or more recesses 11. B is the average value of thicknesses measured for two or more recesses 11 at positions on annular region C that is 50 to 60% of radius r of the opening of recess 11. In the cell culture vessel 1, the region inside the circumference of a circle with a radius of 0.5r when viewed from above in the recess 11 (inside the annular region C) becomes the culture region 12 where spheroids are cultured.

[0018] Thickness ratio t A / t BIf the ratio is 0.80 to 1.20, the thickness of the recesses 11 in the culture section 12 is uniform, so that the background is clear when observing the spheroids, and noise is reduced during fluorescent observation, resulting in excellent observation of the spheroids. t A t B If it is less than t A / t B is 0.80 or more and less than 1.00, preferably 0.85 or more and less than 1.00, more preferably 0.90 or more and less than 1.00, and even more preferably 0.95 or more and less than 1.00. Also, t A t B If it is greater than t A / t B is greater than 1.00 and not greater than 1.20, preferably greater than 1.00 and not greater than 1.15, more preferably greater than 1.00 and not greater than 1.10, and even more preferably greater than 1.00 and not greater than 1.05. Most preferably, t A and B is the same value, i.e., t A / t B =1.00.

[0019] The average thickness t of the bottom of the recess 11 A is preferably 0.025 to 0.350 mm, more preferably 0.05 to 0.30 mm, and further preferably 0.10 to 0.25 mm. The average thickness t of the recess 11 at the position on the annular region C B is preferably 0.025 to 0.35 mm, more preferably 0.05 to 0.30 mm, and further preferably 0.10 to 0.25 mm.

[0020] The maximum height roughness Rz of the surface of the culture section 12 (the inner surface 11a of the recess 11) is 0.170 μm or less. This makes it easier for cells to gather toward the bottom in the culture section 12, making it easier to form spheroids. In addition, since the cells are less likely to get caught on uneven surfaces in the culture section 12 and tend to gather smoothly, the shape of the formed spheroids when viewed from above approaches a perfect circle. The maximum height roughness Rz of the surface of the culture section 12 is preferably 0.150 μm or less, more preferably 0.120 μm or less, and further preferably 0.090 μm or less. The maximum height roughness Rz (μm) is the difference in height between the highest and lowest points when a reference length L is extracted from the roughness curve while avoiding large scratches, and the value specified in the present invention is a value measured in a straight line along the concave shape. The value of Rz can be measured, for example, by a three-dimensional measuring machine UA3P (manufactured by Panasonic Production Engineering Co., Ltd.).

[0021] The maximum ten-point height roughness Rzjis of the surface (inner surface 11a of recess 11) of culture section 12 is preferably 0.150 μm or less, more preferably 0.100 μm or less, and further preferably 0.080 μm or less. If the surface roughness Ra of inner surface 11a of recess 11 is equal to or less than the upper limit, cells tend to gather toward the bottom in culture section 12 and spheroids tend to be formed more easily. The maximum ten-point height roughness Rzjis is obtained by taking the peaks and valley bottoms of the roughness curve, taking the values ​​of the top five highest peak heights and the top five lowest valley bottoms, averaging the deviations from the median of the top five highest values ​​and the deviations from the median of the bottom five lowest values, and adding these two values ​​together.It can be measured, for example, by a three-dimensional measuring machine UA3P (manufactured by Panasonic Production Engineering Co., Ltd.).

[0022] The surface roughness Ra of the surface of the culture section 12 (the inner surface 11a of the recess 11) is preferably 0.024 μm or less, more preferably 0.020 μm or less, and further preferably 0.018 μm or less. If the surface roughness Ra of the surface of the culture section 12 (the inner surface 11a of the recess 11) is equal to or less than the upper limit, noise tends to be reduced during fluorescent observation of the formed spheroids, and the observation of the spheroids tends to be excellent. The surface roughness Ra can be measured, for example, by a three-dimensional measuring machine UA3P (manufactured by Panasonic Production Engineering Co., Ltd.).

[0023] Examples of materials constituting the bottom portion 10 include glass and resin, with glass being preferred because it allows spheroids to be formed more easily and also allows the formation of a culture portion that allows excellent observation of the spheroids. The glass that can be used for the bottom part 10 is not particularly limited, and examples thereof include quartz glass, borosilicate glass, phosphate glass, aluminosilicate glass, and reinforced glass. The glass used for the bottom part 10 may be one type or two or more types.

[0024] Examples of resins that can be used for the bottom 10 include acrylic resin, polystyrene resin, polyester resin, polycarbonate resin, polypropylene resin, silicone resin, polyethylene terephthalate (PET) resin, polyvinyl chloride resin, high-density polyethylene, polyethersulfane, PET copolymer, Permanox (trademark), cycloolefin polymer resin, Cytop (trademark), etc. The resin used for the bottom 10 may be one type or two or more types.

[0025] In this example, the inner surface 11a of the recess 11 is a low cell adhesion surface. "The inner surface of the recess is a cell adhesion surface" means that the inner surface of the recess is a surface that has been treated to inhibit cell adhesion, such as a surface on which a film is formed by applying a cell adhesion inhibitor. If the inner surface of the recess (culture section) is a low cell adhesion surface, cells will aggregate together in the recess to easily form spheroids.

[0026] Examples of cell adhesion inhibitors include phospholipid polymers such as 2-methacryloyloxyethyl phosphorylcholine (MPC), polyhydroxyethyl methacrylate, fluorine-containing compounds, polyethylene glycol, and silane coupling agents having MPC polymer chains. One type of cell adhesion inhibitor may be used alone, or two or more types may be used in combination.

[0027] The shape and thickness of the frame 20 are not particularly limited as long as it has a through-hole 21 corresponding to the recess 11 of the bottom 10, and can be appropriately designed according to the shape of the intended cell culture vessel 1.

[0028] The shape of the through-hole 21 can be appropriately set depending on the opening shape of the recess 11, and is typically cylindrical. The height of the through hole 21 corresponds to the thickness of the frame body 20 . The diameter of the opening of the through hole 21 is typically the same as the diameter of the opening of the recess 11, but may be different as long as the effect of the present invention is not impaired.

[0029] The material constituting the side wall portion 22 of the frame body 20 is not particularly limited, and examples thereof include resins such as acrylic resin, polystyrene resin, polyester resin, polycarbonate resin, polypropylene resin, silicone resin, PET resin, polyvinyl chloride resin, high density polyethylene, polyethersulfane, PET copolymer, Permanox (trademark), cycloolefin polymer resin, Cytop (trademark), etc. These resins may be used alone or in combination of two or more kinds. Glass may be used as a material forming the side wall portion 22 of the frame body 20. When glass is used, examples of the glass include quartz glass, borosilicate glass, phosphate glass, aluminosilicate glass, and tempered glass. These glasses may be used alone or in combination of two or more kinds.

[0030] The side wall 22 of the frame 20 may be transparent or opaque. For example, when observing spheroids using luminescence such as fluorescence, the side wall 22 is preferably opaque from the viewpoint of blocking light from adjacent wells to reduce noise and improve observation. When the side wall 22 is opaque, the color tone is more preferably black. The method of making the side wall 22 opaque is not particularly limited, and for example, a method of blending a coloring agent such as a pigment can be used.

[0031] In the cell culture vessel 1 of the embodiment, the S / N ratio when the fluorescence of the spheroids stained with a fluorescent staining reagent in the wells 11 is measured by a fluorometric method is preferably 11.5 or more, more preferably 12.0 or more, and even more preferably 12.5 or more. If the S / N ratio is equal to or more than the above value, the observation of the spheroids is excellent. The fluorescence of the spheroids can be measured, for example, by a high-content confocal imaging system (PerkinElmer Operetta). Here, S is the signal amount (fluorescence intensity) in the presence of spheroids stained with a fluorescent staining reagent, and N is the signal amount (fluorescence intensity) in the background (absence of spheroids).

[0032] [Manufacturing method of cell culture vessel] The method for producing the cell culture vessel will now be described. The method for producing a cell culture vessel according to the embodiment includes the following steps (i) and (ii). (i) A plate-like member is placed on a mold having a plurality of holes, and while heating, a vacuum is drawn from the side opposite to the side on which the plate-like member is placed, thereby partially deforming the plate-like member to form a plurality of recesses and obtain a bottom portion. (ii) Joining the bottom and the frame body. Steps (i) and (ii) are described in more detail below.

[0033] (Step (i)) 4, for example, a plate-shaped member 10A is placed on a metal mold (lower mold) 110 having a plurality of holes 111 on its upper surface that communicate with a vacuum chamber 112 so that the plurality of holes 111 are blocked, and a metal mold (upper mold) 120 is further placed on top of the plate-shaped member 10A. In this state, by drawing a vacuum from the vacuum chamber 112 side while heating the plate-shaped member 10A, the portions of the plate-shaped member 10A blocking the holes 111 are pulled toward the vacuum chamber 112 and partially deformed, forming recesses.

[0034] Hole 111 formed in mold 110 has a truncated cone-shaped portion 111a whose diameter decreases from the opening downward, and a cylindrical connecting portion 111b connecting truncated cone-shaped portion 111a and vacuum chamber 112. When drawing a vacuum, the central portion of the portion of plate-like member 10A that deforms at each hole 111 does not come into contact with mold 110, and only the peripheral portion comes into contact with the surface of truncated cone portion 111a. As a result, cutting marks made when forming holes 111 in mold 110 are not transferred to the bottom peripheral portion of the recess that is formed. Therefore, by using the bottom peripheral portion of the recess that is not in contact with mold 110 as the culture portion, spheroids are more easily formed and the formed spheroids are more easily observed.

[0035] The opening shape of the lower end of the truncated cone portion 111a of the hole 111 is typically circular, but is not limited to this. The diameter of the opening at the bottom end of the truncated cone-shaped portion 111a can be appropriately set according to the size of the desired culture portion, and can be, for example, 0.8 to 20.0 mm.

[0036] As the plate-like member, for example, a glass sheet, a resin sheet, or the like can be used, and from the viewpoint of the formability and observability of spheroids, a glass sheet is preferable. The thickness of the plate-like member is not particularly limited and may be, for example, 0.03 to 1.0 mm, 0.04 to 0.8 mm, or 0.05 to 0.6 mm, and is most preferably 0.18 mm, which is the same thickness as a cover glass for ordinary microscope observation.

[0037] The mold temperature in step (i) is set according to the material of the plate-like member 10A within a range in which the plate-like member 10A can be sufficiently deformed by evacuation to form recesses. Specifically, the temperature is set so that the log η of the plate-like member 10A is 6.0 to 10.0 poise, preferably 7.0 to 9.0 poise, and more preferably 7.5 to 8.5 poise. For example, when the plate-like member 10A is a glass sheet made of borosilicate glass, the mold temperature is preferably 600 to 800°C, more preferably 630 to 780°C, and even more preferably 650 to 750°C. The time for vacuum suction may be appropriately set so that recesses having the desired shape are formed, and may be, for example, 1 to 4 minutes.

[0038] When the inner surface of the well is to be made a surface with low cell adhesion, for example, a known coating method is used to coat the inner surface of the well with a cell adhesion inhibitor to form a coating.

[0039] (Step (ii)) The method of joining the bottom obtained by forming a recess in the plate-like member to the frame is not particularly limited, and examples of the method include pressure bonding, welding, adhesion with an adhesive, etc. Specifically, for example, a method of attaching the bottom to the frame using a double-sided tape having adhesive layers on both sides of a base sheet can be mentioned. The adhesive is not particularly limited, and examples thereof include silicone-based, instantaneous, epoxy-based, ultraviolet curing, polypropylene-based, acrylic-based, rubber-based, and polyethylene-based adhesives.

[0040] As described above, in the cell culture vessel of the present invention, the maximum height roughness Rz of the surface of the culture part in the well and t A / t B By controlling the amount of the spheroids in the cell culture vessel to be cultured, the cell culture vessel can be efficiently produced by the method including the above-mentioned steps (i) and (ii).

[0041] The cell culture vessel of the present invention is not limited to the cell culture vessel 1 described above. For example, the inner surface of the well (culture section) does not have to be a low cell adhesion surface. In other words, the inner surface of the well (culture section) does not have to be coated with a cell adhesion inhibitor. In this case, the formed spheroids tend to adhere to the inner surface of the well, but the formed spheroids are easily observable.

[0042] If the inner surface of the recess is not a low cell adhesion surface, the thickness ratio t A / t B If the ratio is 0.80 to 1.20, the thickness of the recesses in the culture area is uniform, making the background clear when observing the spheroids, and noise is reduced during fluorescent observation, resulting in excellent observation of the spheroids. t A t B If it is less than t A / t B is 0.80 or more and less than 1.00, preferably 0.85 or more and less than 1.00, more preferably 0.90 or more and less than 1.00, and even more preferably 0.95 or more and less than 1.00. Also, t A t B If it is greater than t A / t B is greater than 1.00 and not greater than 1.20, preferably greater than 1.00 and not greater than 1.15, more preferably greater than 1.00 and not greater than 1.10, and even more preferably greater than 1.00 and not greater than 1.05. Most preferably, t A and B is the same value, i.e., t A / t B =1.00.

[0043] When the inner surface of the well is not a low cell-adhesive surface, the maximum height roughness Rz of the surface of the culture part (inner surface of the well) is 0.170 μm or less, preferably 0.150 μm or less, more preferably 0.120 μm or less, and even more preferably 0.090 μm or less.

[0044] When the inner surface of the well is not a low cell-adhesive surface, the maximum ten-point height roughness Rzjis of the surface of the culture part (inner surface of the well) is preferably 0.150 μm or less, more preferably 0.100 μm or less, and even more preferably 0.080 μm or less.

[0045] When the inner surface of the well is not a low cell-adhesive surface, the surface roughness Ra of the surface of the culture part (inner surface of the well) is preferably 0.024 μm or less, more preferably 0.020 μm or less, and even more preferably 0.018 μm or less.

[0046] In addition, within the scope of the invention, the components in the above-described embodiments may be replaced with well-known components, and the above-described modified examples may be combined as appropriate. EXAMPLES

[0047] The present invention will be specifically described below with reference to examples, but the present invention is not limited to the following description.

[0048] [Experimental Example 1] A glass sheet (thickness: 0.3 mm) of borosilicate glass measuring 111 mm×75 mm was prepared as a plate-like member, and a recess was formed using the molds 110 and 120 illustrated in FIG. 4 to obtain a bottom portion. Specifically, a glass sheet (plate-like member 10A) was placed on top of a mold (lower mold) 110 so that multiple holes 111 were covered, and then a mold (upper mold) 120 was placed on top of that, and the mold was heated to 715°C while being evacuated, thereby obtaining a bottom having 96 recesses. A silane coupling agent having an MPC polymer chain, which is a cell adhesion inhibitor, was applied to the inner surface of each recess to form a coating. Next, the bottom was attached to a polystyrene frame using double-sided tape to prepare a 96-well cell culture vessel.

[0049] [Experimental Example 2] A 96-well cell culture vessel was prepared in the same manner as in Experimental Example 1, except that a coating of the cell adhesion inhibitor was not formed on the inner surface of each well.

[0050] [Experimental Example 3] For comparison, a commercially available polystyrene bottom having 96 recesses was prepared, and a 96-well cell culture vessel was fabricated in the same manner as in Experimental Example 1.

[0051] [Maximum height roughness Rz] The maximum height roughness Rz was measured by a three-dimensional measuring machine UA3P (manufactured by Panasonic Production Engineering Co., Ltd.).

[0052] [Maximum ten-point height roughness Rzjis] The maximum ten-point height roughness Rzjis was measured using a three-dimensional measuring machine UA3P (manufactured by Panasonic Production Engineering Co., Ltd.).

[0053] [Surface roughness Ra] The surface roughness Ra was measured by a three-dimensional measuring machine UA3P (manufactured by Panasonic Production Engineering Co., Ltd.).

[0054] Recess Thickness The thickness of the bottom of the recess and the thickness of the recess at a position on a circular region that is 50 to 60% of the radius of the opening of the recess from the bottom when viewed from above were measured with a dial gauge (manufactured by Mitutoyo Corporation).

[0055] For each concave portion in the experiment, the average thickness of the bottom t A , the average thickness t of the recess at a position on the annular region that is 50 to 60% of the radius of the opening of the recess from the bottom when viewed from above B , thickness ratio t A / t B The maximum height roughness Rz, surface roughness Ra, and maximum ten-point height roughness Rzjis of the culture area surface were measured and the results are shown in Table 1.

[0056] [Culture test 1] Using a 96-well cell culture vessel for each experiment, HepG2 cells (human hepatoma-derived cell line) were cultured in each well at a cell density of 5 × 10 3 The cells were seeded so that each cell was 100% and cultured at 37°C for 2 days in E-MEM / serum 10% medium. After the culture, the cell clusters were fluorescently stained using DRAQ5 (Cosmo Bio), and then fluorescent images were obtained using a high content confocal imaging system (PerkinElmer Operetta). The micrograph of Experimental Example 1 is shown in Figure 5 (Experimental Example 1), and the micrograph of Experimental Example 3 is shown in Figure 6. In addition, image analysis software (Image Analysis Software Harmony TM The size (area) and circularity of the spheroids were measured by the NMR spectroscopy (NMR spectroscopy) and the results are shown in Table 1.

[0057] [Table 1]

[0058] As shown in Fig. 5 and Fig. 6, the maximum height roughness Rz of the surface of the culture part and the thickness ratio t A / t BThe cell culture vessel in Experimental Example 1 that satisfies the conditions is the maximum height roughness Rz and t A / t B Compared to the cell culture vessel of Experimental Example 3 which did not satisfy the above condition, the background was clear and the cultured spheroids were easily observed. Furthermore, as shown in Table 1, spheroids with a higher circularity rate were formed in the cell culture vessel of Experimental Example 1 than in the cell culture vessel of Experimental Example 3. This indicates that the cells in Experimental Example 1 gathered more smoothly toward the bottom in the culture area, making it easier to form spheroids, compared to Experimental Example 3.

[0059] [Culture test 2] Using the 96-well cell culture vessels of Experimental Examples 1 and 3, HepG2 cells (human hepatoma-derived cell line) were placed in each well at a cell density of 5 × 10 3 The cells were seeded so that each cell was 100x the size of a single cell, and cultured at 37°C in E-MEM / 10% serum medium for 2 days. After culture, the cell clusters were fluorescently stained using DRAQ5 (Cosmo Bio), and then fluorescent images were obtained using a high-content confocal imaging system (PerkinElmer Operetta). The signal level of the spheroids (S) and the signal level of the background (N) were obtained, and the S / N ratio was calculated. The results are shown in Figure 7.

[0060] 7, the cell culture vessel of Experimental Example 1 had a higher S / N ratio and better observability of spheroids in fluorescent observation than the cell culture vessel of Experimental Example 3. The minimum S / N ratio in Experimental Example 1 was 11.90 and the maximum was 19.17, while the minimum S / N ratio in Experimental Example 3 was 6.48 and the maximum was 11.14. [Explanation of symbols]

[0061] 1...cell culture vessel, 10...bottom, 11...recess, 11a...inner surface, 12...culture section, 20...frame, 21...through hole, 22...side wall, 30...well, 110...mold (lower mold), 111...hole, 111a...frustum-shaped section, 111b...connecting section, 112...vacuum chamber.

Claims

1. A cell culture vessel for use in culturing and observing cells, a transparent bottom portion on which a plurality of recesses are formed, and a frame body provided on the bottom portion and having a side wall portion which forms a plurality of through holes at positions corresponding to the plurality of recesses when viewed from above; a plurality of wells are formed by the recesses and the through holes, The recess has a rounded shape at least at the bottom, the inner surface of the recess is a low cell-adhesion surface, The average thickness of the bottom of the recess is t Ａ (mm), and the average thickness of the recess at a position on a circular region that is 50 to 60% of the radius of the opening of the recess from the bottom when viewed from above is t Ｂ (mm), the thickness ratio t Ａ / t Ｂ is 0.80 to 1.20, A region inside the annular region when viewed from above in the recess is a culture region, A cell culture vessel, wherein the maximum height roughness Rz of the surface of the culture portion is 0.170 μm or less.

2. The cell culture vessel according to claim 1 , wherein the surface roughness Ra of the culture portion is 0.024 μm or less.

3. 3. The cell culture vessel according to claim 1, wherein the maximum ten-point height roughness Rzjis of the surface of the culture portion is 0.150 μm or less.

4. 3. The cell culture vessel according to claim 1, wherein the S / N ratio when fluorescence measurement is performed on spheroids stained with a fluorescent staining reagent in the culture section is 11.5 or more.

5. The cell culture vessel according to claim 1 or 2, wherein the bottom is made of glass.

6. The cell culture vessel according to claim 1 or 2, wherein the side wall portion of the frame contains a coloring agent.

7. A cell culture vessel for use in culturing and observing cells, a transparent bottom portion on which a plurality of recesses are formed, and a frame body provided on the bottom portion and having a side wall portion which forms a plurality of through holes at positions corresponding to the plurality of recesses when viewed from above; a plurality of wells are formed by the recesses and the through holes, The recess has a rounded shape at least at the bottom, The average thickness of the bottom of the recess is t Ａ (mm), and the average thickness of the recess at a position on a circular region that is 50 to 60% of the radius of the opening of the recess from the bottom when viewed from above is t Ｂ (mm), the thickness ratio t Ａ / t Ｂ is 0.80 to 1.20, A region inside the annular region when viewed from above in the recess is a culture region, A cell culture vessel, wherein the maximum height roughness Rz of the surface of the culture portion is 0.170 μm or less.

8. The cell culture vessel according to claim 7 , wherein the surface roughness Ra of the culture portion is 0.024 μm or less.

9. 9. The cell culture vessel according to claim 7, wherein the maximum ten-point height roughness Rzjis of the surface of the culture portion is 0.150 μm or less.

10. The cell culture vessel according to claim 7 or 8, wherein the bottom is made of glass.

11. The cell culture vessel according to claim 7 or 8, wherein the side wall portion of the frame contains a coloring agent.

12. a transparent bottom portion on which a plurality of recesses are formed, and a frame body provided on the bottom portion and having a side wall portion which forms a plurality of through holes at positions corresponding to the plurality of recesses when viewed from above; A method for manufacturing a cell culture vessel for use in culturing and observing cells, in which a plurality of wells are formed by each of the recesses and the through-holes, comprising: a plate-like member is placed on a mold having a plurality of holes, and while heating, a vacuum is drawn from the side opposite to the side where the plate-like member is placed in each hole, thereby partially deforming the plate-like member and forming a plurality of recesses to obtain the bottom portion; and joining the bottom and the frame.

13. The method for producing a cell culture vessel according to claim 12 , wherein the plate-like member is a glass sheet.

Images