Cell culture device

Abstract

To provide a cell culture device that, with simple operation, can suppress leakage of liquid from around a cell culture insert into a well and can allow a liquid to flow through the well.SOLUTION: A cell culture device comprises: a well including a bottom part and a body part joined to the bottom part, the well having a through-hole penetrating the body part; a cylindrical part forming an opening in the well; a communication part connecting an inner wall surface of the well and the cylindrical part at a position closer to the bottom part than an end surface of the cylindrical part opposite the bottom part; a flow path through which a liquid containing a physiologically active substance flows to the well; and a cell culture insert having, on a bottom surface thereof, a membrane permeable to the physiologically active substance and inserted into the cylindrical part from a body part side through the well, wherein a gap between an inner wall surface of the cylindrical part and an outer wall surface of the cell culture insert is configured to be very small.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] The present invention relates to a cell culture device, and more particularly to a cell culture device for culturing cells using a cell culture insert. [Background technology]

[0002] Conventionally, cell culture inserts have been known as devices used for culturing cells to construct in vitro (outside the body) evaluation systems that mimic the in vivo environment (see, for example, Patent Document 1 below). The cell culture insert described in Patent Document 1 is a cylindrical member having a membrane on the bottom that is permeable to physiologically active substances and the like. [Prior art documents] [Patent documents]

[0003] [Patent Document 1] Patent No. 7265243 Summary of the Invention [Problem to be solved by the invention]

[0004] For example, a cell culture insert is inserted into a well through which a culture medium flows via a flow channel. By seeding cells on the membrane of the cell culture insert, physiologically active substances contained in the culture medium can be supplied to the cells via the membrane. As an example, an evaluation system can be constructed in which the cells are liver-derived cells. In this case, the culture medium corresponds to blood, and the flow channel corresponds to blood vessels.

[0005] However, simply inserting a cell culture insert into a well may result in the culture medium leaking into the well from around the cell culture insert, preventing the culture medium from flowing through the well.

[0006] In response to this, a cell culture device has been proposed in which the space within the well in which the cell culture insert is placed is sealed by providing a lid on top of the well to seal the inside of the well. In this case, the space within the well becomes a sealed space, which prevents the culture medium from leaking into the well from around the cell culture insert when the culture medium is passed through the well.

[0007] However, the device requires the insertion of a cell culture insert into a well and then the installation of a lid on the well, which complicates the process of constructing a cell evaluation system. In particular, when culturing cells, cells are typically cultured under multiple conditions, for example, by varying the concentration of a physiologically active substance in the culture medium. Furthermore, to improve the accuracy of the evaluation, multiple cell samples cultured under the same conditions are prepared. The need to close the lid on each well significantly complicates the process of constructing an evaluation system.

[0008] It is also anticipated that the effects of drug administration will be evaluated every time a specified culture period has elapsed. However, with the above device, the lid must be opened and closed each time a drug is administered. When administering drugs to a large number of samples, opening and closing the lid of each well takes a significant amount of time, which raises concerns that the timing of drug administration cannot be accurately determined.

[0009] As described above, conventional cell culture devices using cell culture inserts require complicated operations to pass liquids such as culture medium through the wells, and there is room for improvement.

[0010] In view of the above circumstances, an object of the present invention is to provide a cell culture device that can prevent leakage of liquid from around the cell culture insert into the well with a simple operation and allows liquid to flow through the well. [Means for solving the problem]

[0011] The cell culture device according to the present invention comprises: 1. A cell culture device comprising: a bottom portion; and a body portion joined to the bottom portion, a well formed by a through-hole penetrating the main body; a cylindrical portion that is located inside the through hole when the well is viewed from the main body portion side and forms an opening in the well; a connecting portion that connects the inner wall surface of the well and the cylindrical portion at a position closer to the bottom than an end surface of the cylindrical portion opposite to the bottom; a flow channel formed in a part of the joining surface between the bottom and the main body portion, for passing a liquid containing a physiologically active substance through the well; a cell culture insert having a membrane permeable to the physiologically active substance on a bottom surface, the cell culture insert being inserted into the cylindrical portion from the main body side via the well; The gap between the inner wall surface of the cylindrical portion and the outer wall surface of the cell culture insert is formed very small.

[0012] According to the above configuration, the cell culture insert is supported by a cylindrical portion formed within the well. A minute gap is formed between the inner wall surface of the cylindrical portion and the outer wall surface of the cell culture insert, allowing the liquid to be sucked up toward the main body portion by capillary force when flowing through the flow path. The liquid then reaches the end face of the cylindrical portion. Since the connecting portion is formed at a position closer to the bottom than the end face of the cylindrical portion, the cylindrical portion has an end in the radial direction extending from the center of the well to the outside. As will be described in detail later, the surface tension of the liquid retains the liquid at least at the end position, or at a position closer to the well than the end position. This prevents the liquid from leaking into the well.

[0013] In other words, with the above configuration, by simply inserting the cell culture insert into the cylindrical portion, it is possible to pass the liquid through the well while preventing the liquid from leaking into the well.

[0014] To reproduce the in vivo environment, culture media typically utilize a bicarbonate buffer system. By utilizing a bicarbonate buffer system, the mechanism that maintains the pH of blood in vivo can be reproduced. For example, a bicarbonate buffer system can be constructed using a CO2 incubator. In other words, contact between the gas in the CO2 incubator and the culture media maintains the equilibrium of the carbon dioxide concentration in the culture media, thereby maintaining the pH of the culture media at a predetermined value. If the space within the wells were sealed to allow the flow of liquid, such as culture media, through the wells, CO2 would no longer be supplied to the wells, making the pH of the culture media more unstable. In contrast, in the cell culture device described above, the space within the wells does not necessarily need to be sealed in order to allow the flow of liquid through the wells. In other words, the above configuration also facilitates the construction of a bicarbonate buffer system.

[0015] In the cell culture device, The gap between the cylindrical portion and the cell culture insert may be 50 μm or more and 500 μm or less. For example, the gap is 300 μm. The gap may be any size as long as it generates a capillary force between the inner wall surface of the cylindrical portion and the outer wall surface of the cell culture insert.

[0016] In the cell culture device, The cylindrical portion may extend from the end of the communication portion to the opposite side from the bottom portion.

[0017] As long as the liquid can be sucked up by capillary force, the location of the tubular portion can be determined as desired, but the above configuration is advantageous in that it makes it easy to form a small gap between the inner wall surface of the tubular portion and the outer wall surface of the cell culture insert.

[0018] In the cell culture device, The thickness of the cylindrical portion in the direction from the center of the well to the outside may be 500 μm or less.

[0019] In order for the liquid to generate surface tension at the end of the cylindrical portion, the liquid must reach that end. In light of this, the thickness of the cylindrical portion in the direction from the center of the well outward is preferably small, 500 μm or less, more preferably 200 μm or less.

[0020] In the cell culture device, The end face of the cylindrical portion opposite the bottom may have a portion where the distance from the bottom is constant or decreases as the end face moves toward the outside of the well. Specific configuration examples of the cylindrical portion are described in detail in the section "Mode for Carrying Out the Invention."

[0021] The cell culture device is a plurality of the wells formed in the body portion; A plurality of the cylindrical portions and the communication portions formed in each of the wells; A plurality of the cell culture inserts may be provided in each of the cylindrical portions.

[0022] According to the above configuration, the simple operation of inserting a cell culture insert into each well allows liquid to flow into each well while suppressing leakage of the liquid. In other words, the above configuration is preferable because it allows for more efficient construction of an evaluation system for a large number of samples.

[0023] In addition, in the cell culture device, The plurality of wells may be connected to each other by the flow path.

[0024] The above configuration makes it easy to construct a more complex evaluation system. Because liquid can be passed through each well with a simple operation while preventing leakage of the liquid into each well, interactions between multiple cells via the flow path can be more easily evaluated.

[0025] In the cell culture device, The main material constituting the main body and the bottom may be selected from the group consisting of cycloolefin polymer (COP), polystyrene (PS), polymethyl methacrylate (PMMA), cycloolefin copolymer (COC), polycarbonate (PC), polyether ether ketone (PEEK), fluororesin, glass material, and polymethylsiloxane.

[0026] In addition, in the cell culture device, The main material constituting the membrane of the cell culture insert may be selected from the group consisting of polyethylene terephthalate (PET), PC, COP, COC, polytetrafluoroethylene (PTFE), mixed cellulose esters, and collagen. [Effects of the Invention]

[0027] According to the present invention, a cell culture device is provided that can prevent leakage of liquid from around the cell culture insert into the well with a simple operation and allows liquid to flow through the well. [Brief explanation of the drawings]

[0028] [Figure 1] FIG. 1 is a perspective view of a cell culture device according to a first embodiment. [Figure 2] 2 is an exploded view of the cell culture device according to FIG. 1. [Figure 3A] 2 is a cross-sectional view taken along the line AA in FIG. 1. [Figure 3B] 3B is an enlarged view of the well in FIG. 3A. [Figure 3C] This is a drawing in which the cell culture insert is not shown in FIG. 3B. [Figure 4] This is a plan view of the well as seen from the +Z side. [Figure 5] 10 is an enlarged view showing the vicinity of a cylindrical portion in a state where a liquid is flowing through it; [Figure 6] 6 is a view similar to FIG. 5, showing another example of the configuration of the cylindrical portion. [Figure 7]10 is a diagram showing yet another example of the configuration of the cylindrical portion. [Figure 8] 10 is a diagram showing yet another example of the configuration of the cylindrical portion. [Figure 9] 10 is a diagram showing yet another example of the configuration of the cylindrical portion. [Figure 10] 10 is a diagram showing yet another example of the configuration of the cylindrical portion. [Figure 11] 10 is a diagram showing another example of the configuration of the cylindrical portion and the connecting portion. [Figure 12A] 10 is a diagram showing another embodiment of the cell culture device. [Figure 12B] 12B is a cross-sectional view of FIG. 12A. [Figure 13] FIG. 10 is a cross-sectional view showing yet another configuration example of the cell culture device. [Figure 14] FIG. 10 is a cross-sectional view showing another example of the configuration of the cell culture insert. DETAILED DESCRIPTION OF THE INVENTION

[0029] The cell culture device according to the present invention will be described with reference to the drawings. Note that the drawings are merely schematic illustrations. That is, the dimensional ratios in the drawings do not necessarily match the actual dimensional ratios, and the dimensional ratios between the drawings do not necessarily match.

[0030] [First embodiment] A first embodiment of a cell culture device will be described below with reference to the drawings. FIG. 1 is a perspective view of a cell culture device according to the first embodiment. As shown in FIG. 1, the cell culture device 1 includes a bottom portion 2, a main body portion 3 joined to the bottom portion 2, and a cell culture insert 7. FIG. 2 is a diagram showing the cell culture device 1 of FIG. 1 exploded into the bottom portion 2, the main body portion 3, and the cell culture insert 7. As shown in FIG. 2, the main surface 2a on the +Z side of the bottom portion 2 is joined to the main surface 3a on the -Z side of the main body portion 3 (not shown in FIG. 2; see FIG. 3A described below). Here, the "main surface" refers to a surface that is much larger in area than the other surfaces of the surface constituting a plate-like object.

[0031] In the following description, an XYZ coordinate system will be referred to where appropriate, with the direction perpendicular to the main surface 2a of the bottom 2 being the Z direction and the plane parallel to the main surface 2a being the XY plane (see FIG. 1, etc.). In the following description, when a positive or negative direction is to be distinguished when expressing a direction, the direction will be described with a positive or negative sign, such as "+X direction" and "-X direction." In addition, when a direction is to be expressed without distinguishing between positive and negative directions, the direction will be simply described as "X direction." In other words, in this specification, when simply referring to the "X direction," both the "+X direction" and the "-X direction" are included. The same applies to the Y direction and the Z direction.

[0032] As shown in Fig. 2, the bottom 2 has a recess 20 formed on the main surface 2a, recesses (21, 22) formed on the +X side and -X side of the recess 20, a groove 23 extending in the X direction to connect the recess 20 and the recess 21, and a groove 24 connecting the recess 20 and the recess 21. Fig. 3A is a cross-sectional view taken along the line AA in Fig. 1. As shown in Fig. 3A, the recesses (20, 21, 22) and the grooves (23, 24) all have a shape recessed from the main surface 2a toward the -Z side.

[0033] As shown in FIG. 3A, the main body 3 has a through hole 4 that penetrates the main body 3 in the Z direction, and through holes (31, 32) formed on the +X side and the −X side of the through hole 4.

[0034] 3A, through-hole 4 is formed at a position corresponding to recess 20, and bottom portion 2 and main body portion 3 are joined to form well 10. When viewed in the -Z direction, well 10 exposes bottom portion 2 (see FIG. 3C, described later).

[0035] FIG. 3B is an enlarged view of well 10 in FIG. 3A. FIG. 3C is a view of FIG. 3B without the cell culture insert 7. As shown in FIG. 3B, grooves 23 and 24 are connected to recess 20 (see also FIG. 2). Therefore, by joining bottom 2 and main body 3, through-hole 4 is connected to grooves 23 and 24 via recess 20. As a result, recess 20, grooves 23, and grooves 24 form flow path 6 that allows liquid to flow through well 10. As shown in FIG. 3A, flow path 6 is formed including joining surface 15 between bottom 2 and main body 3.

[0036] As an example, the flow path 6 is formed by etching the main surface 2a of the bottom portion 2 so that the areas where the recesses 20 and grooves (23, 24) are to be formed are recessed in the -Z direction from the area where the main body portion 3 is to be bonded. The bottom portion 2 may be embossed or injection molded to the desired shape. The groove depth is set according to the height H1 of the flow path 6 (see FIG. 3A). As an example, the height H1 of the flow path 6 is 100 μm or more and 1 mm or less. As an example, the width (dimension in the Y direction) of the flow path 6 is 100 μm or more and 20 mm or less.

[0037] Furthermore, through-hole 31 is formed at a position corresponding to recess 21, and through-hole 32 is formed at a position corresponding to recess 22 (see FIG. 3A). By joining bottom portion 2 and main body portion 3, through-hole 31 and well 10 are connected via groove 23. Similarly, through-hole 32 and well 10 are connected via groove 24. This makes it possible, for example, to introduce a liquid into well 10 through through-hole 32 and discharge the liquid through through-hole 31.

[0038] As shown in FIG. 3C, the well 10 has a cylindrical portion 11 and a connecting portion 12.

[0039] Fig. 4 is a plan view of the well 10 as viewed from the +Z side. As in Fig. 3C, the cell culture insert 7 is not shown in Fig. 4. As shown in Fig. 4, the cylindrical portion 11 forms an opening 5 on the inside of the through-hole 4 when viewed from the main body portion 3 side, i.e., the +Z side.

[0040] Also, as shown in FIG. 3C , the end face 11a of the cylindrical portion 11 opposite the bottom portion 2, i.e., on the +Z side, is located on the -Z side of the +Z side surface of the main body portion 3. In other words, the opening 5 is located within the well 10. As an example, the end face 11a of the cylindrical portion 11 has a flat shape, and when facing the outside of the well 10 in the XY plane, the distance in the Z direction between the end face 11a and the bottom portion 2 is constant. Note that the shape of the end face 11a is not limited to this. Configuration examples of the end face 11a will be described later in the section "Other Embodiments."

[0041] As shown in FIG. 3C , the connecting portion 12 connects the inner wall surface 10a of the well 10 to the cylindrical portion 11 at a position on the -Z side of the end surface 11a of the cylindrical portion 11. As a result of the connecting portion 12 being formed at a position on the -Z side of the end surface 11a of the cylindrical portion 11, an end portion 11b is formed in the cylindrical portion 11. Furthermore, in this embodiment, the cylindrical portion 11 is formed so as to extend from the end portion of the connecting portion 12 in the +Z direction, i.e., on the side opposite to the bottom portion 2. Note that the "end portion of the connecting portion 12" refers to a position corresponding to the inner wall surface 11c of the cylindrical portion 11.

[0042] As shown in FIG. 4, the communication portion 12 is formed over the entire area of ​​the cylindrical portion 11 in the circumferential direction.

[0043] The cylindrical portion 11 and the communication portion 12 can be realized by forming the main body portion 3 by, for example, injection molding. The same applies to the through-hole 4 that forms the well 10 and the through-holes (31, 32).

[0044] For example, the diameter of the through-hole 4 is 8 mm or more and 35 mm or less, and the diameter of the opening 5 is 4 mm or more and 26 mm or less. Also, for example, the diameter of the through-holes (31, 32) is 1 mm or more and 10 mm or less.

[0045] The bottom 2 and the main body 3 can be made of a thermoplastic resin. More specifically, the main material of the bottom 2 and the main body 3 can be selected from the group consisting of COP, PS, PMMA, COC, PC, PEEK, and fluororesin. In addition to the above-mentioned thermoplastic resins, glass materials such as quartz glass and borosilicate glass, or polydimethylsiloxane may also be used as the main material of the bottom 2 and the main body 3. To facilitate observation of the cultured cells, the bottom 2 and the main body 3 are typically made of a transparent material. Among the above-mentioned materials, COP, which is designated as a medical-grade resin, is particularly preferred. COP is a thermoplastic resin that exhibits high transparency, low autofluorescence, and low drug adsorption.

[0046] Here, the "main material" may refer to the material that makes up the object in the highest proportion of the materials.

[0047] The bottom portion 2 and the main body portion 3 can be joined using a method that utilizes surface modification using light or plasma, thermal bonding, adhesion using an adhesive, or solvent bonding. One example is a method of irradiating ultraviolet light onto the area where the bottom portion 2 and the main body portion 3 are to be joined. More specifically, vacuum ultraviolet light (VUV) with a wavelength of 200 nm or less can be used as the ultraviolet light. A suitable ultraviolet light source is an Xe excimer lamp with a peak wavelength of approximately 172 nm. The bottom portion 2 and the main body portion 3 can then be joined by pressing them together using a press or the like while they are in contact with each other through the area irradiated with ultraviolet light.

[0048] As shown in Figure 3B, the cell culture insert 7 is inserted into the cylindrical part 11 from the +Z side through the well 10. The cell culture insert 7 is cylindrical (see also Figure 2) and has a membrane 8 permeable to physiologically active substances on the bottom surface on the -Z side.

[0049] The main material constituting the membrane 8 can be selected from the group consisting of PET, PC, COP, COC, PTFE, mixed cellulose esters, and collagen. PET is preferred from the viewpoint of high versatility. The material constituting the membrane 8 can be appropriately selected depending on the type of cultured cells and the purpose of cell evaluation.

[0050] As an example, the main material of the cell culture insert 7 is a thermoplastic resin such as PS or COP.

[0051] For example, a culture medium (not shown) containing cells is injected into the cell culture insert 7 inserted into the cylindrical portion 11 (see also FIG. 3B), whereby the cells are seeded on the membrane 8 of the cell culture insert 7.

[0052] Then, for example, a liquid L1 (see FIG. 5 described later) containing a predetermined physiologically active substance is introduced into the flow channel 6 through the through-hole 32. This brings the liquid L1 into contact with the -Z side surface of the membrane 8. As described above, the membrane 8 is permeable to physiologically active substances. When the liquid L1 comes into contact with the -Z side surface of the membrane 8, the physiologically active substance contained in the liquid L1 is supplied to the culture solution in the cell culture insert 7. This allows cells to be cultured while the predetermined physiologically active substance is being supplied.

[0053] Fig. 5 is an enlarged view of the vicinity of the cylindrical portion 11 with the liquid L1 flowing therethrough. Fig. 5 shows an enlarged view of the -X side of the cylindrical portion 11. Fig. 5 also shows a gap D1 between the inner wall surface 11c of the cylindrical portion 11 and the outer wall surface 7a of the cell culture insert 7. In the cell culture device 1, the gap D1 is formed very minutely.

[0054] By forming a minute gap D1, as shown in Fig. 5, a capillary force is generated between the inner wall surface 11c of the cylindrical portion 11 and the outer wall surface 7a of the cell culture insert 7, which sucks up the liquid L1 flowing through the flow path 6 toward the +Z side. The liquid L1 sucked up by the capillary force reaches the end surface 11a of the cylindrical portion 11. After reaching the end surface 11a, the liquid L1 spreads on the end surface 11a in the -X direction and stops at the end 11b on the -X side of the end surface 11a.

[0055] The reason why the liquid L1 that has spread out stops at the end 11b is as follows. A predetermined contact angle θ1 is formed between the liquid L1 and the end surface 11a. The contact angle θ1 is determined depending on the relationship between the surface tension of the liquid L1 and the surface energy of the end surface 11a. Here, in order for the liquid L1 to spread out beyond the end 11b, the contact angle θ1 needs to increase. However, at the end 11b, the surface tension of the liquid L1 tries to keep the contact angle θ1 at a predetermined value. In other words, the liquid L1 is held at the end 11b by the surface tension of the liquid L1.

[0056] That is, in the cell culture device 1, when the liquid L1 flows in the X direction, the liquid L1 is sucked up to the +Z side by capillary force and reaches the end 11b. Here, the surface tension of the liquid L1 prevents the liquid L1 from spreading beyond the end 11b. As a result, the liquid L1 flowing in the X direction is prevented from leaking into the well 10.

[0057] In other words, in the cell culture device 1, by simply inserting the cell culture insert 7 into the cylindrical portion 11, the liquid L1 can be passed through the well 10 while preventing the liquid L1 from leaking from the flow path 6 into the well 10.

[0058] In the above description, the liquid L1 is assumed to wet and spread on the end surface 11a in the −X direction, but the same discussion can be applied to directions parallel to the XY plane.

[0059] The gap D1 can be any size as long as it generates a capillary force between the outer wall surface 7a of the cell culture insert 7 and the inner wall surface 11c of the cylindrical portion 11, but is preferably 50 μm or more and 500 μm or less.

[0060] In order to generate surface tension at end 11b, liquid L1 must reach end 11b. In view of this, it is preferable that thickness D2 of cylindrical portion 11 in the direction from the center of well 10 to the outside be small. Specifically, thickness D2 is preferably 500 μm or less, and more preferably 300 μm or less.

[0061] [verification] The results of actually forming a minute gap D1 and passing a liquid L1 through the flow path 6 will be described.

[0062] The configuration of the cell culture device used in this verification is the same as that described with reference to the above embodiment. Detailed conditions of the cell culture device 1 are shown below. Main material of bottom 2: COP Main material constituting the main body 3: COP Gap D1: 300μm Thickness D2 of the cylindrical portion 11: 1.5 mm Diameter of well 10: 20 mm Diameter of bottom of Cell Culture Insert 7: 14 mm Height of channel 6 H1: 100 μm Width of channel 6 (dimension in the Y direction): 1 mm

[0063] Phosphate buffered saline (PBS) was introduced as liquid L1 into the flow channel 6 of the cell culture device. The flow rate of PBS was set to 100 μL / min.

[0064] When liquid L1 was introduced, it was confirmed that liquid L1 could be passed through flow path 6 without leaking into well 10. The inventors also confirmed that liquid L1 remained at end 11b of tubular portion 11 while liquid L1 was being passed through flow path 6. In other words, a minute gap D1 was formed, and after liquid L1 was sucked up to the +Z side, surface tension of liquid L1 was generated at end 11b, thereby preventing liquid L1 from leaking into well 10.

[0065] In the cell culture device, the gap D1 is formed minutely, so that the liquid L1 can be sucked up to the +Z side by capillary force and reach the end 11b. In this verification, PBS was flowed as the liquid L1, but the fact that it can be sucked up by capillary force and that surface tension is generated at the end 11b is not limited to PBS. In other words, even if the liquid L1 is a culture solution containing a physiologically active substance, it is possible to flow the liquid L1 through the flow channel 6 while suppressing leakage into the well 10.

[0066] In other words, this verification confirmed that in the cell culture device 1 according to the above embodiment, the liquid L1 can be passed through the well 10 while preventing leakage of the liquid L1 into the well 10 by simply inserting the cell culture insert 7 into the cylindrical portion 11.

[0067] In addition, in the above test, the contact angle θ1 was about 60°. In view of this, it is estimated that the contact angle θ1 between the end surface 11a and the liquid L1 is preferably in the range of 50° to 70°.

[0068] [Another embodiment] Another embodiment of the cell culture device 1 will be described below.

[0069] <1> In the above description, it has been assumed that the end surface 11a of the cylindrical portion 11 has a flat shape, and that the distance in the Z direction between the end surface 11a and the bottom portion 2 is constant when extending outward from the well 10 within the XY plane. However, the configuration of the end surface 11a of the cylindrical portion 11 is not limited to the above. Figure 6 is a diagram showing another example of the configuration of the cylindrical portion 11, following Figure 5. As shown in Figure 6, the end surface 11a may be formed of a curved surface.

[0070] 6, the end surface 11a of the cylindrical portion 11 includes a portion p1 where the distance in the Z direction between the end surface 11a and the bottom 2 increases and a portion p2 where the distance decreases as the end surface 11a moves toward the outside of the well 10. In FIG. 6, a minute gap D1 is formed, and the liquid L1 flowing through the flow path 6 is sucked up to the +Z side by capillary force between the inner wall surface 11c of the cylindrical portion 11 and the outer wall surface 7a of the cell culture insert 7, as described with reference to FIG.

[0071] 6, the liquid L1 sucked up by capillary force spreads along a portion p1 of the end surface 11a, but does not spread to a portion p2 of the end surface 11a and is retained when it reaches the portion p2. This is because the portion p2 has a shape in which the end surface 11a approaches the bottom 2, and therefore, in order for the liquid L1 to spread beyond the portion p1 and to wet the portion p2, the contact angle θ1 between the end surface 11a and the liquid L1 must be large. In other words, the surface tension of the liquid L1 tries to keep the contact angle θ1 at a predetermined value, so the liquid L1 is retained when it reaches the portion p2 due to the surface tension of the liquid L1.

[0072] 7 is a diagram showing yet another example of the configuration of the cylindrical portion 11. As shown in FIG. 7, the end surface 11a may include curved portions (p1, p2) and a flat portion. In the example of FIG. 7, the wetting and spreading of the liquid L1 stops when it reaches the portion p2, as in the case of FIG. 6.

[0073] 8 and 9 are diagrams showing yet another example configuration of the cylindrical portion 11. As shown in FIGS. 8 and 9, the end surface 11a may be configured to have either a portion p1 where the distance in the Z direction between the end surface 11a and the bottom 2 increases as the end surface 11a moves outward from the well 10, or a portion p2 where the distance decreases. In the example of FIG. 8, the liquid L1 stops flowing when it reaches the end 11b of the cylindrical portion 11, as discussed with reference to FIG. 5. In the example of FIG. 9, the end surface 11a approaches the bottom 2 as the end surface 11a moves outward from the well 10, so the liquid L1 stops flowing when it reaches the end surface 11a. Note that the end surface 11a may be linear in FIGS. 8 and 9.

[0074] 10, the cylindrical portion 11 may have a protruding portion 11d that extends so as to approach the inner wall surface 10a of the well 10.

[0075] <2> Figure 11 is a diagram showing another example of the configuration of the cylindrical portion 11 and the connecting portion 12. In the above, the cylindrical portion 11 has been described as extending from the end of the connecting portion 12 toward the +Z side. However, as shown in Figure 11, the cylindrical portion 11 may be formed at a position closer to the inner wall surface 10a of the well 10 than the end of the connecting portion 12. The location where the cylindrical portion 11 is formed is arbitrary, as long as the gap D1 is formed minutely and the liquid L1 can be sucked up by capillary force.

[0076] From the viewpoint of easily forming the gap D1 minute, it is preferable that the cylindrical portion 11 extends from the end of the communication portion 12 toward the +Z side.

[0077] <3> Fig. 12A is a diagram showing another embodiment of the cell culture device 1. As shown in Fig. 12A, the cell culture device 1 may have a plurality of through-holes 4 formed in the main body 3, and may have a plurality of wells 10. Fig. 12B is a cross-sectional view taken along the line BB of Fig. 12A. As shown in Fig. 12B, a flow path 6 is formed in each of the plurality of wells 10. The method of forming the flow path 6 for each well 10 is the same as that described in the first embodiment.

[0078] In FIG. 12A, a cylindrical portion 11 and a connecting portion 12 are formed in each well 10, as described with reference to FIG. 3C. Also, as shown in FIG. 12B, a cell culture insert 7 is inserted into each well 10. According to this embodiment, by simply inserting the cell culture insert 7 into each well 10, the liquid L1 can be passed through each well 10 while suppressing leakage of the liquid L1. This makes it possible to more efficiently construct an evaluation system for a large number of samples. The number of wells 10 formed in the cell culture device 1 is arbitrary.

[0079] FIG. 13 is a cross-sectional view showing yet another example of the configuration of the cell culture device 1. In FIG. 13, the cross section of the cell culture device 1 is shown following FIG. 12B. As shown in FIG. 13, the cell culture device 1 may have multiple wells 10 connected by channels 6. As an example, in the example of FIG. 12B, a groove 23 is formed to connect a well 10 on the -X side with a well 10 on the +X side, thereby allowing the multiple wells 10 to be connected to each other by the channels 6. This configuration allows for the construction of a more complex evaluation system, making it possible to evaluate interactions between multiple cells via the channels 6. Note that the number of other wells 10 connected to one well 10 is arbitrary.

[0080] <4> Figure 14 is a cross-sectional view showing another example of the configuration of the cell culture insert 7. Similar to Figure 5, Figure 14 shows an enlarged view of the vicinity of the cylindrical portion 11 on the -X side. As shown in Figure 14, the cell culture insert 7 may have a tapered shape in which the outer wall surface 7a tapers toward the bottom 2. In this case, the gap D1 may be the distance from the position on the end surface 11a that is closest to the cell culture insert 7 in the X direction to the outer wall surface 7a of the cell culture insert 7.

[0081] It is more preferable that the gap between the inner wall surface 11c of the cylindrical portion 11 and the outer wall surface 7a of the cell culture insert 7 is formed very small over the entire area in the Z direction.

[0082] <5> Furthermore, to prevent microorganisms, dust, and the like present in the working environment from entering and affecting the cell evaluation system, the cell culture device 1 may be provided with a lid covering the +Z side of the well 10. As described above, in the cell culture device 1, when the liquid L1 is passed through, the operation of inserting the cell culture insert 7 into the cylindrical portion 11 prevents the liquid L1 from leaking into the well 10. That is, the lid may be configured to cover at least the +Z side of the well 10; for example, it may simply be placed on the +Z side of the main body 3. The lid may have any configuration. When a bicarbonate buffer system is constructed using a CO2 incubator, it is preferable that the atmosphere in the well 10 be replaced by the atmosphere in the CO2 incubator by natural diffusion, at least during the cell culture period. Furthermore, for example, in the example of FIG. 12A, a lid may be placed for each of the multiple wells 10, or a lid may be configured to cover all of the multiple wells 10 at once.

[0083] <6> The configurations of the above-described embodiments can be realized by appropriately combining them.

[0084] <7> The configuration of the cell culture device 1 described above is merely an example, and the present invention is not limited to the illustrated configurations. [Explanation of symbols]

[0085] 1: Cell culture device 2 : Bottom 3: Main body 4: Through hole 5 : Aperture 6: Flow path 7: Cell culture insert 8 : Membrane 10: Well 10a: Inner wall surface 11: Cylindrical part 12: Liaison Department 15: Joint surface 20, 21, 22: recesses 23,24 : Groove 31, 32: Through holes D1: Gap D2: Thickness H1: Height L1: Liquid θ1: Contact angle

Claims

1. 1. A cell culture device comprising: a bottom portion; and a body portion joined to the bottom portion, a well formed by a through-hole penetrating the main body; a cylindrical portion that is located inside the through hole when the well is viewed from the main body portion side and forms an opening in the well; a connecting portion that connects the inner wall surface of the well and the cylindrical portion at a position closer to the bottom than an end surface of the cylindrical portion opposite to the bottom; a flow channel formed in a part of the joining surface between the bottom and the main body portion, for passing a liquid containing a physiologically active substance through the well; a cell culture insert having a membrane permeable to the physiologically active substance on a bottom surface, the cell culture insert being inserted into the cylindrical portion from the main body side via the well; A cell culture device characterized in that a minute gap is formed between the inner wall surface of the cylindrical portion and the outer wall surface of the cell culture insert.

2. The cell culture device according to claim 1 , wherein the gap between the cylindrical portion and the cell culture insert is 50 μm or more and 500 μm or less.

3. The cell culture device according to claim 1 or 2, wherein the cylindrical portion extends from an end of the communication portion to a side opposite to the bottom portion.

4. 3. The cell culture device according to claim 1, wherein the thickness of the cylindrical portion in the direction from the center of the well to the outside is 500 [mu]m or less.

5. 3. The cell culture device according to claim 1, wherein the end face of the cylindrical portion opposite the bottom has a portion where the distance from the bottom is constant or the distance from the bottom decreases as the end face moves toward the outside of the well.

6. a plurality of the wells formed in the body portion; A plurality of the cylindrical portions and the communication portions formed in each of the wells; The cell culture device according to claim 1 or 2, comprising a plurality of the cell culture inserts disposed in each of the cylindrical portions.

7. The cell culture device according to claim 6 , wherein the plurality of wells are connected to each other by the flow channels.

8. 3. The cell culture device according to claim 1, wherein the main material constituting the main body and the bottom is selected from the group consisting of COP, PS, PMMA, COC, PC, PEEK, fluororesin, glass material, and polymethylsiloxane.

9. The cell culture device according to claim 1 or 2, characterized in that the main material constituting the membrane of the cell culture insert is selected from the group consisting of PET, PC, COP, COC, cellulose mixed esters, PTFE, and collagen.

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