Structure

Abstract

This structure constitutes a culture container having a plurality of accommodation sections in which cells are cultured. The structure comprises: a base that includes a plurality of through holes opening in the vertical direction; and an adherence layer which is provided on the bottom surface of the base and onto the bottom surface of which a sheet for closing the through holes is adhered. The adherence layer has openings that are located below the through holes and that have an outline greater than an outline of the through holes in plan view.

Description

Technical Field

[0001] The present invention relates to a structure constituting a container and a container.

Background Art

[0002] Conventionally, as disclosed in Patent Document 1, cell culture has been performed using a culture container having a housing portion for culturing cells.

[0003] In addition, as a cell culture container, a container having a configuration in which a transparent resin sheet (or film) is attached to the bottom surface of a cylindrical substrate to form a container, or a transparent resin sheet is attached to the upper surface of a well to form a sealed container is known. Also known is a container having a configuration in which a plurality of substrates are attached to form a container shape such as a flow path shape in which liquid flows or a well shape in which liquid does not flow.

[0004] When manufacturing these containers, adhesives or adhesives are used for attaching the transparent resin sheet or the substrate. For example, Patent Document 2 describes that polydimethylsiloxane (PDMS) can be used as an adhesive for attaching a plurality of substrates.

[0005] Also, for example, as described in Patent Document 3, in order to observe substances such as proteins produced by cultured cells or to confirm the state of the cultured cells, a drug such as a fluorescent dye capable of detecting a biologically-derived substance may be introduced into the culture solution in the container.

Prior Art Documents

Patent Documents

[0006] m

Patent Document 1

Patent Document 2

Patent Document 3

Summary of the Invention

[0007] Regarding the culture vessel disclosed in Patent Document 1 mentioned above, there has been a growing demand in recent years for culture vessels with superior culture capabilities.

[0008] In view of these circumstances, the present invention aims, in its first aspect, to provide a structure that constitutes a culture vessel with high culture function, and a culture vessel.

[0009] Furthermore, regarding the culture vessel disclosed in Patent Document 2 mentioned above, the use of a non-curing adhesive makes the fabrication of the cell culture vessel simpler than using an adhesive that requires curing after application and attachment. In addition, according to the inventors' findings, adhesives may shrink during curing, which can cause the attached transparent resin sheet to warp. When the transparent resin sheet warps, the observability of the inside of the container decreases, thus reducing the efficiency of observing bio-derived substances with drugs. Moreover, the shrunk adhesive itself loses its transparency, which can also reduce the efficiency of observing bio-derived substances.

[0010] In contrast, using an adhesive that does not harden eliminates the problem of reduced observation efficiency due to shrinkage during hardening. However, according to the inventors' findings, when cell culture or other processes are performed in containers using adhesives, the observation efficiency may not be as high as expected, for example, because the fluorescence intensity of fluorescent dyes decreases over time.

[0011] In view of these circumstances, the present invention aims, in its second aspect, to provide a structure for manufacturing a container in which different members are attached with an adhesive, thereby improving the efficiency of observing biologically derived substances, and a container manufactured from said structure. [Means for solving the problem]

[0012] One aspect of the structure relating to the first aspect of the present invention is, A structure comprising a culture vessel having multiple containment sections for culturing cells, A base material having multiple through holes that open in the vertical direction, It comprises an adhesive layer provided on the lower surface of the base material, to which a sheet that closes the through hole is attached to the lower surface, The adhesive layer has an opening below the through-hole, the outer shape of which in plan view is larger than the outer shape of the through-hole. When implementing the above-described structure, preferably, the adhesive layer is formed by attaching a test piece cut to a size of 10 mm × 10 mm to the bottom surface of a tissue culture polystyrene well with an opening diameter of 16.2 mm, a well depth of 18 mm, and a well volume of 3.5 mL, filling the well with 0.5 mL of phosphate-buffered saline solution containing 5 μM rhodamine B, and allowing it to stand at 23°C for 48 hours, with an adhesive that leaves a rhodamine B residue of 20% or more in the solution.

[0013] Furthermore, one embodiment of a container relating to the first aspect of the present invention is, The above structure and, It comprises a sheet that closes the through-hole and, together with the through-hole, constitutes a housing section.

[0014] Furthermore, one embodiment of the structure relating to the second aspect of the present invention is, A substrate having through holes or recesses, It has an adhesive layer provided on one surface of the substrate, A structure comprising a container capable of observing biologically derived materials, wherein another substrate or sheet is adhered to it via an adhesive layer to close at least one side or recess of a through-hole, The adhesive that forms the adhesive layer is A test specimen cut to a size of 10 mm x 10 mm was attached to the bottom surface of a tissue culture polystyrene well with an opening diameter of 16.2 mm, a well depth of 18 mm, and a well volume of 3.5 mL. 0.5 mL of phosphate-buffered saline solution containing 5 μM rhodamine B was filled into the well, and after standing at 23°C for 48 hours, the remaining rhodamine B in the solution was 20% or more, and, For a 5mm wide adhesive test piece, the resulting load when applying a 0.05% dynamic strain using a solid viscoelasticity measuring device is 20g or less.

[0015] Furthermore, one embodiment of a container relating to a second aspect of the present invention is: The structure relating to the second aspect of the present invention described above, It comprises another substrate or sheet attached to the structure via an adhesive layer. [Effects of the Invention]

[0016] According to the first aspect of the present invention, a culture vessel with high culture function can be provided.

[0017] Furthermore, according to a second aspect of the present invention, a structure for manufacturing a container in which different members are attached with an adhesive, thereby improving the efficiency of observing biologically derived substances, and a container manufactured from said structure are provided. [Brief explanation of the drawing]

[0018] [Figure 1] Figure 1 is a perspective view of a culture vessel according to the first embodiment. [Figure 2] Figure 2 is a cross-sectional view of a portion of the culture vessel. [Figure 3] Figure 3 is a plan view of the adhesive layer. [Figure 4] Figure 4 is a diagram illustrating the shape of the through-hole in the cylindrical portion and the opening in the adhesive layer. [Figure 5] Figure 5 is a diagram illustrating the amount of displacement between the through-hole in the cylindrical portion and the opening in the adhesive layer. [Figure 6] Figure 6 is a diagram illustrating the amount of displacement between the through-hole in the cylindrical portion and the opening in the adhesive layer. [Figure 7] Figure 7 is a cross-sectional view of a portion of the adhesive layer. [Figure 8] Figure 8 is a plan view of an adhesive layer according to an example of a modified example. [Figure 9] Figure 9 is a perspective view of a culture vessel according to the second embodiment. [Figure 10] Figure 10 is a cross-sectional view of a portion of the culture vessel. [Figure 11] Figure 11 is a plan view of the adhesive layer. [Figure 12] Figure 12 is a cross-sectional view of a part of the culture vessel according to the third embodiment. [Modes for carrying out the invention]

[0019] The structures and culture vessels according to the present invention will be described in detail below with reference to the drawings. Note that the structures and culture vessels described below are examples of the structures and culture vessels according to the present invention, and the present invention is not limited to the embodiments described later.

[0020] [First Embodiment] A structure and culture vessel according to the first embodiment of the present invention will be described with reference to Figures 1 to 7.

[0021] Figure 1 is a perspective view of a culture vessel 1 according to the first embodiment of the present invention. Figure 2 is a cross-sectional view of a part of the culture vessel 1.

[0022] Culture vessel 1 is used, for example, to culture human-derived cells. Such culture vessel 1 has a plurality of containment sections 10 (see Figure 2) for culturing cells. Culture vessel 1 is placed in the culture space of a culture device (for example, an incubator) with the drug (culture medium) and the cells to be cultured (hereinafter referred to as "target cells") contained in the containment sections 10.

[0023] The culture vessel 1 of this embodiment can be suitably used, for example, for culturing spheroids (cell aggregates) of target cells. Note that the culture vessel 1 does not need to be used while housed in a culture device. The culture vessel 1 may be used in various situations depending on the target cells.

[0024] The following describes the specific configuration of culture vessel 1.

[0025] The culture vessel 1 has a structure 2 and a gas permeable sheet 5.

[0026] Furthermore, when describing the structure of culture vessel 1, the Cartesian coordinate system (X, Y, Z) shown in Figure 1 may be used. The X direction corresponds to the front-to-back direction of culture vessel 1. The X-direction + side corresponds to the front side of culture vessel 1. The X-direction - side corresponds to the rear side of culture vessel 1. The Y direction corresponds to the left-to-right direction of culture vessel 1. The Y-direction + side corresponds to the left side of culture vessel 1. The Y-direction - side corresponds to the right side of culture vessel 1. The Z direction corresponds to the up-and-down direction of culture vessel 1. The Z-direction + side corresponds to the top side of culture vessel 1. The Z-direction - side corresponds to the bottom side of culture vessel 1. Note that the left, right, front, and rear sides of culture vessel 1 differ depending on the direction from which culture vessel 1 is viewed, and are not limited to the left, right, front, and rear sides of culture vessel 1 shown in Figure 1.

[0027] Structure 2 has a main body 3 and an adhesive layer 4.

[0028] The main body 3 is an example of a base material and has a frame 30 and a main body side housing 31. The main body 3 is, for example, made of synthetic resin and is a one-piece molded product made by injection molding.

[0029] The synthetic resin constituting the main body 3 may be, for example, polystyrene or polyolefin. The polyolefin may be a cyclic olefin (co)polymer, a 4-methyl-1-pentene (co)polymer, polypropylene, or polyethylene.

[0030] From the viewpoint of gas permeability, a 4-methyl-1-pentene (co)polymer is preferred as the synthetic resin constituting the main body 3. Furthermore, from the viewpoint of heat resistance, a 4-methyl-1-pentene (co)polymer, polystyrene, polypropylene, or a cyclic olefin (co)polymer is preferred as the synthetic resin constituting the main body 3.

[0031] The instantaneous Shore D hardness of the main body 3 is, for example, 60 to 90. The instantaneous Shore D hardness is obtained by using a durometer hardness tester (Type D) to immediately read the scale after bringing the indenter into contact with a part of the main body 3 (in accordance with ASTM D2240).

[0032] If the instantaneous value of the Shore D hardness is in the range of 60 to 90, the gas permeable sheet 5, described later, can be attached to the main body 3 in a good appearance.

[0033] The frame portion 30 is composed of rectangular frame-shaped members. The frame portion 30 constitutes the outer shape of the culture container 1.

[0034] The frame portion 30 has a lower frame portion 300 and an upper frame portion 301.

[0035] The lower frame portion 300 is a rectangular frame-shaped member and constitutes the lower half of the frame portion 30. The lower frame portion 300 is an example of the lower peripheral wall portion.

[0036] The lower frame portion 300 is the part that, when the culture container 1 is in use, is placed on a mounting section provided on, for example, a culture device (not shown). The lower frame portion 300 has a height such that the lower surface of the gas permeable sheet 5, described later, does not come into contact with the mounting surface of the mounting section.

[0037] The lower frame portion 300 is provided so as to surround the entire perimeter of the space located below the main body side housing portion 31, which will be described later. The space located below the main body side housing portion 31 is also the space located below the gas permeable sheet 5, which will be described later (hereinafter referred to as the "space below the gas permeable sheet 5").

[0038] The lower frame portion 300 may have multiple notches 300a at its lower end. The notches 300a connect the space on the inner circumferential side of the lower frame portion 300 (i.e., the space below the gas permeable sheet 5) with the space on the outer circumferential side of the lower frame portion 300 (i.e., the external space) when the culture container 1 is in use.

[0039] Therefore, when the culture vessel 1 is in use, air from the external space is supplied to the area below the gas permeable sheet 5 through the notch 300a.

[0040] As described above, the configuration in which the lower frame portion 300 has a notch portion 300a contributes to improving the oxygen supply to the lower region of the gas permeable sheet 5. Furthermore, the oxygen supplied to the lower region of the gas permeable sheet 5 is supplied to the target cells contained in the containment portion 10 of the culture vessel 1 via the gas permeable sheet 5, thereby promoting the culture of the target cells. Note that the position and / or number of the notches are not limited to the position and / or number of the notches 300a described above. Also, the notches may be omitted.

[0041] The upper frame portion 301 is a rectangular frame-shaped member and constitutes the upper half of the frame portion 30. The upper frame portion 301 is provided above the lower frame portion 300. The upper frame portion 301 is an example of the upper peripheral wall portion.

[0042] The upper frame portion 301 is provided so as to surround the main body side housing portion 31 all around.

[0043] The upper end of the upper frame portion 301 has a shape that fits with the lower end of the lower frame portion 300 of another culture container 1. In other words, the culture container 1 is configured so that multiple culture containers 1 can be stacked vertically.

[0044] The upper frame portion 301 of the culture container 1 positioned below and the lower frame portion 300 of the culture container 1 positioned above are fitted together, allowing the culture container 1 positioned below and the culture container 1 positioned above to be stacked without wobbling.

[0045] The main body side housing section 31 is provided in a space enclosed by the frame section 30. The main body side housing section 31 is provided integrally with the frame section 30.

[0046] The main body side housing section 31 has a plurality of cylindrical sections 310 and a bottom plate section 311.

[0047] The cylindrical portion 310 is cylindrical with an opening in the vertical direction. In this embodiment, the shape of the opening of the cylindrical portion 310 (in other words, the outer shape in a plan view) is circular. The cylindrical portions 310 are arranged side by side in the left-right and front-back directions.

[0048] The number of tubular sections 310 corresponds to the number of containment sections 10 for culturing cells. The number of tubular sections 310 may be, for example, 6, 24, 96, or 384. Of course, the number of tubular sections 310 may be other than 6, 24, 96, and 384.

[0049] Adjacent cylindrical sections 310 are connected by connecting sections (not shown). In addition, the leftmost cylindrical section 310, the rightmost cylindrical section 310, the frontmost cylindrical section 310, and the rearmost cylindrical section 310 are connected to the inner circumferential surface of the frame section 30 via connecting sections (not shown).

[0050] The space defined by the inner circumferential surface of the cylindrical portion 310 (hereinafter referred to as the "inner space of the cylindrical portion 310") corresponds to an example of a through-hole in the substrate. The inner space of the cylindrical portion 310, together with the gas permeable sheet 5 described later, constitutes the containment section 10 for culturing cells. Hereinafter, the inner space of the cylindrical portion 310 may also be simply referred to as the through-hole 310a.

[0051] In this embodiment, the outer shape of the through-hole 310a in plan view is circular. That is, the inner circumferential surface of the through-hole 310a is cylindrical.

[0052] Note that a plan view means viewing the culture vessel 1 and structure 2 from above (Z-direction + side). Unless otherwise specified, the term "outer shape of the through-hole 310a" refers to the outer shape of the through-hole 310a in a plan view.

[0053] The outer shape of the through-hole 310a is preferably circular or square. From the viewpoint of improving the adhesion strength between the gas permeable sheet 5 and the area around the through-hole 310a, the outer shape of the through-hole 310a is more preferably circular. This is because, when the outer shape of the through-hole 310a is circular, adhesion can be more evenly achieved at the point of contact between the area around the through-hole 310a and the gas permeable sheet 5.

[0054] Furthermore, from the viewpoint of increasing the volume occupied by the through-holes 310a (in other words, the volume of the storage section 10), a rectangular shape is preferable for the through-holes 310a. For example, if there are many through-holes 310a (for example, 384), the volume of the storage section 10 may become smaller if the through-holes 310a are circular. In this case, if the through-holes 310a are polygonal, such as a rectangle, it becomes easier to increase the volume of the storage section 10 compared to the case where the through-holes 310a are circular.

[0055] As shown in Figure 2, the bottom plate portion 311 is a plate-like member that connects the lower ends of the cylindrical portions 310. The outer edge of the bottom plate portion 311 is connected to the inner surface of the frame portion 30 around its entire circumference. The lower surface of the bottom plate portion 311 is also the lower surface of the main body side housing portion 31. Furthermore, the lower surface of the bottom plate portion 311 is also the lower surface of the main body portion 3. Therefore, the lower surface of the bottom plate portion 311 is an example of the lower surface of the base material.

[0056] The adhesive layer 4 will be described below with reference to Figures 2 to 7. The adhesive layer 4 is an example of an adhesive layer and is provided on the lower surface of the main body 3. Specifically, the adhesive layer 4 is provided on the lower surface of the main body side housing 31. Such an adhesive layer 4 is composed of an adhesive. The adhesive layer 4 is a component for attaching the gas permeable sheet 5, which will be described later, to the lower surface of the main body side housing 31.

[0057] The adhesive layer 4 has an adhesive layer body 40 and a plurality of openings 41.

[0058] The adhesive layer body 40 is a sheet-like member and is the portion that adheres to the lower surface of the main body side housing portion 31 (specifically, the bottom plate portion 311) and to the gas permeable sheet 5.

[0059] The opening 41 is provided in the adhesive layer body 40. The opening 41 penetrates the adhesive layer body 40 in the vertical direction.

[0060] As shown in Figure 2, the opening 41 is positioned below the through-hole 310a of the main body housing 31 when it is attached to the lower surface of the main body housing 31. As shown in Figures 2 to 4, the outer shape of the opening 41 in plan view is larger than the outer shape of the through-hole 310a. In Figures 3 and 4, the opening 41 is shown by a solid line. In Figures 3 and 4, the through-hole 310a is shown by a dashed line.

[0061] In this embodiment, the outer shape of the opening 41 in plan view is circular, as shown in Figures 3 and 4. Hereafter, when referring to the outer shape of the opening 41, it means the outer shape of the opening 41 in plan view. The outer shape of the through hole 310a is also circular. The outer shape of the opening 41 is similar to the outer shape of the through hole 310a.

[0062] In Figure 4, the center O of the outer shape of the opening 41 41 and the center of the outer shape of the through hole 310a O 310a This is consistent with the above. In this state, the peripheral edge of the opening 41 does not overlap with the through hole 310a in a plan view.

[0063] If the periphery of the opening 41 overlaps with the through-hole 310a in a plan view, the contact area between the adhesive layer 4 and the drug contained in the containment section 10 may increase when the culture vessel 1 is in use. When the contact area between the adhesive layer 4 and the drug contained in the containment section 10 increases, the amount of drug adsorbed to the adhesive layer 4 increases, which may decrease the concentration of the drug in the containment section 10. A decrease in the concentration of the drug in the containment section 10 is undesirable because it can cause a decrease in culture function.

[0064] On the other hand, in this embodiment, the peripheral edge of the opening 41 does not overlap with the through hole 310a in a plan view. Therefore, the contact area between the adhesive layer 4 and the drug when the culture container 1 is in use is small. In other words, the amount of drug adsorbed by the adhesive layer 4 is small. As a result, the deterioration of the culture function is suppressed. Hereinafter, the effect exhibited by the culture container 1 of this embodiment will be referred to as the drug adsorption suppression effect of the culture container 1.

[0065] Furthermore, the positional relationship between the opening 41 and the through-hole 310a is most preferably the positional relationship between the opening 41 and the through-hole 310a shown in FIGS. 3 and 4.

[0066] However, as shown in FIG. 5, if the peripheral edge of the opening 41 and the through-hole 310a do not overlap, the center O of the outer shape of the opening 41 41 and the center O of the through-hole 310a 310a may be offset.

[0067] If the positional relationship between the peripheral edge of the opening 41 and the through-hole 310a is the positional relationship between the peripheral edge of the opening 41 and the through-hole 310a shown in FIG. 5, the above-described drug adsorption suppression effect can be obtained.

[0068] Also, as shown in FIG. 6, even if the configuration is such that a part of the peripheral edge of the opening 41 and the through-hole 310a overlap, a part of the above-described drug adsorption suppression effect can be obtained.

[0069] The difference between the radius R of the opening 41 41 and the radius R of the through-hole 310a 310a may be smaller than 1 / 2 of the distance L between the ends of adjacent openings 41. In the case of the present embodiment, the distance between the ends of adjacent openings 41 in the vertical direction in FIG. 3 is equal to the distance between the ends of adjacent openings 41 in the horizontal direction in FIG. 3. 41 It should be noted that in the case of the present embodiment, the distance between the ends of adjacent openings 41 in the vertical direction in FIG. 3 is equal to the distance between the ends of adjacent openings 41 in the horizontal direction in FIG. 3. When the distance between the ends of adjacent openings 41 varies depending on the adjacent direction, the smallest distance is the distance between the ends of adjacent openings 41.

[0071] Also, the distance L1 between the center O of the opening 41 41 and the center O of the through-hole 310a 310a may be at most 1 / 2 of the difference between the radius R of the opening 41 41 and the radius R of the through-hole 310a 310a It should be noted that the distance between the center O of the opening 41 41 and the center O of the through-hole 310a 310a is the distance between the center O of the opening 41 <00000!4>and the center O of the through-hole 310a 310a ​It is also the amount of deviation from that.

[0072] The adhesive layer 4 has a laminated structure in which multiple sheet-like members are stacked. Specifically, as shown in Figure 7, the adhesive layer 4 has a base layer 420, an upper adhesive layer 421, and a lower adhesive layer 422. The thickness dimension H of the adhesive layer 4 may be, for example, 20 μm ≤ H ≤ 150 μm.

[0073] Figure 7 schematically shows a cross-sectional view of a portion of the adhesive layer 4. Furthermore, Figure 7 shows the upper protective film 6a provided on the upper surface of the upper adhesive layer 421, and the lower protective film 6b provided on the lower surface of the lower adhesive layer 422, indicated by dashed lines.

[0074] The adhesive layer 4 constitutes the culture container 1 when the upper protective film 6a and the lower protective film 6b are peeled off. The adhesive layer 4 also constitutes the structure 2 when only the upper protective film 6a of the upper protective film 6a and the lower protective film 6b is peeled off.

[0075] The base layer 420 is a sheet-like member made of resin. The resin constituting the base layer 420 may be, for example, polyethylene terephthalate (PET).

[0076] The base layer 420 has a base layer side body 420a and a base layer side opening 420b.

[0077] The base layer body 420a is a sheet-like member made of resin. The upper adhesive layer 421 is attached to the upper surface of the base layer body 420a. The lower adhesive layer 422 is attached to the lower surface of the base layer body 420a. The base layer body 420a constitutes an intermediate layer of the adhesive layer body 40.

[0078] The base layer side opening 420b is provided in the base layer side body 420a. The base layer side opening 420b penetrates the base layer side body 420a in the vertical direction. The base layer side opening 420b constitutes the middle portion of the opening 41.

[0079] The upper adhesive layer 421 is an example of a first adhesive layer and is a sheet-like member having adhesive properties.

[0080] The upper adhesive layer 421 has an upper body 421a and an upper opening 421b.

[0081] The upper body 421a is a sheet-like member made of adhesive. The upper body 421a is attached to the upper surface of the base body 420a. The adhesive constituting the upper body 421a may be, for example, an acrylic or silicone-based adhesive. The upper body 421a constitutes the upper portion of the adhesive layer body 40.

[0082] The upper-side opening 421b is provided in the upper-side body 421a. The upper-side opening 421b penetrates the upper-side body 421a in the vertical direction. The inner diameter of the upper-side opening 421b is equal to the inner diameter of the base-side opening 420b.

[0083] With the upper adhesive layer 421 attached to the upper surface of the base layer body 420a, the central axis of the base layer opening 420b and the central axis of the upper layer opening 421b coincide. The upper layer opening 421b constitutes the upper end of the opening 41.

[0084] The lower adhesive layer 422 is an example of a second adhesive layer and is an adhesive sheet-like member.

[0085] The lower adhesive layer 422 has a lower body 422a and a lower opening 422b.

[0086] The lower layer body 422a is a sheet-like member made of adhesive. The lower layer body 422a is attached to the lower surface of the base layer body 420a. The lower layer body 422a constitutes the lower part of the adhesive layer body 40.

[0087] The adhesive constituting the lower body 422a is the same as the adhesive constituting the upper body 421a. However, the adhesive constituting the lower body 422a may be different from the adhesive constituting the upper body 421a.

[0088] The lower-side opening 422b is provided in the lower-side body 422a. The lower-side opening 422b penetrates the lower-side body 422a in the vertical direction. The inner diameter of the lower-side opening 422b is equal to the inner diameter of the base-side opening 420b.

[0089] With the lower adhesive layer 422 attached to the lower surface of the base layer body 420a, the central axis of the base layer opening 420b and the central axis of the lower layer opening 422b coincide. The lower layer opening 422b constitutes the lower end of the opening 41.

[0090] Although not shown in the diagram, the adhesive layer may have a single-layer structure. In this case, the adhesive layer may consist of, for example, a base layer made of a nonwoven fabric and an adhesive composition attached to or impregnated into the base layer.

[0091] Furthermore, the outer shape of the opening in the adhesive layer is not limited to a circle. The outer shape of the opening may be, for example, a circle, a rectangle, or a regular polygon.

[0092] Figure 8 shows an example of a modified adhesive layer. The adhesive layer 4a shown in Figure 8 has an adhesive layer body 40a and a plurality of openings 41a.

[0093] In this example, the outer shape of the opening 41a is square. As shown in Figure 8, the outer shape of the opening 41a in plan view is larger than the outer shape of the through hole 310b. Figure 8 is the same as Figure 3. In Figure 8, the opening 41a is shown by a solid line. Also in Figure 8, the through hole 310b is shown by a dashed line. The other components of the adhesive layer 4a are almost the same as those of the adhesive layer 4 in the first embodiment described above.

[0094] Furthermore, the adhesive layer is not limited to the adhesive layer 4 described above. For example, the adhesive layer may consist of an adhesive layer containing an adhesive.

[0095] The gas permeable sheet 5 is a flat, sheet-like member. The gas permeable sheet 5 is attached to the lower surface of the main body 3 via an adhesive layer 4. Specifically, the gas permeable sheet 5 is attached to the lower surface of the bottom plate portion 311 in the main body side housing portion 31 via an adhesive layer 4.

[0096] Gas permeable sheet 5 has an oxygen permeability of 200 to 60,000 cm². 3 / (m 2 It is a sheet-like member with a temperature of ×24hr×atm. The gas permeable sheet 5 is a sheet-like member containing a polymer having a constituent unit derived from, for example, 4-methyl-1-pentene.

[0097] The oxygen permeability values ​​mentioned above were measured using a differential pressure type gas permeability meter MT-C3 manufactured by Toyo Seiki Seisakusho Co., Ltd., at a temperature of 23°C and 0% humidity [cm²]. 3 / (m 2 The measured value was calculated as (×24h × atm). The measurement sample was prepared by cutting a 90 × 90 mm test piece from gas permeability sheet 5. The diameter of the measurement area was 70 mm (permeability area was 38.46 cm²). 2 ) was used. When high oxygen permeability is expected, an aluminum mask was placed over the measurement sample beforehand, and the actual permeable area was set to 5 cm². 2 It is preferable to do so.

[0098] The gas permeable sheet 5 closes the lower opening of the through-hole 310a in the main body side housing section 31. The upper surface of the gas permeable sheet 5, together with the through-hole 310a, constitutes the housing section 10 of the culture container 1.

[0099] The following briefly describes a method for culturing spheroids using the culture vessel 1 of the above-described embodiment. The operator places the drug (culture medium) and target cells into the containment section 10 of the culture vessel 1. The target cells may be, for example, cells collected from a human. The culture medium may be appropriately selected depending on the target cells. The target cells settle at the lower end of the containment section 10 (i.e., on the upper surface of the gas permeable sheet 5).

[0100] Next, the operator places the culture vessel 1 into the culture space of a culture device (not shown), such as an incubator. The environmental conditions of the culture space are set to an environment suitable for culturing the target cells. After a predetermined time has elapsed, the cells are cultured in the containment section 10 of the culture vessel 1.

[0101] In this embodiment, the culture vessel 1 has a gas permeable sheet 5 in the lower region of the containment section 10. Because the gas permeable sheet 5 has excellent oxygen permeability, a large amount of oxygen is supplied to the target cells that have settled at the lower end of the containment section 10. As a result, the culture of the target cells is promoted.

[0102] Furthermore, cultured cells tend to accumulate at the lower end of the containment section 10. Therefore, spheroids (cell aggregates) of the target cells are easily formed at the lower end of the containment section 10. As described above, the culture vessel 1 of this embodiment can be suitably used for culturing spheroids (cell aggregates) of target cells.

[0103] (Operation and effects of this embodiment) According to the culture vessel 1 of this embodiment, which has the configuration described above, a culture vessel with high culture performance can be provided. The reasons for this will be explained below.

[0104] First, in the case of the culture container 1 according to this embodiment, the adhesive layer 4 has an opening 41 whose outer shape in plan view is larger than the outer shape of the through hole 310a, as shown in Figures 3 and 4. Therefore, the periphery of the opening 41 does not easily overlap with the through hole 310a in plan view. Thus, the contact area between the adhesive layer 4 and the drug when the culture container 1 is in use is reduced, and the amount of drug adsorbed to the adhesive layer 4 can be suppressed. As a result, the decrease in drug concentration can be suppressed, and the culture function of the culture container 1 can be improved. Furthermore, as will be described later, the culture container 1 may also contain reagents for fluorescence observation of cultured cells. According to the culture container 1 according to this embodiment, the amount of such reagents adsorbed to the adhesive layer 4 can be suppressed. As a result, the culture container 1 according to this embodiment can obtain high observation efficiency.

[0105] In particular, in this embodiment, as shown in Figures 3 and 4, the peripheral edge of the opening 41 does not overlap with the through hole 310a in a plan view. This configuration minimizes the contact area between the adhesive layer 4 and the drug. Therefore, the amount of drug adsorbed onto the adhesive layer 4 can be significantly reduced. Other effects and benefits obtained from the culture vessel 1 of this embodiment are as described above.

[0106] (Preferred embodiment of adhesive layer 4) In this embodiment, the adhesive layer 4 may satisfy one or more of the following conditions.

[0107] (Condition 1) A test piece cut to a size of 10 mm x 10 mm is attached to the bottom surface of a tissue culture polystyrene (TCPS) well with an opening diameter of 16.2 mm, a well depth of 18 mm, and a well volume of 3.5 mL. 0.5 mL of phosphate-buffered saline (PBS) solution containing 10 μmol / L rhodamine B is then filled into the well, and after standing at 23°C for 48 hours, the remaining rhodamine B in the aqueous solution is 20% or more.

[0108] According to the inventors' findings, the adhesive layer 4 readily adsorbs the drug, which can reduce the concentration of the drug introduced into the containment section 10.

[0109] Based on the above findings, the inventors investigated the adsorption amounts of various drugs for multiple adhesives and found that rhodamine B can serve as an indicator for predicting the adsorption amount of various drugs by adhesives. In other words, compared to other drugs, rhodamine derivatives tend to show differences in adsorption amounts depending on the adhesive. Furthermore, by using an adhesive with low rhodamine B adsorption, high observation efficiency can be obtained even when observing with other drugs.

[0110] Condition 1 is based on the aforementioned findings and identifies that the adhesive layer 4 has a low adsorption capacity for rhodamine B. The adhesive layer 4 that satisfies Condition 1 is less likely to adsorb various drugs in the portion exposed to the inner surface of the containment section 10.

[0111] Regarding condition 1, in this embodiment, the adhesive layer 4 is cut to a size of 10 mm x 10 mm to serve as a test piece. The thickness of the test piece is the thickness of the adhesive itself if it is a commercially available product, and is also the thickness of the adhesive layer 4. The test piece is then attached to the bottom surface of a tissue culture polystyrene well with an opening diameter of 16.2 mm, a well depth of 18 mm, and a well volume of 3.5 mL. 0.5 mL of phosphate-buffered saline (PBS) solution containing 5 μM rhodamine B is filled into the well and left to stand at 23°C for 48 hours. If the remaining percentage of rhodamine B in the aqueous solution after standing is 20% or more (i.e., the concentration of rhodamine B in the aqueous solution after standing is 1 μM or more), then the adhesive layer 4 is considered to satisfy condition 1.

[0112] Furthermore, the amount of rhodamine B in the aqueous solution after standing shall be the value measured by fluorescence analysis using the following procedure. Measuring device: FP-6600 manufactured by JASCO Corporation Cell used: Quartz microcell Bandwidth: Excitation side: 5nm, Fluorescence side: 6nm Sensitivity (PMT voltage): 400V Excitation wavelength: 555 nm (Rhodamine B) Fluorescence measurement wavelength: 580nm (Rhodamine B) Scan speed: 2000nm / min

[0113] From the above perspective, the remaining percentage of rhodamine B in Condition 1 is preferably 30% or more, and more preferably 40% or more. While there is no particular upper limit to the remaining percentage of rhodamine B in Condition 1, it can be 100%.

[0114] (Condition 2) For a test specimen with a 5 mm wide adhesive layer 4, the resulting load when applying a 0.05% dynamic strain using a solid viscoelasticity measuring device is 20 g or less.

[0115] Condition 2 means that the adhesive layer 4 is easily deformable even with a small force. Such an adhesive layer 4 can easily conform to the shape of the bottom surface (underside) of the housing section 31 on the main body side, and is less likely to create minute gaps or flexures when attached. Therefore, an adhesive layer 4 that satisfies condition 2 is less likely to cause a decrease in observation efficiency due to unintended refraction of light caused by these gaps or flexures. In addition, an adhesive layer 4 that satisfies condition 2 reduces the contact area with the liquid (culture medium, etc.) filled inside the housing section 10, thereby reducing the amount of various drugs adsorbed by the adhesive layer 4.

[0116] Regarding condition 2, in this embodiment, the adhesive layer 4 is cut to a size of 5 mm in width and 30 mm in length to serve as a test specimen. The thickness of the test specimen is the thickness of the adhesive layer 4. The adhesive layer 4 is deemed to satisfy condition 2 when, under the following conditions, the load required to impart a 0.05% dynamic strain to the test specimen is 20 g or less when solid viscoelasticity measurement is performed. Measurement device: RSA-III, manufactured by T.A. Instruments Co., Ltd. Deformation mode: Tension Temperature: 23℃ Frequency: 1Hz Distortion: 0.05% Atmospheric environment: Nitrogen atmosphere

[0117] From the above viewpoint, the resulting load when applying a 0.05% dynamic strain under Condition 2 is preferably 18g or less, and more preferably 15g or less. While there is no particular lower limit to the resulting load when applying a 0.05% dynamic strain under Condition 2, from the viewpoint of facilitating application, it can be 2g or more, and preferably 5g or more.

[0118] (Condition 3) The glass transition temperature (Tg) is -122°C or higher.

[0119] Condition 3 means that the adhesive forming the adhesive layer 4 does not have excessively high molecular mobility. In other words, if the molecules constituting the adhesive have high mobility, the adhesive will be more likely to adsorb drugs. In contrast, an adhesive that satisfies condition 3 has moderately low molecular mobility, so it is less likely to adsorb various drugs at the parts exposed on the inner surface of the containment section 10. In particular, when the adhesive is an acrylic adhesive, the higher the Tg, the more likely the amount of drug adsorbed will decrease.

[0120] Regarding condition 3, in this embodiment, a solid viscoelastic temperature dispersion measurement of the adhesive is performed under the following conditions, and the peak top value of the obtained loss tangent (tanδ) is taken as the glass transition temperature (Tg) of the adhesive. (Measurement conditions) Measurement device: RSA-III, manufactured by T.A. Instruments Co., Ltd. Deformation mode: Tension Temperature range: -100℃ to 25℃ Silence rate: 3℃ / min Frequency: 1Hz Distortion: 0.05% Atmospheric environment: Air

[0121] Furthermore, if the goal is to increase adhesiveness, a lower Tg of the adhesive is preferable. On the other hand, from the viewpoint of improving the observation efficiency of bio-derived materials based on the above findings, a higher Tg of the adhesive is preferable. From the viewpoint of achieving both of these, the Tg of the adhesive under condition 3 is more preferably -121°C to -30°C, even more preferably -80°C to -30°C, and particularly preferably -35°C to -30°C.

[0122] (Condition 4) It is acrylic-based and contains an adhesive having a structure derived from an aromatic ring-containing compound.

[0123] Acrylic adhesives having a structure derived from aromatic ring-containing compounds have reduced molecular mobility due to the aromatic ring (benzene ring). Therefore, adhesives that satisfy condition 3 have low molecular mobility, making it difficult for them to adsorb various drugs at the parts exposed on the inner surface of the containment section 10. Note that the aromatic ring-containing compound is not particularly limited as long as it is a compound containing an aromatic ring (benzene ring). Examples of aromatic ring-containing compounds include styrene, styrene sulfonic acid and its salts, α-methylstyrene, pt-butylstyrene, butoxystyrene, vinyltoluene, chlorostyrene, and aromatic vinyl compounds such as vinylnaphthalene.

[0124] Regarding condition 3, in this embodiment, the surface of the adhesive layer 4 was measured by Fourier transform infrared spectrophotometric (FT / IR) under the following conditions, and the obtained IR spectrum contained 700 cm² corresponding to the styrene-derived structure. -1 When the peak is confirmed, the adhesive layer 4 is deemed to satisfy condition 4. (Measurement conditions) Measurement device: Thermo Fisher Scientific Nicoleti S50 Measurement method: Total internal reflection measurement method (ATR method) using a Ge prism Resolution: 4cm -1 Total number of times: 32

[0125] (Condition 5) It is a silicone-based material, and in the IR spectrum, the 1068 cm⁻¹ originates from Si-O. -1 843 cm⁻¹ of Si-CH3 relative to the peak intensity -1 The adhesive includes a peak intensity ratio ((Si-CH3) / (Si-O)) of 0.500 or more and 0.600 or less.

[0126] (Condition 6) It is a silicone-based material, and in the IR spectrum, the 1068 cm⁻¹ originates from Si-O. -1 756 cm² of the peak intensity originating from Si-CH3 -1The adhesive includes a peak intensity ratio ((Si-CH3) / (Si-O)) of 0.280 or more and 0.300 or less.

[0127] Conditions 5 and 6 specify the amount of terminal methyl groups relative to the siloxane bonds in the silicone adhesive, meaning that the molecular weight of the silicone adhesive is of an appropriate size. Adhesives that satisfy condition 5 or condition 6 have moderately low molecular mobility, making them less likely to adsorb various drugs at the portion exposed on the inner surface of the containment section 10. On the other hand, adhesives that satisfy condition 5 or condition 6 have sufficient molecular mobility, resulting in sufficiently high adhesiveness.

[0128] Furthermore, an adhesive that satisfies condition 5 or condition 6 may satisfy only one of these conditions or both of these conditions.

[0129] Furthermore, conditions 5 and 6 are the ratios of the respective peak intensities for the IR spectra obtained by measurement under the same conditions as condition 3.

[0130] (Condition 7) A test piece cut to a size of 10 mm x 10 mm is attached to the bottom surface of a tissue culture polystyrene well with an opening diameter of 16.2 mm, a well depth of 18 mm, and a well volume of 3.5 mL. 0.5 mL of a PBS solution containing 5 μM rhodamine 6 G is then filled into the well, and after standing at 23°C for 48 hours, the remaining rhodamine 6 G in the aqueous solution is 20% or more.

[0131] According to our findings, similar to rhodamine B under condition 1, rhodamine 6G is also prone to differences in adsorption amounts depending on the adhesive compared to other drugs. Furthermore, adhesives with low adsorption of rhodamine 6G tend to have low adsorption amounts for other drugs as well. Therefore, adhesive layer 4 that satisfies condition 7 is less likely to adsorb various drugs in the portion exposed on the inner surface of the containment section 10.

[0132] Condition 7 is the remaining percentage of rhodamine 6G in the aqueous solution after standing, obtained by measuring under the same conditions as Condition 1, except that a 5 μM rhodamine 6G aqueous solution was used instead of a 0.5 mL / well rhodamine B aqueous solution.

[0133] From the above perspective, the remaining percentage of rhodamine 6G in condition 1 is preferably 30% or more, more preferably 40% or more, and even more preferably 50% or more. While there is no particular upper limit to the remaining percentage of rhodamine 6G in condition 7, it can be 100%.

[0134] (Condition 8) The weight-average molecular weight (Mw) is between 1,000 and 5,000,000.

[0135] Condition 8 means that the molecular weight of the adhesive is of an appropriate size. Adhesives that satisfy condition 8 have moderately low mobility of the molecules that make up the adhesive, so they do not easily adsorb various drugs at the parts exposed on the inner surface of the containment section 10. On the other hand, adhesives that satisfy condition 8 have sufficient mobility of the molecules that make up the adhesive, so the adhesiveness of the adhesive is also sufficiently high. From the above perspective, the weight-average molecular weight (Mw) of the adhesive under condition 8 is preferably 2,000 or more and 2,000,000 or less, and more preferably 3,000 or more and 1,000,000 or less.

[0136] Condition 8 is the weight-average molecular weight of the adhesive measured by gel permeation chromatography (GPC) performed under the following conditions. Column: Agilent Technologies, 2xPLgel 5μ MIXED-C (7.5mm x 300mm) Column temperature: 40℃ Mobile phase: Tetrahydrofuran for HPLC (containing stabilizer) (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) Flow rate: 1mL / min. Injection volume: 100μL Detection: RI (Refractive Index) Column calibration: Monodisperse polystyrene (Agilent Technologies, EasiCal PS-1) Molecular weight calibration: Relative calibration method (polystyrene equivalent) Equipment: Waters Japan Ltd. 515 HPLC pump / 717plus (automatic injection system) / Shodex RI-101 (differential refractometer)

[0137] (Condition 9) Storage modulus (E') is 1 × 10 5 Pa or more 1×10 10 It is below Pa. (Condition 10) The loss tangent (tanδ), which is the ratio (E'' / E') of the storage modulus (E') to the loss modulus (E''), is between 0.0100 and 1.0000. (Condition 11) Loss modulus (E'') is 1 × 10 6 Pa or more 5×10 8 It is below Pa.

[0138] For conditions 9, 10, and 11, in this embodiment, solid viscoelasticity measurements are performed under the same measurement conditions as for condition 2, and the obtained values ​​are used. (Measurement conditions) Measurement device: RSA-III, manufactured by T.A. Instruments Co., Ltd. Deformation mode: Tension Temperature: 23℃ Frequency: 1Hz Distortion: 0.05% Atmospheric environment: Nitrogen atmosphere

[0139] The storage modulus (E') of the adhesive layer 4 under condition 9 is, for example, 1 × 10⁻⁶ 6 Pa or more, 1×10 7 Pa or more, 1×10 8 Pa or higher or 1 × 10 9 It can be set to Pa or higher, and also 1 × 10 9 Pa or less, 1×10 8 Pa or less, 1×10 7 Pa or less or 1 × 10⁻⁶ 6 It can be set to Pa or less.

[0140] The loss loss tangent (tanδ) of the adhesive layer 4 under condition 10 is, for example, 0.0500 or more, 0.1000 or more, 0.1500 or more, 0.2000 or more, 0.2500 or more, 0.3000 or more, 0.3500 or more, 0.4000 or more, 0.4500 or more, 0.5000 or more, 0.5500 or more, 0.6000 or more, 0.6500 or more, 0.7000 or more, 0.7500 or more, 0.8000 or more, 0.8500 or more, 0.9000 or more. It can be 0.9500 or greater, and can also be 0.9500 or less, 0.9000 or less, 0.8500 or less, 0.8000 or less, 0.7500 or less, 0.7000 or less, 0.6500 or less, 0.6000 or less, 0.5500 or less, 0.5000 or less, 0.4500 or less, 0.4000 or less, 0.3500 or less, 0.3000 or less, 0.2500 or less, 0.2000 or less, 0.1500 or less, or 0.1000 or less.

[0141] The loss modulus of elasticity (E'') of the adhesive under condition 10 is preferably 1 × 10⁻⁶ 6 Pa or more 5×10 8 Pa or less, more preferably 5 × 10 6 Pa or more 1×10 8 It is less than or equal to Pa. When the loss modulus of elasticity (E'') of the adhesive is within this range, the low viscosity of the adhesive makes it difficult for the drug to remain inside the adhesive, resulting in better drug adsorption.

[0142] (Condition 11) The peel strength of the adhesive layer at -50°C is 0.1 N / 25 mm or more.

[0143] The peel strength of the adhesive layer under condition 11 can be, for example, 0.2 N / 25 mm or more, 0.3 N / 25 mm or more, 1 N / 25 mm or more, 5 N / 25 mm or more, 10 N / 25 mm or more, 15 N / 25 mm or more, or 20 N / 25 mm or more.

[0144] If the peel strength of the adhesive layer at -50°C is above the lower limit mentioned above, the structure and the substrate or sheet will be firmly bonded even at low temperatures, thus preventing delamination due to impact, etc. In other words, impact resistance at low temperatures can be improved.

[0145] Furthermore, there is no particular upper limit to the peel strength of the adhesive layer at -50°C; for example, it can be 200 N / 25 mm or less.

[0146] [Second Embodiment] Figure 9 is a perspective view of a culture vessel 1B according to a second embodiment of the present invention. Figure 10 is a cross-sectional view of a part of the culture vessel 1B.

[0147] Culture vessel 1B is used, for example, to culture human-derived cells. Such a culture vessel 1B has multiple containment sections 10B (see Figure 10) for culturing cells. Culture vessel 1B is placed in the culture space of a culture device (for example, an incubator) with the drug (culture medium) and the cells to be cultured (hereinafter referred to as "target cells") contained in the containment sections 10B.

[0148] The culture vessel 1B of this embodiment is suitably used for culturing spheroids (cell aggregates) of target cells. Note that the culture vessel 1B does not need to be used while housed in a culture device. The culture vessel 1B may be used in various situations depending on the target cells.

[0149] The following describes the specific configuration of culture vessel 1B.

[0150] The culture vessel 1B has a structure 2B and a transparent resin sheet 5B.

[0151] The structure 2B has a main body 3B and an adhesive layer 4B.

[0152] The main body 3B has a frame 30B and a main body side housing 31B. The main body 3B is made of synthetic resin, for example, and is an integrally molded product made by injection molding. Examples of synthetic resins that make up the main body 3B include polystyrene and polyolefin. Examples of polyolefins include cyclic olefin (co)polymers, 4-methyl-1-pentene (co)polymers, polypropylene, and polyethylene. Of these, 4-methyl-1-pentene (co)polymers are preferred from the viewpoint of improving the gas permeability of the main body 3B. Alternatively, 4-methyl-1-pentene (co)polymers, polystyrene, polypropylene, and cyclic olefin (co)polymers are preferred from the viewpoint of improving the heat resistance of the main body 3B.

[0153] The instantaneous Shore D hardness of the main body 3B is, for example, 60 to 90. The instantaneous Shore D hardness is obtained by using a durometer hardness tester (Type D) to immediately read the scale after bringing the indenter into contact with a part of the main body 3B (in accordance with ASTM D2240). When the instantaneous Shore D hardness is in the range of 60 to 90, the transparent resin sheet 5B described later can be attached to the main body 3B with a good appearance.

[0154] The frame portion 30B is composed of rectangular frame-shaped members. The frame portion 30B constitutes the outer shape of the culture container 1B.

[0155] The main body side housing section 31B is provided in a space enclosed by the frame section 30B. The main body side housing section 31B is provided integrally with the frame section 30B.

[0156] The main body side housing section 31B has a plurality of cylindrical sections 310B and a bottom plate section 311B.

[0157] The cylindrical portion 310B is cylindrical with an opening in the vertical direction. In this embodiment, the shape of the opening of the cylindrical portion 310B (in other words, the outer shape in a plan view) is circular. The cylindrical portions 310B are arranged in the left-right and front-back directions. The number of cylindrical portions 310B corresponds to the number of containment portions 10B for culturing cells. The number of cylindrical portions 310B may be, for example, 6, 24, 96, or 384. Of course, the number of cylindrical portions 310B may be other than 6, 24, 96, and 384.

[0158] Adjacent cylindrical sections 310B are connected by connecting sections (not shown). In addition, the leftmost cylindrical section 310B, the rightmost cylindrical section 310B, the frontmost cylindrical section 310B, and the rearmost cylindrical section 310B are connected to the inner circumferential surface of the frame section 30B via connecting sections (not shown).

[0159] The space defined by the inner circumferential surface of the cylindrical portion 310B (hereinafter referred to as the "inner space of the cylindrical portion 310B") corresponds to an example of a through-hole in the substrate. The inner space of the cylindrical portion 310B, together with the transparent resin sheet 5B described later, constitutes the containment section 10B for culturing cells.

[0160] As shown in Figure 10, the bottom plate portion 311B is a plate-like member that connects the lower ends of the cylindrical portions 310B. The outer edge of the bottom plate portion 311B is connected to the inner surface of the frame portion 30B around its entire circumference. The lower surface of the bottom plate portion 311B is also the lower surface of the main body side housing portion 31B. Furthermore, the lower surface of the bottom plate portion 311B is also the lower surface of the main body portion 3B. Therefore, the lower surface of the bottom plate portion 311B is an example of the lower surface of the base material.

[0161] The transparent resin sheet 5B is attached to the lower surface of the main body portion 3B via an adhesive layer 4B. Specifically, the transparent resin sheet 5B is attached to the lower surface of the bottom plate portion 311B in the main body side storage portion 31B via an adhesive layer 4B. As a result, the transparent resin sheet 5B closes the bottom surface of the cylindrical portion 310B and forms the bottom surface of the container 1B.

[0162] Transparent resin sheet 5B is a sheet of transparent resin. Examples of transparent resins include acrylic resin, cyclic olefin (co)polymer, 4-methyl-1-pentene (co)polymer, olefin resins including polypropylene and polyethylene, polyester resins including polyolefin terephthalate (PET), polystyrene resin, ethylene-vinyl alcohol (EVOH) resin, polyvinyl alcohol (PVOH) resin, and polyvinylidene chloride (PVDC).

[0163] From the perspective of creating aerobic conditions inside the containment section 10B to enhance the metabolic activity of cells, the transparent resin sheet 5B has an oxygen permeability of 200 to 60,000 cm². 3 / (m 2 It is preferable that the temperature is (x24hr x atm). From the viewpoint of effectively increasing oxygen permeability, it is preferable that the transparent resin sheet 5B is a sheet-like member containing a polymer having structural units derived from 4-methyl-1-pentene. Furthermore, according to the inventors' findings, polymers having structural units derived from 4-methyl-1-pentene have a lower adsorption capacity for drugs such as fluorescent dyes than conventionally used polydimethylsiloxane (PDMS). Therefore, if the transparent resin sheet 5B is a sheet-like member containing a polymer having structural units derived from 4-methyl-1-pentene, a decrease in observation efficiency due to the adsorption of drugs by the transparent resin sheet 5B is less likely to occur.

[0164] The oxygen permeability values ​​mentioned above were measured using a differential pressure type gas permeability meter MT-C3 manufactured by Toyo Seiki Seisakusho Co., Ltd., at a temperature of 23°C and 0% humidity [cm²]. 3 / (m 2 The measured value is (×24h×atm). The measurement sample was prepared by cutting a 90×90mm test piece from a transparent resin sheet 5, with a measurement area diameter of 70mm (transmission area of ​​38.46cm²). 2 ) When high oxygen permeability is expected, an aluminum mask should be applied to the measurement sample beforehand, and the actual permeable area should be 5.0 cm². 2 It is preferable to do so.

[0165] The adhesive layer 4B is attached to the lower surface of the bottom plate portion 311B of the main body side housing portion 31B by adhering a transparent resin sheet 5B to it. Known adhesives such as acrylic, silicone, urethane, and rubber can be used for the adhesive layer 4B. Of these, acrylic adhesives and silicone adhesives are preferred, and acrylic adhesives are more preferred, because their adhesive properties can be easily adjusted.

[0166] Figure 11 is a plan view of the adhesive layer 4B. The adhesive layer 4B has an adhesive layer body 40B and a plurality of openings 41B.

[0167] The adhesive layer body 40B is a sheet-like member and is the portion that adheres to the lower surface of the main body side housing portion 31B (specifically, the bottom plate portion 311B) and the transparent resin sheet 5B.

[0168] The opening 41B is provided in the adhesive layer body 40B. The opening 41B penetrates the adhesive layer body 40B in the vertical direction. As shown in Figure 10, when the opening 41B is attached to the lower surface of the body-side housing 31B, it is positioned below the through-hole 310c of the body-side housing 31B. As shown in Figures 10 and 11, the outer shape of the opening 41B in plan view is larger than the outer shape of the through-hole 310c. In Figure 11, the opening 41B is shown by a solid line. In Figure 11, the through-hole 310c is shown by a dashed line.

[0169] In this embodiment, the outer shape of the opening 41B in plan view is circular, as shown in Figure 11. Hereafter, when referring to the outer shape of the opening 41B, it means the outer shape of the opening 41B in plan view. The outer shape of the through hole 310c is also circular. The outer shape of the opening 41B is similar to the outer shape of the through hole 310c. In this embodiment, the center of the outer shape of the opening 41B and the center of the outer shape of the through hole 310c coincide. Therefore, the peripheral edge of the opening 41B does not overlap with the through hole 310c in plan view.

[0170] In this embodiment, the adhesive layer 4B is formed of an adhesive that satisfies the following conditions 1 and 2.

[0171] (Condition 1) A test piece cut to a size of 10 mm x 10 mm is attached to the bottom surface of a tissue culture polystyrene (TCPS) well with an opening diameter of 16.2 mm, a well depth of 18 mm, and a well volume of 3.5 mL. 0.5 mL of phosphate-buffered saline (PBS) solution containing 10 μmol / L rhodamine B is then filled into the well, and after standing at 23°C for 48 hours, the remaining rhodamine B in the aqueous solution is 20% or more.

[0172] (Condition 2) When a 0.05% dynamic strain is applied to a 5 mm wide test piece of the adhesive using a solid viscoelasticity measuring device, the resulting load is 20 g or less.

[0173] According to the inventors' findings, adhesives readily adsorb drugs such as fluorescent dyes, which can reduce the concentration of drugs introduced into the containment section 10B. Therefore, in conventional culture vessels using adhesives, when attempting to examine the viability and activity of cells after culturing, it is difficult to sufficiently detect signals from drugs used to detect biological substances, resulting in low observation efficiency. Similarly, drugs that assist in cell culture can also be adsorbed, which can prevent the expected improvement in cell culture efficiency.

[0174] Based on the above findings, the inventors investigated the adsorption amounts of various drugs for multiple adhesives and found that rhodamine B can serve as an indicator for predicting the adsorption amount of various drugs by adhesives. In other words, compared to other drugs, rhodamine derivatives tend to show differences in adsorption amounts depending on the adhesive. Furthermore, by using an adhesive with low rhodamine B adsorption, high observation efficiency and culture efficiency can be obtained even when observing with other drugs, and culture efficiency can be sufficiently increased when promoting cell culture with other drugs.

[0175] Condition 1 is based on the aforementioned findings and identifies that the adhesive forming the adhesive layer 4B has a low adsorption capacity for rhodamine B. An adhesive that satisfies Condition 1 is less likely to adsorb various drugs in the portion exposed on the inner surface of the containment section 10B. This improves the signal detection capability from drugs in the containment section 10B, thereby increasing the efficiency of observing bio-derived substances and improving the efficiency of cell culture using drugs.

[0176] Regarding condition 1, in this embodiment, the adhesive is cut to a size of 10 mm x 10 mm to serve as a test piece. The thickness of the test piece is the thickness of the adhesive itself if it is a commercially available product, and is the thickness of the adhesive when used in the adhesive layer 4B. The test piece is then attached to the bottom surface of a tissue culture polystyrene well with an opening diameter of 16.2 mm, a well depth of 18 mm, and a well volume of 3.5 mL. 0.5 mL of a phosphate-buffered saline (PBS) solution containing 5 μM rhodamine B is filled into the well and left to stand at 23°C for 48 hours. If the remaining percentage of rhodamine B in the aqueous solution after standing is 20% or more (i.e., the concentration of rhodamine B in the aqueous solution after standing is 1 μM or more), the adhesive forming the adhesive layer 4B is considered to satisfy condition 1.

[0177] The amount of rhodamine B in the aqueous solution after standing shall be the value measured by fluorescence analysis using the following procedure. Measuring device: FP-6600 manufactured by JASCO Corporation Cell used: Quartz microcell Bandwidth: Excitation side: 5nm, Fluorescence side: 6nm Sensitivity (PMT voltage): 400V Excitation wavelength: 555nm Fluorescence measurement wavelength: 580nm Scan speed: 2000nm / min

[0178] From the above perspective, the remaining percentage of rhodamine B in Condition 1 is preferably 30% or more, and more preferably 40% or more. While there is no particular upper limit to the remaining percentage of rhodamine B in Condition 1, it can be 100%.

[0179] Condition 2 means that the adhesive forming the adhesive layer 4B is easily deformable even with a small force. Such an adhesive easily conforms to the shape of the bottom surface (underside) of the main body housing section 31B, and is less likely to create minute gaps or flexures when attached. Therefore, an adhesive that satisfies condition 2 is less likely to cause a decrease in observation efficiency due to these gaps or flexures causing the transparent resin sheet 5B to flex or unintended refraction of light at the periphery of the through-hole 310c. In addition, an adhesive that satisfies condition 2 reduces the contact area with the liquid (culture medium, etc.) filled inside the housing section 10B, thereby reducing the amount of various drugs adsorbed by the adhesive layer 4B. As a result, the detectability of signals from drugs in the housing section 10B can be improved, and the observation efficiency of biological substances can be increased.

[0180] Regarding condition 2, in this embodiment, the adhesive is cut to a size of 5 mm in width and 30 mm in length to form a test specimen. The thickness of the test specimen is the thickness of the adhesive when used in the adhesive layer 4B. The adhesive is deemed to satisfy condition 2 if, when solid viscoelasticity measurement is performed under the following conditions, the load generated to impart a dynamic strain of 0.05% to the test specimen is 20 g or less. Measurement device: RSA-III, manufactured by T.A. Instruments Co., Ltd. Deformation mode: Tension Temperature: 23℃ Frequency: 1Hz Distortion: 0.05% Atmospheric environment: Nitrogen atmosphere

[0181] From the above viewpoint, the resulting load when applying a 0.05% dynamic strain under condition 2 is preferably 18g or less, and more preferably 15g or less. Although there is no particular lower limit to the resulting load when applying a 0.05% dynamic strain under condition 2, from the viewpoint of facilitating application, it can be 2g or more, and preferably 5g or more.

[0182] The adhesive layer 4B is preferably formed of an adhesive that satisfies the following conditions. Note that any one of these conditions may be satisfied individually, or a combination of these conditions may be satisfied.

[0183] (Condition 3) The glass transition temperature (Tg) is -122°C or higher.

[0184] Condition 3 means that the adhesive forming the adhesive layer 4B does not have excessively high molecular mobility. In other words, if the molecules constituting the adhesive have high mobility, the adhesive will more easily adsorb drugs. In contrast, adhesives that satisfy condition 3 have moderately low molecular mobility, making it difficult for them to adsorb various drugs at the parts exposed on the inner surface of the containment section 10B. This improves the detection of signals from drugs in the containment section 10B, thereby increasing the efficiency of observing bio-derived substances and improving the efficiency of cell culture using drugs. In particular, when the adhesive is an acrylic adhesive, a significant tendency is observed where the amount of drug adsorbed decreases as the Tg increases, leading to improved efficiency in observing bio-derived substances and cell culture.

[0185] Regarding condition 3, in this embodiment, a solid viscoelastic temperature dispersion measurement of the adhesive is performed under the following conditions, and the peak top value of the obtained loss tangent (tanδ) is taken as the glass transition temperature (Tg) of the adhesive. (Measurement conditions) Measurement device: RSA-III, manufactured by T.A. Instruments Co., Ltd. Deformation mode: Tension Temperature range: -100℃ to 25℃ Silence rate: 3℃ / min Frequency: 1Hz Distortion: 0.05% Atmospheric environment: Air

[0186] Furthermore, if the goal is to increase adhesiveness, a lower Tg of the adhesive is preferable. On the other hand, from the viewpoint of improving the observation efficiency of bio-derived materials based on the above findings, a higher Tg of the adhesive is preferable. From the viewpoint of achieving both of these, the Tg of the adhesive under condition 3 is more preferably -121°C to -30°C, even more preferably -80°C to -30°C, and particularly preferably -35°C to -30°C.

[0187] (Condition 4) It is an acrylic adhesive with a structure derived from an aromatic ring-containing compound.

[0188] Acrylic adhesives with structures derived from aromatic ring-containing compounds have reduced molecular mobility due to the aromatic ring (benzene ring). Therefore, adhesives that satisfy condition 3 have low molecular mobility, making it difficult for them to adsorb various drugs at the parts exposed on the inner surface of the containment section 10B. This improves the detection of signals from drugs in the containment section 10B, thereby increasing the efficiency of observing bio-derived substances and improving the efficiency of cell culture using drugs.

[0189] Regarding condition 3, in this embodiment, the surface of the adhesive was measured by Fourier transform infrared spectrophotometric (FT / IR) under the following conditions, and the 700 cm² corresponding to the structure derived from the aromatic ring-containing compound was found in the obtained IR spectrum. -1 When the peak is confirmed, the adhesive is deemed to satisfy condition 4. (Measurement conditions) Measurement device: Thermo Fisher Scientific Nicoleti S50 Measurement method: Total internal reflection measurement method (ATR method) using a Ge prism Resolution: 4cm -1 Total number of times: 32

[0190] (Condition 5) It is a silicone-based adhesive, and in the IR spectrum, the Si-O originates from 1068 cm⁻¹. -1 843 cm⁻¹ of Si-CH3 relative to the peak intensity-1 The peak intensity ratio ((Si-CH3) / (Si-O)) is between 0.500 and 0.600.

[0191] (Condition 6) It is a silicone-based adhesive, and in the IR spectrum, the Si-O originates from 1068 cm⁻¹. -1 756 cm² of the peak intensity originating from Si-CH3 -1 The ratio of peak intensities ((Si-CH3) / (Si-O)) is between 0.280 and 0.300.

[0192] Conditions 5 and 6 specify the amount of terminal methyl groups relative to the siloxane bonds in the silicone adhesive, meaning that the molecular weight of the silicone adhesive is of an appropriate size. Adhesives that satisfy condition 5 or condition 6 have moderately low molecular mobility, making them less likely to adsorb various drugs at the portion exposed on the inner surface of the containment section 10B. This improves the signal detection capability from drugs in the containment section 10B, thereby increasing the efficiency of observing bio-derived substances and improving the efficiency of cell culture using drugs. On the other hand, adhesives that satisfy condition 5 or condition 6 have sufficient molecular mobility, resulting in sufficiently high adhesiveness.

[0193] Furthermore, an adhesive that satisfies condition 5 or condition 6 may satisfy only one of these conditions, or both of these conditions.

[0194] Furthermore, conditions 5 and 6 are the ratios of the respective peak intensities for the IR spectra obtained by measurement under the same conditions as condition 3.

[0195] (Condition 7) A test piece cut to a size of 10 mm x 10 mm is attached to the bottom surface of a tissue culture polystyrene well with an opening diameter of 16.2 mm, a well depth of 18 mm, and a well volume of 3.5 mL. 0.5 mL of a PBS solution containing 5 μM rhodamine 6 G is then filled into the well, and after standing at 23°C for 48 hours, the remaining percentage of rhodamine 6 G in the aqueous solution is 20% or more.

[0196] According to our findings, similar to rhodamine B under condition 1, rhodamine 6G is also prone to differences in adsorption amounts depending on the adhesive compared to other drugs. Furthermore, adhesives with low adsorption of rhodamine 6G tend to have low adsorption amounts of other drugs as well. Therefore, adhesives that satisfy condition 7 are less likely to adsorb various drugs in the portion exposed on the inner surface of the containment section 10B. This improves the signal detection capability from drugs in the containment section 10B, thereby increasing the efficiency of observing biologically derived substances and improving the efficiency of cell culture using drugs.

[0197] Condition 7 is the remaining percentage of rhodamine 6G in the aqueous solution after standing, obtained by measuring under the same conditions as Condition 1, except that a 5 μM rhodamine 6G aqueous solution was used instead of a 0.5 mL / well rhodamine B aqueous solution.

[0198] From the above viewpoint, the remaining percentage of rhodamine 6G in condition 1 is preferably 30% or more, more preferably 40% or more, and even more preferably 50% or more. While there is no particular upper limit to the remaining percentage of rhodamine 6G in condition 7, it can be 100%.

[0199] (Condition 8) The weight-average molecular weight (Mw) is between 1,000 and 5,000,000.

[0200] Condition 8 means that the molecular weight of the adhesive is of an appropriate size. Adhesives that satisfy condition 8 have moderately low molecular mobility, making it difficult for them to adsorb various drugs at the parts exposed to the inner surface of the containment section 10B. This improves the detection of signals from drugs in the containment section 10B, thereby increasing the efficiency of observing bio-derived substances and improving the efficiency of cell culture using drugs. On the other hand, adhesives that satisfy condition 8 also have sufficiently high adhesiveness because the molecules that make up the adhesive have sufficient mobility.

[0201] From the above perspective, the weight-average molecular weight (Mw) of the adhesive under condition 8 is preferably 2,000 or more and 2,000,000 or less, and more preferably 3,000 or more and 1,000,000 or less.

[0202] Condition 8 is the weight-average molecular weight of the adhesive measured by gel permeation chromatography (GPC) performed under the following conditions. Column: Agilent Technologies, 2xPLgel 5μ MIXED-C (7.5mm x 300mm) Column temperature: 40℃ Mobile phase: Tetrahydrofuran for HPLC (containing stabilizer) (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) Flow rate: 1mL / min. Injection volume: 100μL Detection: RI (Refractive Index) Column calibration: Monodisperse polystyrene (Agilent Technologies, EasiCal PS-1) Molecular weight calibration: Relative calibration method (polystyrene equivalent) Equipment: Waters Japan Ltd. 515 HPLC pump / 717plus (automatic injection system) / Shodex RI-101 (differential refractometer)

[0203] (Condition 9) Storage modulus (E') is 1 × 10 5 Pa or more 1×10 10 It is below Pa. (Condition 10) The loss tangent (tanδ), which is the ratio (E'' / E') of the storage modulus (E') to the loss modulus (E''), is between 0.0100 and 1. (Condition 11) Loss modulus (E'') is 1 × 10 6 Pa or more 5×10 8 It is below Pa.

[0204] For conditions 9, 10, and 11, in this embodiment, solid viscoelasticity measurements are performed under the same measurement conditions as for condition 2, and the obtained values ​​are used.

[0205] The storage modulus (E') of the adhesive under condition 9 is, for example, 1 × 10⁻⁶ 6 Pa or more, 1×10 7 Pa or more, 1×10 8 Pa or higher or 1 × 10 9 It can be set to Pa or higher, and also 1 × 10 9 Pa or less, 1×10 8 Pa or less, 1×10 7 Pa or less or 1 × 10⁻⁶ 6 It can be set to Pa or less.

[0206] The loss tangent (tanδ) of the adhesive under condition 10 is, for example, 0.0500 or more, 0.1000 or more, 0.1500 or more, 0.2000 or more, 0.2500 or more, 0.3000 or more, 0.3500 or more, 0.4000 or more, 0.4500 or more, 0.5000 or more, 0.5500 or more, 0.6000 or more, 0.6500 or more, 0.7000 or more, 0.7500 or more, 0.8000 or more, 0.8500 or more, 0.9000 or more It can be 0.9500 or higher, and can also be 0.9500 or lower, 0.9000 or lower, 0.8500 or lower, 0.8000 or lower, 0.7500 or lower, 0.7000 or lower, 0.6500 or lower, 0.6000 or lower, 0.5500 or lower, 0.5000 or lower, 0.4500 or lower, 0.4000 or lower, 0.3500 or lower, 0.3000 or lower, 0.2500 or lower, 0.2000 or lower, 0.1500 or lower, or 0.1000 or lower.

[0207] The loss modulus of elasticity (E'') of the adhesive under condition 10 is preferably 1 × 10⁻⁶ 6 Pa or more 5×10 8 Pa or less, more preferably 5 × 10 6 Pa or more 1×10 8 It is less than or equal to Pa. When the loss modulus of elasticity (E'') of the adhesive is within this range, the low viscosity of the adhesive makes it difficult for the drug to remain inside the adhesive, resulting in better drug adsorption.

[0208] (Condition 11) The peel strength of the adhesive layer at -50°C is 0.1 N / 25 mm or more.

[0209] The peel strength of the adhesive layer under condition 11 can be, for example, 0.2 N / 25 mm or more, 0.3 N / 25 mm or more, 1 N / 25 mm or more, 5 N / 25 mm or more, 10 N / 25 mm or more, 15 N / 25 mm or more, or 20 N / 25 mm or more.

[0210] If the peel strength of the adhesive layer at -50°C is above the lower limit mentioned above, the structure and the substrate or sheet will be firmly bonded even at low temperatures, thus preventing delamination due to impact, etc. In other words, impact resistance at low temperatures can be improved.

[0211] Furthermore, there is no particular upper limit to the peel strength of the adhesive layer at -50°C; for example, it can be 200 N / 25 mm or less.

[0212] (Further explanation about adhesive layer 4B) The adhesive layer 4B may be formed by adhering the adhesive composition to both sides of the base layer (support), or by impregnating the base layer (support), such as a nonwoven fabric, with the adhesive composition. Alternatively, the adhesive layer 4B may be formed from a so-called substrate-less adhesive that does not have a base layer (support). Of these, it is preferable to use an adhesive with a base layer because it facilitates the formation of openings and adhesion to the lower surface of the bottom plate portion 311B in the main body side housing portion 31B. Furthermore, it is preferable that the adhesive is applied to both sides of the base layer (support) because it is easier to reduce the thickness of the adhesive layer 4B.

[0213] The thickness of the adhesive layer 4B is preferably 20 μm to 150 μm, more preferably 30 μm to 100 μm, and even more preferably 40 μm to 70 μm. The thicker the adhesive layer 4B, the easier it is to form an opening and to attach it to the main body side housing. The thinner the adhesive layer 4B, the lower the amount of various drugs adsorbed by the adhesive layer 4B, thereby improving the signal detection from drugs in the housing 10B, increasing the efficiency of observing biological substances, and improving the efficiency of cell culture with drugs.

[0214] From the viewpoint of enhancing the observation efficiency of the bio-derived substance, the adhesive layer 4B is preferably transparent. Specifically, the total light transmittance measured in accordance with JIS K 7361-1 is preferably 50% or more and 100% or less, and more preferably 70% or more and 100% or less.

[0215] In the present embodiment, by using the above-described adhesive layer 4B, adsorption of the drug by the adhesive layer 4B, bending of the adhesive layer 4B, etc. can be suppressed, and a decrease in the observation efficiency of the bio-derived substance derived from the adhesive layer 4B can be suppressed.

[0216] [Third Embodiment] FIG. 12 is a partial cross-sectional view of the culture vessel 1C according to the second embodiment. The culture vessel 1C is different from the culture vessel 1B according to the second embodiment in the configuration of the main body side accommodation portion 31B and the positions of the adhesive layer 4B and the transparent resin sheet 5B. Hereinafter, descriptions overlapping with the second embodiment are omitted.

[0217] The main body side accommodation portion 31B has a plurality of cylindrical portions 310B, a bottom plate portion 311B, and an upper plate portion 312B.

[0218] The cylindrical portion 310B is a recess that is closed downward by the bottom plate portion 311B and opens only upward. The configuration of the other cylindrical portions 310B is the same as that of the first embodiment.

[0219] As shown in FIG. 11, the bottom plate portion 311B is a plate-like member that connects the lower end portions of the cylindrical portions 310B. However, in the present embodiment, no opening is formed in the bottom plate portion 311B, and thus the bottom plate portion 311B constitutes the bottom surface of the accommodation portion 10B.

[0220] As shown in FIG. 11, the upper plate portion 312B is a plate-shaped member that connects the upper end portions of the cylindrical portion 310B. The outer edge portion of the upper plate portion 312B is connected to the inner peripheral surface of the frame portion 30B over the entire circumference. Note that the upper surface of the upper plate portion 312B is also the upper surface of the main body side accommodating portion 31B. Further, the lower surface of the upper plate portion 312B is also the upper surface of the main body portion 3B. Therefore, the upper surface of the upper plate portion 312B corresponds to an example of the upper surface of the base material.

[0221] And in the present embodiment, the transparent resin sheet 5B is adhered to the upper surface of the main body portion 3B via the adhesive layer 4B. Specifically, the transparent resin sheet 5B is adhered to the upper surface of the upper plate portion 312B in the main body side accommodating portion 31B via the adhesive layer 4B. Thereby, the transparent resin sheet 5B closes the upper surface of the container 1B and seals the accommodating portion 10B (the inner space of the cylindrical portion 310B). Further, the adhesive layer 4B adheres the transparent resin sheet 5B to the upper surface of the upper plate portion 312B in the main body side accommodating portion 31B.

[0222] Also in the present embodiment, the adhesive layer 4B has an adhesive layer main body 40B and a plurality of openings 41B (see FIG. 11). And as shown in FIG. 11, the outer shape in plan view of the opening 41B is larger than the outer shape of the through hole 310c. Further, the center of the outer shape of the opening 41B and the center of the outer shape of the through hole 310c coincide with each other. Therefore, the peripheral edge portion of the opening 41B does not overlap the through hole 310c in plan view.

[0223] Also in the present embodiment, as the adhesive layer 4B, an adhesive layer formed of the same adhesive as the adhesive layer 4B in the second embodiment is used. Thereby, adsorption of the drug by the adhesive layer 4B, bending of the adhesive layer 4B, etc. can be suppressed, and a decrease in the observation efficiency of biomaterials derived from the adhesive layer 4B, a decrease in the cell culture efficiency, etc. can be suppressed. D

[0224] [Other Embodiments] Note that each of the above-described embodiments shows an example of the present invention, and the present invention is not limited to each of the above-described embodiments. Needless to say, various other embodiments are possible within the scope of the idea of the present invention.

[0225] For example, although the above embodiment described a culture vessel for culturing cells, the present invention is not limited thereto. Insofar as other components are attached to the substrate via an adhesive layer to form a containment section 10B to which biological substances (including cells, etc.) are applied, the present invention can also be applied to cell storage containers and containers for the reaction and measurement of biological substances such as fluid channel chips.

[0226] Furthermore, in the second and third embodiments described above, the opening 41B of the adhesive layer 4B was assumed to be larger than the outer shape of the through hole 310c, but they may be the same size, and there is no impediment to making the opening 41B of the adhesive layer 4B smaller than the outer shape of the through hole 310c. However, by making the opening 41B of the adhesive layer 4B larger than the outer shape of the through hole 310c, the effect of suppressing the adsorption of the drug by the adhesive layer 4B is significantly enhanced.

[0227] Furthermore, there is no preclude using other non-transparent materials instead of the transparent resin sheet 5B. [Examples]

[0228] The present invention will be described in detail based on examples, but the present invention is not limited to these examples.

[0229] 1. Preparation and measurement of adhesive. For commercially available industrial adhesives, various physical properties were investigated by measuring the residual rate of the chemical agent, performing solid viscoelasticity measurements, infrared spectroscopy measurements, molecular weight measurements, differential scanning calorimetry, and low-temperature peel tests according to the following procedures.

[0230] 1-1. Drug retention rate Each adhesive was cut to a size of 10 mm x 10 mm to form a test specimen. This specimen was attached to the bottom surface of a tissue culture polystyrene well with an opening diameter of 16.2 mm, a well depth of 18 mm, and a well volume of 3.5 mL. 0.5 mL of an aqueous solution of the drug was then filled into the well and left to stand at 23°C for 48 hours. The amount of drug in the aqueous solution after standing was measured by fluorescence analysis for rhodamine B and by LC / MS analysis for the other samples, using the following procedure. The ratio of the amount of drug in the aqueous solution after standing to the amount of drug in the filled aqueous solution (before standing) (amount of drug after standing / amount of drug before standing) was then calculated and defined as the remaining drug rate for each adhesive. (Measurement conditions for fluorescence analysis) Measuring device: FP-6600 manufactured by JASCO Corporation Cell used: Quartz microcell Bandwidth: Excitation side: 5nm, Fluorescence side: 6nm Sensitivity (PMT voltage): 400V Excitation wavelength: 555 nm (Rhodamine B) Fluorescence measurement wavelength: 580nm (Rhodamine B) Scan speed: 2000nm / min (LC / MS measurement conditions) Measurement device: TQ-S micro(water) manufactured by Acquity UPLC I-class system Ionization methods: Electrospray ionization (ESI), positive and negative ion detection Detection method: Selective reaction monitoring (SRM)

[0231] The following solutions were used to measure the residual rate for each sample. Rhodamine B: PBS solution of rhodamine B at a concentration of 5 μM. Triglitazone: A 50 μM solution of triglitazone in dimethyl sulfoxide (DSMO). Ticlopidine hydrochloride: DSMO solution of ticlopidine hydrochloride at a concentration of 50 μM. Isoproterenol hydrochloride: DSMO solution of isoproterenol hydrochloride at a concentration of 50 μM. Leflunomide: DMSO solution of leflunomide at a concentration of 50 μM Cyclosporin A: DMSO solution of cyclosporin A at a concentration of 10 μM

[0232] 1 - 2. Solid viscoelasticity measurement Each adhesive was cut into test pieces with a width of 5 mm and a length of 30 mm. Using a solid dynamic viscoelasticity measuring device, a dynamic strain of 0.05% was applied to each test piece under the following conditions, and the generated load at this time was measured to obtain the 0.05% strain stress for each adhesive. Also, from the measurement results at this time, the storage modulus (E'), loss modulus (E''), and loss tangent (tanδ), which is the ratio of the storage modulus (E') to the loss modulus (E'') (E'' / E'), were determined. Measuring device: RSA-III manufactured by TA Instruments Co., Ltd. Deformation mode: Tensile Temperature: 23°C Frequency: 1 Hz Strain: 0.05% Ambient environment: Nitrogen atmosphere

[0233] Also, the test pieces of each adhesive were subjected to solid viscoelastic temperature dispersion measurement using a solid dynamic viscoelasticity measuring device, and the peak top value of the obtained loss modulus was taken as the glass transition temperature (Tg) of the adhesive. (Measurement conditions) Measuring device: RSA-III manufactured by TA Instruments Co., Ltd. Deformation mode: Tensile Temperature range: -100°C to 25°C Cooling rate: 3°C / min Frequency: 1 Hz Strain: 0.05% Ambient environment: Air

[0234] 1 - 3. Infrared spectroscopic measurement The surface of each adhesive was measured by Fourier transform infrared spectrophotometric (FT / IR) under the following conditions, and IR spectra were obtained. For acrylic adhesives, if a peak corresponding to a styrene-derived structure was confirmed in the obtained IR spectrum, the adhesive was determined to have a styrene-derived structure. For silicone adhesives, the peak corresponding to 1068 cm⁻¹ in the IR spectrum, which originates from Si-O, was identified. -1 843 cm⁻¹ of Si-CH3 relative to the peak intensity -1 The ratio of peak intensities ((Si-CH3) / (Si-O)) and the 1068 cm⁻¹ derived from Si-O. -1 756 cm² of the peak intensity originating from Si-CH3 -1 The peak intensity ratio ((Si-CH3) / (Si-O)) was determined and set as the 843 / 1068 ratio and 756 / 1068 ratio for each adhesive. (Measurement conditions) Measurement device: Thermo Fisher Scientific Nicoleti S50 Measurement method: Total internal reflection measurement method (ATR method) using a Ge prism Resolution: 4cm -1 Total number of times: 32

[0235] 1-4. Measurement of Molecular Weight The molecular weight of each adhesive was measured by gel permeation chromatography (GPC) under the following conditions. Column: Agilent Technologies, 2xPLgel 5μ MIXED-C (7.5mm x 300mm) Column temperature: 40℃ Mobile phase: Tetrahydrofuran for HPLC (containing stabilizer) (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) Flow rate: 1mL / min. Injection volume: 100μL Detection: RI (Refractive Index) Column calibration: Monodisperse polystyrene (Agilent Technologies, EasiCal PS-1) Molecular weight calibration: Relative calibration method (polystyrene equivalent) Equipment: Waters Japan Ltd. 515 HPLC pump / 717plus (automatic injection system) / Shodex RI-101 (differential refractometer)

[0236] 1-5. Low-temperature peel test The peel strength of the adhesive layer at -50°C was measured by the following method in accordance with JIS Z 0237.

[0237] Test specimens were prepared by fixing a 50 μm thick film (4-methyl-1-pentene copolymer, manufactured by Mitsui Chemicals, Inc., TPX) to a stainless steel plate via various adhesives in an atmosphere of 20°C and 50% RH. The prepared test specimens were stored at -40°C to -50°C for 24 hours. Using the test specimens immediately after storage, the peel strength was measured in an atmosphere of -50°C by peeling the film 180° from the stainless steel plate (180° peel method). Approximately 10 cm was peeled off, and the average value was calculated excluding the first and last 15%, which was used as the peel strength value of the adhesive layer at -50°C.

[0238] 2.Measurement results Table 1 shows the residual rate of the drug in each adhesive. Table 2 shows other physical properties of each adhesive. Table 3 shows the results of the low-temperature peel test.

[0239] [Table 1]

[0240] [Table 2]

[0241] [Table 3]

[0242] 3. Observational Test Culture vessel 1B, as shown in Figures 9 and 10, was prepared. Using Toyo Styrene's Toyo Styrofoam G-200C as the polystyrene, a 24-well plate frame with open well bottoms was prepared by injection molding. A transparent resin sheet 5B made of 4-methyl-1-pentene copolymer (Mitsui Chemicals, Inc., TPX) was attached to this using adhesive 3B, adhesive 4B, or adhesive 9.

[0243] 0.5 mL of a 1.5 mg / mL collagen coating solution was added to the inner surface of the containment section 10B of culture vessel 1B, and then the excess collagen coating solution was removed. After standing at room temperature for 30-60 minutes, it was washed with Dulbecco's PBS(-) and dried overnight at room temperature.

[0244] Next, a culture medium (0.5 mL) containing primary frozen rat hepatocytes was seeded onto the culture surface of container 10B using a micropipette, covered with a polystyrene lid, and placed in an incubator. Cultivation was started at 37°C under 5% CO2 conditions. After 5 days of cultivation, the cells were stained by introducing a 5 μM PBS solution of rhodamine B into container 10B.

[0245] After staining, the cells were observed using a phase-contrast fluorescence microscope to confirm whether the cultured cells were sufficiently stained and clearly visible, and whether there was any decrease in visibility due to the bending of the adhesive (such as blurring at the edges).

[0246] [Table 4]

[0247] The results shown in Table 4 indicate that there are differences in the amount of drugs adsorbed by the adhesives. Furthermore, adhesives that adsorb less rhodamine B (higher retention rate) also adsorb less other drugs (higher retention rate).

[0248] Furthermore, the results shown in Tables 1 to 4 indicate that using an adhesive that exhibits low rhodamine B adsorption and generates a load of 20g or less when applying a 0.05% dynamic strain to a 5mm wide test piece using a solid viscoelasticity measuring device improves the efficiency of observing cells (specifically, in Table 4, bio-derived substances to which each drug reacts).

[0249] (Note) When implementing the culture vessel 1 of the first embodiment described above, a gas-blocking sheet (not shown) that blocks the permeation of gas (specifically, oxygen) may be provided on the lower surface of the gas-permeable sheet 5. A culture vessel 1 having such a configuration can switch between a state in which air is not supplied to the containment section 10 via the gas-permeable sheet 5 (hereinafter also referred to as the "first state of culture vessel 1") and a state in which air is supplied to the containment section 10 via the gas-permeable sheet 5 (hereinafter also referred to as the "second state of culture vessel 1").

[0250] The first state of culture vessel 1 corresponds to a state in which a gas barrier sheet is provided on the underside of the gas permeable sheet 5. The second state of culture vessel 1 corresponds to a state in which the gas barrier sheet has been removed from the underside of the gas permeable sheet 5.

[0251] [Example of structural configuration for the second aspect] According to a second aspect of the present invention, A substrate having through holes or recesses, The substrate has an adhesive layer provided on one surface, A structure comprising a container capable of observing biologically derived substances, wherein another substrate or sheet is adhered to the adhesive layer to close at least one side of the through-hole or the recess, and the structure comprises: The adhesive forming the adhesive layer is A test specimen cut to a size of 10 mm x 10 mm was attached to the bottom surface of a tissue culture polystyrene well with an opening diameter of 16.2 mm, a well depth of 18 mm, and a well volume of 3.5 mL. 0.5 mL of a phosphate-buffered saline solution containing 5 μM rhodamine B was then filled into the well and left to stand at 23°C for 48 hours. The remaining percentage of rhodamine B in the solution after this period was 20% or more, and, When a 5 mm wide test piece of the adhesive is subjected to a 0.05% dynamic strain using a solid viscoelasticity measuring device, the resulting load is 20 g or less. A structure is provided.

[0252] Furthermore, according to a second aspect of the present invention, The adhesive has a remaining content of rhodamine B of 30% or more. A structure is provided.

[0253] Furthermore, according to a second aspect of the present invention, The aforementioned adhesive layer is a layer formed of an adhesive having a glass transition temperature (Tg) of -122°C or higher. A structure is provided.

[0254] Furthermore, according to a second aspect of the present invention, The adhesive layer is a layer formed of an acrylic adhesive or a silicone adhesive. A structure is provided.

[0255] Furthermore, according to a second aspect of the present invention, The aforementioned adhesive layer is a layer formed from an acrylic adhesive having a styrene-derived structure. A structure is provided.

[0256] Furthermore, according to a second aspect of the present invention, The adhesive layer is found in the IR spectrum at 1068 cm², which originates from Si-O. -1 843 cm⁻¹ of Si-CH3 relative to the peak intensity -1 The layer is formed with a silicone-based adhesive in which the peak intensity ratio ((Si-CH3) / (Si-O)) is between 0.500 and 0.600. A structure is provided.

[0257] Furthermore, according to a second aspect of the present invention, The adhesive layer is found in the IR spectrum at 1068 cm², which originates from Si-O. -1 756 cm² of the peak intensity originating from Si-CH3-1 The layer is formed with a silicone-based adhesive in which the peak intensity ratio ((Si-CH3) / (Si-O)) is between 0.280 and 0.300. A structure is provided.

[0258] Furthermore, according to a second aspect of the present invention, The peel strength of the adhesive layer at -50°C, as measured by a method conforming to JIS Z0237, is 0.1 N / 25 mm or more. A structure is provided.

[0259] Furthermore, according to a second aspect of the present invention, The adhesive layer comprises a base layer and an adhesive composition attached to or impregnated in the base layer. A structure is provided.

[0260] Furthermore, according to a second aspect of the present invention, The adhesive layer has a thickness of 20 μm or more and 150 μm or less. A structure is provided.

[0261] Furthermore, according to a second aspect of the present invention, The substrate has a plurality of through holes, The adhesive layer has an opening below the through hole, the outer shape of which in a plan view is larger than the outer shape of the through hole. A structure is provided.

[0262] Furthermore, according to a second aspect of the present invention, The structure relating to the second aspect of the present invention described above, Having another substrate or sheet attached to the structure via the adhesive layer, A container capable of observing biologically derived substances is provided.

[0263] Furthermore, according to a second aspect of the present invention, The aforementioned other substrate or sheet is an oxygen-permeable sheet. A container is provided.

[0264] Furthermore, according to a second aspect of the present invention, The oxygen-permeable sheet comprises a polymer having a structural unit derived from 4-methyl-1-pentene. A container is provided.

[0265] Furthermore, according to a second aspect of the present invention, The aforementioned other substrate or sheet constitutes the bottom surface of the container. A container is provided.

[0266] Furthermore, according to a second aspect of the present invention, Cell culture vessel, A container is provided.

[0267] Furthermore, according to the first and second aspects of the present invention, A structure comprising a culture vessel having multiple containment sections for culturing cells, A base material having multiple through holes that open in the vertical direction, The substrate comprises an adhesive layer provided on the lower surface of the substrate, to which a sheet that closes the through hole is attached to the lower surface, The peel strength of the adhesive layer at -50°C, as measured by a method conforming to JIS Z0237, is 0.1 N / 25 mm or more. A structure is provided.

[0268] Structures whose adhesive layer peel strength at -50°C is within the above range exhibit high impact resistance at low temperatures. In other words, because the structure and the sheet are firmly bonded even at low temperatures, delamination due to impacts can be prevented.

[0269] All disclosures in the specifications, drawings, and abstracts contained in the Japanese applications 2023-26893 and 2023-26897, filed on 24 February 2023, are incorporated herein by reference. [Industrial applicability]

[0270] The culture vessel according to the present invention can be applied to the culture of various cells. [Explanation of Symbols]

[0271] 1, 1B, 1C culture vessel 10, 10B Storage area 2, 2B structure 3, 3B Main body 30, 30B frame 300 Lower frame section 300a Notch 301 Upper frame section 31, 31B Main body side housing 310, 310B Cylindrical section 310a, 310b, 310c through hole 311, 311B Bottom plate part 312 Upper plate section 4, 4a, 4B adhesive layer 40, 40a, 40B Adhesive layer body 41, 41a, 41B opening 420 base layer 420a Base layer side main body 420b Base layer side opening 421 Upper adhesive layer 421a Upper main body 421b Upper layer side opening 422 Lower adhesive layer 422a Lower main body 422b Lower layer opening 5, 5B Gas permeable sheet 6a Upper protective film 6b Lower protective film

Claims

[Claim 1] A structure comprising a culture vessel having multiple containment sections for culturing cells, A base material having multiple through holes that open in the vertical direction and constitute the housing portion, The substrate comprises an adhesive layer provided on the lower surface of the substrate, to which a sheet that closes the through hole is attached to the lower surface, The adhesive layer is a sheet-like member having an opening below the through-hole, the outer shape of which in a plan view is larger than the outer shape of the through-hole. structure.

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