Cell culture devices, cell culture sets, and cell characteristic detection sets

Abstract

It effectively applies mechanical stimulation to cell aggregates during contraction. [Solution] The cell culture device 10 comprises a substrate 20 having at least one opening 22, a pair of support bodies 30 for supporting cell aggregates 15, the pair of support bodies 30 being vertically extended from each of two opposing positions 22a of the opening 22 and elastically deformable, and a contact body 70 having a base 71 that locks into the opening 22, and a pair of contact portions 72 that extend from the base 71 and are inserted into the opening 22 and abut against each of the pair of support bodies 30, wherein when the contact body 70 is inserted into the pair of support bodies 30, the distance L4 between the pair of contact portions 72 at the contact position P with the support bodies 30 is greater than the distance L3 between the two positions 22a of the opening 22.

Description

[Technical Field]

[0001] This invention relates to a cell culture device, a cell culture set, and a cell characteristic detection set. [Background technology]

[0002] Output devices are known for evaluating the characteristics of muscle cells. For example, Patent Document 1 discloses an output device capable of detecting the contractile characteristics of muscle cells held on a support mainly composed of an extracellular matrix containing collagen, which has a long portion, using a strain gauge connected to a connecting portion at one end of the long portion.

[0003] According to Patent Document 1, the output device can effectively contract a complex including a support when muscle cells are stimulated to contract, and as a result, it can output tension, etc. According to the output device in Patent Document 1, it is possible to detect the contractile ability of muscle cells when stimulated to induce muscle contraction, so the function, responsiveness, and degree of differentiation of the muscle cells themselves can be evaluated, and drug screening can also be performed. Examples of the above-mentioned muscle contraction stimuli include physical stimuli such as electrical stimulation and mechanical stimulation, and chemical stimuli such as drugs, and it is stated that in the case of cardiac muscle, etc., it is useful to evaluate the output when the muscle is stretched, which is a mechanical stimulus. [Prior art documents] [Patent Documents]

[0004] [Patent Document 1] Japanese Patent Publication No. 2011-030574 [Overview of the Initiative] [Problems that the invention aims to solve]

[0005] However, in the output device described in Patent Document 1, muscle cells are cultured in a sheet-like (layered) manner on the long portion of the support and are composited with the support, which limits the method of applying mechanical stimulation. Therefore, there is still room for improvement in devices that apply mechanical stimulation to muscle cells, including the structure of the support that supports the muscle cells.

[0006] Therefore, there is a need for a cell culture device that can effectively apply mechanical stimulation to cell aggregates during contraction. [Means for solving the problem]

[0007] The cell culture device according to the present invention comprises a substrate having at least one opening; a pair of supports for supporting cell aggregates, the pair of supports being vertically extended from each of two opposing positions of the opening and elastically deformable; and a contact body having a base that locks into the opening and a pair of contact portions extending from the base and being inserted into the opening and in contact with each of the pair of supports, wherein, when the contact body is inserted into the pair of supports, the distance between the pair of contact portions at the contact position with the supports is greater than the distance between the two positions of the opening.

[0008] The cell culture set according to the present invention is characterized by comprising the cell culture device and a container capable of accommodating at least a portion of a pair of supports of the cell culture device together with a culture medium.

[0009] With these configurations, in a cell culture device, the distance between the pair of supports that support the cell aggregate can be restricted to a distance greater than the distance between two opposing positions of the openings in the substrate. This ensures that a load is reliably applied to the expanding and contracting cell aggregate, allowing for the culture of cell aggregates with strong contractile forces.

[0010] The cell characteristic detection set according to the present invention is a cell characteristic detection set using the cell culture set described above, characterized in that a pair of supports holding cell aggregates are housed in the container into which the culture medium is introduced, and the characteristics of the cell aggregates can be detected by detecting the amount of displacement of the supports from outside the cell culture set.

[0011] With this configuration, since the above-mentioned cell culture set is used, a load can be reliably applied to the cell aggregates during contraction, making it easier to detect the characteristics of the cell aggregates. In other words, by applying a load during expansion and contraction, the characteristics can be made more pronounced, enabling more accurate detection of those characteristics.

[0012] Preferred embodiments of the present invention will be described below. However, the scope of the present invention is not limited by the examples of preferred embodiments described below.

[0013] In the cell culture device according to the present invention, the pair of contact portions are preferably a pair of elastic pieces suspended from the base, and the pair of elastic pieces each have a pair of protruding portions extending toward the pair of supports.

[0014] This configuration allows a pair of elastic pieces to be inserted into the opening of the substrate in an elastically deformed state, and facilitates the attachment of a contact body between a pair of supports.

[0015] In the cell culture device according to the present invention, the pair of contact portions are a pair of overhanging portions that extend toward the pair of supports from the side walls on both sides of a plate-shaped body suspended from the base, and it is preferable that the plate-shaped body has a notch in the side wall between the base and the overhanging portion.

[0016] With this configuration, the notch can be inserted while rotating along the edge of the opening in the substrate, making it easy to attach the contact body between the pair of supports.

[0017] In the cell culture device according to the present invention, the contact body is detachable from the opening of the substrate and replaceable with another contact body, and it is preferable that the shortest distance between the locking surface of the substrate at the base portion and the straight line connecting the two contact positions of the pair of contact portions is different from that of the contact body.

[0018] According to this configuration, since different resistances can be imparted to the shrinking cell aggregates by the other contact body, it is easy to impart resistances according to the culture conditions of the cell aggregates to the expanding and shrinking cell aggregates.

[0019] In the cell culture device according to the present invention, it is preferable that the pair of supports has a support main body portion extending downward from the substrate and a bent end portion formed by bending the lower end portion of the support main body portion inside the pair of supports.

[0020] According to this configuration, it is easy to stably hold the cell aggregates by the bent end portion.

[0021] In the cell culture device according to the present invention, it is preferable that the pair of supports is a pair of film bodies arranged opposite to each other.

[0022] According to this configuration, it is easy to easily change the resolution of displacement measurement and the maximum measurable displacement amount by changing the thickness of the pair of film bodies.

[0023] In the cell culture device according to the present invention, it is preferable that the cell aggregates are skeletal muscle cell aggregates, cardiomyocyte aggregates, or smooth muscle cell aggregates.

[0024] According to this configuration, by applying a load to the cell aggregates when the cell aggregates contract, it becomes possible to more accurately detect the muscle contraction characteristics of skeletal muscle cell aggregates, cardiomyocyte aggregates, or smooth muscle cell aggregates.

[0025] Further features and advantages of the present invention will become more apparent from the following description of exemplary and non-limiting embodiments described with reference to the drawings. [Brief explanation of the drawing]

[0026] [Figure 1] Decomposed perspective view of the cell characteristic detection set of the first embodiment. [Figure 2] This is a cross-sectional view illustrating the cell characteristic detection set of the first embodiment, showing a relaxed state of cell aggregates. [Figure 3] This is a cross-sectional view illustrating the cell characteristic detection set of the first embodiment, showing a state in which the cell aggregate has contracted. [Figure 4] Perspective view of the support of the first embodiment [Figure 5] This is a cross-sectional view illustrating the cell characteristic detection set of the first embodiment, showing the contact body attached to the substrate. [Figure 6] Perspective view of the contact body of the first embodiment [Figure 7] This is a cross-sectional view illustrating the cell characteristic detection set of the first embodiment, and shows the procedure for attaching the contact body to the substrate. [Figure 8] This is a cross-sectional view illustrating the cell characteristic detection set of the first embodiment, showing the state in which other contact bodies are attached to the substrate. [Figure 9] Perspective view of the contact body of the second embodiment [Figure 10] This is a cross-sectional view illustrating the cell characteristic detection set of the second embodiment, showing the procedure for attaching the contact body to the substrate. [Figure 11] Examples of other supports [Figure 12] Examples of other supports [Modes for carrying out the invention]

[0027] 1. First Embodiment In the following description, the cell characteristic detection set 100 according to the first embodiment will be explained with reference to Figures 1 to 8. The cell characteristic detection set 100 of the present invention is used by placing the bottom surface of the container 50 of the cell culture set 5 on a workbench or the like. In the following description of this specification, when terms indicating direction are used, unless otherwise specified, the vertical direction, up-down direction and height direction refer to the up-down direction of the paper in each figure.

[0028] Figure 1 is an exploded perspective view of the cell characteristic detection set 100. The cell characteristic detection set 100 uses the cell culture set 5, and as shown in Figure 5, the displacement of the support 30 that supports the cell aggregates 15 can be detected from outside the cell culture set 5 using a measuring instrument such as an optical microscope 60. The cell characteristic detection set 100 detects the characteristics of the cell aggregates 15, specifically the characteristics when the cell aggregates 15 move (for example, the myocardial beating characteristics of cardiomyocyte aggregates).

[0029] The cell culture set 5 is used to culture at least a portion of cell aggregates 15 in order to detect cell characteristics, and comprises a cell culture device 10 and a container 50 capable of housing at least a portion of a pair of supports 30 of the cell culture device 10 along with culture medium. The cell culture device 10 has a pair of supports 30 suspended from a substrate 20 to support the cell aggregates 15, and a contact body 70 inserted between the pair of supports 30 to provide resistance when the cell aggregates 15 contract. In the cell culture set 5, the pair of supports 30 holding the cell aggregates 15 are housed in a container 50 into which culture medium has been introduced.

[0030] Figures 2 and 3 show the cell aggregate 15 held between a pair of supports 30 in the cell characteristic detection set 100. Both Figures 2 and 3 show the state before the contact body 70 is attached. Figure 2 shows the cell aggregate 15 in a relaxed state, and Figure 3 shows the cell aggregate 15 in a contracted state. With the cell aggregate 15 held between the pair of supports 30 in this state, the displacement of the supports 30, for example the displacement ΔW shown in Figure 3, can be detected from the outside using a measuring instrument such as an optical microscope 60. A cell aggregate refers to a mass of cells formed by the aggregation of multiple cells.

[0031] [Cell culture devices] The substrate 20 constituting the cell culture device 10 according to this embodiment is a rectangular resin plate material and has four openings 22 that penetrate the thickness direction of the substrate 20 at equal intervals along the longitudinal direction of the substrate 20. Each opening 22 is rectangular in shape, with the longitudinal direction of the substrate 20 being the elongated direction. A pair of support members 30 are suspended from each of the opposing positions 22a of the openings 22 of the substrate 20. Specifically, as shown in Figure 2, the substrate 20 has a support member fixing portion 20a near the opposing positions 22a of the openings 22, to which one end of the support member 30 can be fixed, and the support member 30 is fixed to the support member fixing portion 20a. In addition, the substrate 20 has stepped portions on two sides other than the two sides having the two opposing positions 22a of the openings 22, on the side opposite to the side where the container 50 is placed. The upper surface of these stepped portions becomes the locking surface 24 of the base portion 71 of the contact body 70, which will be described later, and the contact body 70 is locked to the substrate 20.

[0032] As shown in Figure 1, in this embodiment, opposing positions 22a of one opening 22, support members 30 suspended from 22a are paired, and in this embodiment, four sets of pairs of support members 30 are provided. Here, "suspended" means that they are provided in a way that they hang down. In the example of Figure 1, a pair of support members 30 are provided hanging vertically, but this is not limited to this, and they may be provided at an inclination of ±10 degrees to ±20 degrees relative to the vertical, for example.

[0033] The support 30 may be integrated with the substrate 20, or it may be detachably attached to the substrate 20. Here, as shown in Figures 1 and 2, the direction in which the pair of support 30 are aligned is defined as direction X1. In this embodiment, the longitudinal direction of the substrate 20 is the same as direction X1. The pair of support 30 are preferably made of polystyrene resin, polypropylene resin, or polyethylene resin.

[0034] As shown in Figures 1 and 2, the container 50 is provided with recesses 52 for holding liquid 55. The container 50 in this embodiment is a mold container formed from plastic, metal, or glass. A mold container is a container for holding cell aggregates 15 in a device, and is a container for agglomerating cells to form the shape of aggregates. The container 50 is provided with a plurality of recesses 52. The number of recesses 52 is preferably a multiple of the number of pairs of supports 30 per cell culture set 5 (in this example, "4"), and in this embodiment, four recesses 52 are provided. In this case, one cell culture device 10 is attached to one container 50.

[0035] Figures 2 and 3 illustrate the detection of the displacement ΔW of the support 30, i.e., the measurement of the displacement of the cell aggregates 15. The distance between the tips of the pair of support 30 changes due to the relaxation and contraction of the cell aggregates 15. Figure 2 shows the support in a state where the tip is not as bent as when the cell aggregates 15 are contracted due to relaxation. In Figure 3, the cell aggregates 15 and the support 30 in a state where the tip is bent due to the contraction of the cell aggregates 15 are shown by solid lines, while the support in Figure 2 in a state where the tip is not bent due to the relaxation of the cell aggregates 15 is shown by a dashed line. The cell aggregates 15 are, for example, skeletal muscle cell aggregates, cardiomyocyte aggregates, or smooth muscle cell aggregates cultured after differentiation induction from induced pluripotent stem cells, and after culture, the cell aggregates 15 enclose the lower ends of the pair of support 30. Thus, in this embodiment, the cell culture set 5 is used in at least part of the cell culture process of the cell aggregates 15 in order to detect cell characteristics. Preferably, the cell culture set 5 is used for differentiation induction and maturation of cell aggregates 15. In the cell culture set 5, for example, cell aggregates 15 before differentiation induction are attached to the respective ends of a pair of supports 30 of the cell culture device 10, and then the ends of the pair of supports 30 and the cell aggregates 15 before differentiation induction are immersed in a liquid culture medium of a culture vessel (not shown) to induce differentiation while crosslinking the cell aggregates 15 between the pair of supports 30. In this way, the target cell aggregates 15 (e.g., cardiomyocyte aggregates) are held between the pair of supports 30 before inserting the contact body 70 between the pair of supports 30.

[0036] The cell culture device 10 according to this embodiment is characterized by comprising a contact body 70 inserted into an opening 22 of a substrate 20, as shown in Figures 1 and 5. The contact body 70 has a pair of elastic pieces 72 (an example of a pair of contact parts) that contact each of a pair of support bodies 30, and provides resistance, which is a mechanical stimulus, when the cell aggregate 15 contracts. For example, when a healthy cell strain is used for the cell aggregate 15, the resistance applied to the expanding and contracting cell aggregate 15 causes the cell aggregate 15 to grow strongly. On the other hand, when a diseased cell strain is used for the cell aggregate 15, the resistance applied to the expanding and contracting cell aggregate 15 causes the cell aggregate 15 to grow weakly. In this way, by using the contact body 70, it is possible to make clear whether or not the cell aggregate 15 can grow strongly under those culture conditions. This makes it possible to culture cell aggregates 15 that can grow strongly.

[0037] Next, the support 30 and contact body 70 of the cell culture device 10 will be described in detail.

[0038] (Support) In this embodiment, as shown in Figure 4, the support 30 has a support body portion 32 extending downward from the support fixing portion 20a of the substrate 20, and a bent end portion 34 formed by bending the lower end of the support body portion 32. In the pair of support 30s, the tip portions of each support body portion 32 are bent perpendicularly in a direction that brings them closer together. By bending the tip portions of the support body portions 32 in this way to form the bent end portion 34, the cell aggregates 15 are held at the bent end portion 34. By placing the cell aggregates 15 on the pair of bent end portions 34 and culturing them, the cell aggregates 15 after culturing are made to enclose the pair of bent end portions 34. This configuration makes it possible to hold the cell aggregates 15 more stably. Furthermore, the pair of support 30s in this embodiment do not culture the cell aggregates 15 in a sheet-like manner on a plane as in Patent Document 1, but rather hold the cell aggregates 15 in the space between the pair of support 30s, making it easier to grow the cell aggregates 15 three-dimensionally.

[0039] In this embodiment, the support 30 is a film body, and the pair of support 30s are a pair of film bodies arranged opposite each other. As shown in Figure 4, the pair of support 30s, being film bodies, have a thin film shape with a width B1, a thickness T1, and the shortest distance L1 from the container 50 side of the substrate 20 to the bent end 34. The length L2 of the bent end 34 is, for example, 1 / 15 to 1 / 5 of L1. The pair of support 30s are suspended from the substrate 20 so that their thickness direction is the same as direction X1, and the bent end 34 side of the support body 32 is made elastically deformable in the direction X1 connecting the pair of support 30s by bending in the thickness direction. As a result, as shown in Figure 3, when the cell aggregate 15 held by the pair of support 30s contracts, both ends of the pair of support 30s elastically deform in the direction toward each other, i.e., in direction X1. If the displacement of one support 30 in this direction X1 is ΔW, then the displacement of the cell aggregate 15 in direction X1 is twice ΔW.

[0040] If the maximum displacement that the support 30 can elastically deform is defined as the maximum measurable displacement ΔWmax, then the maximum measurable displacement ΔWmax and the resolution can be changed by changing the thickness T1, without changing the measuring instrument that detects the displacement ΔW, the resin material of the support 30, or the length L1 and width B1 of the support 30. The pair of support bodies 30, which are film bodies, are preferably made of a resin material with a Young's modulus E of 100 MPa to 4500 MPa. They are also preferably made of a resin material with a Young's modulus E of 300 MPa to 4300 MPa. Even more preferably, they are made of a resin material with a Young's modulus E of 400 MPa to 4000 MPa. Furthermore, the pair of support bodies 30 preferably have a thickness T1 of 5 μm to 400 μm. They are also preferably made of a thickness T1 of 7 μm to 200 μm. Even more preferably, they have a thickness T1 of 8 μm to 100 μm. Note that the Young's modulus E in this specification is a measured value at 25°C (room temperature).

[0041] If the pair of support bodies 30, which are film bodies, are made of polystyrene resin, the Young's modulus E is preferably 3000 MPa or more and 4000 MPa or less, and the thickness T1 is preferably 40 μm or more and 55 μm or less. If the pair of support bodies 30, which are film bodies, are made of polypropylene resin, the Young's modulus E is preferably 1500 MPa or more and 2500 MPa or less, and the thickness T1 is preferably 50 μm or more and 65 μm or less.

[0042] Table 1 shows the Young's modulus E, maximum measured displacement ΔWmax, length L1, width B1, and thickness T1 for Test Example 1, which is a support 30 made of polystyrene, and Test Example 2, which is a support 30 made of polypropylene, as specific examples of a pair of film supports 30. Test Examples 1 and 2 were measured using a support 30 consisting only of a support body 32 without a bent end 34. The maximum measured displacement ΔWmax was calculated by placing a weight of known weight (1 to 30 mg) on ​​the support, measuring the displacement from a microscope, and then calculating the displacement under that load. [Table 1]

[0043] The resolution can also be changed by changing the measuring instrument that detects the displacement ΔW. The cell characteristic detection set 100 of this embodiment has a transparent or translucent container 50, and an optical microscope 60 capable of measuring the displacement ΔW of the support 30 is provided below the container 50. The optical microscope 60 corresponds to the measuring instrument that detects the displacement ΔW. The container 50 contains a transparent or translucent liquid 55. Preferably, illumination is provided above the container 50. The liquid 55 is a liquid containing compounds such as low molecular weight compounds, and by measuring the displacement ΔW of the support 30 and obtaining it as tension information, it is possible to evaluate the effect (toxicity) of the compound on cardiomyocyte aggregates, evaluate the efficacy of the compound on disease models of cardiomyocyte aggregates, and search for the optimal compound for inducing differentiation into cardiomyocyte aggregates.

[0044] (Abutting body) As shown in Figures 5 and 6, the contact body 70 has a base portion 71 that locks onto the locking surface 24 of the stepped portion of the opening 22, and a pair of elastic pieces 72 that are perpendicular to the base portion 71. In this embodiment, the base portion 71 of the contact body 70 has two extending portions 71A that extend in a direction perpendicular to the direction in which the pair of elastic pieces 72 are positioned (the direction of W2 in Figure 6) in its surface direction. The extending portions 71A of the base portion 71 are locked onto the locking surface 24 of the substrate 20. The pair of elastic pieces 72 each have a pair of projections 73 that extend toward the pair of support bodies 30. As shown in Figure 5, when the contact body 70 is inserted between the pair of support bodies 30, the shortest distance L4 between the pair of elastic pieces 72 at the contact position with the support bodies 30 is greater than the distance L3 between the two positions 22a of the opening 22. Furthermore, in this embodiment, the shortest distance L4 is set to be greater than the distance between the pair of support bodies 30 on the substrate 20 (i.e., the distance between the support body fixing portions 20a on the substrate 20).

[0045] As shown in Figure 5, the contact body 70 comes into contact with the pair of support bodies 30 from the inside when the cell aggregate 15 contracts. When the cell aggregate 15 relaxes, the contact body 70 does not necessarily have to be in contact with the pair of support bodies 30; it may be in contact with only one of the pair of support bodies 30, or with both.

[0046] The pair of support bodies 30 that deform due to the contraction of the cell aggregate 15 come into contact with the protruding portions 73 of the pair of elastic pieces 72 of the contact body 70, thereby suppressing the contraction deformation of the pair of support bodies 30. In this embodiment of the cell culture device, the contact body 70 can restrict the distance between the pair of support bodies 30 to a distance greater than the distance L3 between two opposing positions 22a of the opening 22 of the substrate 20, so that a stronger load can be applied to the cell aggregate 15 more effectively when it contracts compared to when the contact body 70 is not present. As a result, for example, when using a healthy strain, cell aggregates 15 with strong contractile force can be cultured.

[0047] The dimensions of each part of this embodiment, as shown in Figure 5, are not limited to those shown. For example, in this embodiment, the distance L3 between two opposing positions 22a of the opening 22 on the substrate 20 is 5 to 10 mm (e.g., 7 mm), the shortest distance L4 between the pair of elastic pieces 72 at the contact position with the support 30 is 8 to 10 mm (e.g., 9 mm), and the shortest distance L5 between the locking surface 24 on the base 71 to the substrate 20 and the straight line connecting the two contact positions P of the pair of elastic pieces 72 is 6 to 13 mm (e.g., 12 mm). Also, the distance between the pair of support 30 on the substrate 20 (i.e., the distance between the support fixing portions 20a of the substrate 20) is 5 to 10 mm (e.g., 8 mm).

[0048] In this embodiment, the contact body 70 has a flat contact surface 73A on which a pair of protruding portions 73 contact a pair of support bodies 30, and thus makes surface contact with the pair of support bodies 30. As a result, compared to the case where the pair of support bodies 30 and the contact body 70 make point contact or line contact, the contact surface 73A of the contact body 70 can stably spread out the film-like support bodies 30, making it easier to impart strong resistance to the cell aggregates 15. The contact surface 73A of the pair of protruding portions 73 is preferably shaped to conform to the surface of the support bodies 30, and may be flat as in this embodiment, or it may be a convex curved surface, etc. Also, the width B1 of the support bodies 30 and the width B2 of the contact surface 73A of the contact body 70 may be approximately the same, or it is preferable that the width B2 is smaller than the width B1. From the viewpoint of stably spreading out the film-like support bodies 30, it is preferable that the width B2 is 1 / 2 or more of the width B1, and more preferably 2 / 3 or more. Also, in the case of a film-like support body 30, it is preferable that the width B2 is equal to or less than the width B1. In this embodiment, the pair of elastic pieces 72 make surface contact with the pair of support bodies 30, so the two contact positions P mentioned above refer to the central portion in the vertical direction of the contact surface 73A of the protruding portion 73 of the pair of elastic pieces 72.

[0049] The dimensions of each part of the contact body 70 in this embodiment, as shown in Figure 6, are not limited to those shown. For example, in a free state without load, the shortest distance W1 between the contact surfaces 73A of the projection portion 73 of the contact body 70 is 8 to 10 mm (e.g., 8 mm), and the length W2 in the direction along the direction X1 of the base portion 71 is 6 to 8 mm (e.g., 7 mm). Also, the height H1 from the lower surface of the base portion 71 to the lower surface of the projection portion 73 is 10 to 14 mm (e.g., 12 mm), the height H2 of the contact surface 73A of the projection portion 73 is 0.5 to 3 mm (e.g., 1 mm), and the width B2 of the contact surface 73A is 2 to 3 mm (e.g., 2 mm). The ratio of the width B2 to the width B1 (e.g., 2.5 mm) of the support 30 is, for example, 4 / 5.

[0050] Next, an example of the procedure for attaching the contact body 70 to the opening 22 of the substrate 20 will be described with reference to Figure 7. Because the contact body 70 has the configuration described above, in a free state where no load is applied to the pair of elastic pieces 72, the shortest distance W1 between the contact surfaces 73A of the contact body 70 is greater than the distance L3 between the two opposing positions 22a in the opening 22.

[0051] As shown in S1 of Figure 7, first, one of the pair of elastic pieces 72 of the contact body 70, the protruding portion 73, is inserted into the opening 22 of the substrate 20. Next, as indicated by the arrow in Figure S1, the pair of elastic pieces 72 are elastically deformed using tweezers or the like so that the lower ends of the pair of elastic pieces 72 are close to each other, and the other protruding portion 73 is also inserted into the opening 22, so that the upper surface of the base 71 of the contact body 70 and the upper surface of the substrate 20 are parallel (S2). Next, as indicated by the arrow in Figure S2, the contact body 70 is inserted downward, and the base 71 of the contact body 70 is locked into the opening 22 of the substrate 20 (S3). At this time, the pair of elastic pieces 72 that were restricted between the two opposing positions 22a of the opening 22 are restored to their original shape, and the shortest distance L4 between the pair of elastic pieces 72 at the contact position P with the pair of support bodies 30 is greater than the distance L3 between the two positions 22a of the opening 22. Thus, the contact body 70 according to this embodiment can be inserted into the opening 22 of the substrate 20 while elastically deforming the pair of elastic pieces 72, making it easy to attach the contact body 70 between the pair of support bodies 30.

[0052] It is preferable that the contact body 70 is detachable from the substrate 20. In this case, when resistance is not required for the shrinking cell aggregate 15, the contact body 70 is removed, and the shrinking cell aggregate 15 is not subjected to resistance. Therefore, the presence or absence of resistance for the shrinking cell aggregate 15 can be switched depending on the degree of culture of the cell aggregate 15.

[0053] It is preferable that the contact body 70 is replaceable with another contact body 75. In the case of the other contact body 75, as shown in Figure 8, the shortest distance L7 between the locking surface 24 on the base 71 to the substrate 20 and the straight line connecting the two contact positions P of the pair of elastic pieces 72 is longer than the shortest distance L5 for the contact body 70. That is, the length from the lower surface of the substrate 20 to the pair of contact positions P is longer for the contact body 75 than for the contact body 70. As shown in Figure 8, if the length L7 of the contact body 75 from the locking surface 24 to the substrate 20 is longer than the length L5 of the contact body 70 from the locking surface 24 to the substrate 20 as shown in Figure 5, then when the cell aggregate 15 contracts, the contact body 75 shown in Figure 8 will contact each support 30 at a position closer to the cell aggregate 15 than the contact body 70 shown in Figure 5. Therefore, the contact body 75 shown in Figure 8 provides greater resistance to the contracting cell aggregate 15 than the contact body 70 shown in Figure 5. As shown in Figure 8, when the contact body 75 is inserted between a pair of support bodies 30, the shortest distance L6 between the pair of elastic pieces 72 at the contact position with the support bodies 30 is greater than the distance L3 between the two positions 22a of the opening 22, and the shortest distance L6 is greater than the shortest distance L4 when the contact body 70 is applied.

[0054] Thus, the greater the distance from the bottom surface of the substrate 20 to the pair of contact positions P, the further the support 30 will contact the contact body 70 at a position further from the substrate 20 (i.e., closer to the cell aggregate 15) when the cell aggregate 15 contracts. Therefore, the resistance that the contact body 70 imparts to the contracting cell aggregate 15 varies depending on the length of the contact body 70, and increases as the length of the contact body 70 increases. As a result, by attaching a contact body 70 of a length appropriate to the culture conditions of the cell aggregate 15 to the substrate 20, it is possible to impart an appropriate amount of resistance to the contracting cell aggregate 15 according to the culture conditions of the cell aggregate 15.

[0055] Furthermore, it is preferable that the cell culture device 10 includes one or more other contact bodies that are detachable and replaceable from the substrate 20. In addition, it is preferable that one or more of the other contact bodies have a different length from the contact body 70 from the locking surface 24 to the substrate 20 to the pair of contact positions P, as is the case with the other contact body 75.

[0056] When culturing the cell aggregates 15, gradually increasing the resistance applied when the cell aggregates 15 contract allows them to grow more strongly or weakly in a shorter time. Therefore, when culturing the cell aggregates 15, by sequentially replacing the contact bodies 70, which have a short distance from the locking surface 24 to the substrate 20 to the pair of contact positions P, with contact bodies 75, which have a longer distance from the locking surface 24 to the substrate 20 to the pair of contact positions P, strong or weak cell aggregates 15 can be efficiently cultured.

[0057] [container] Next, the details of the container 50 will be described. The inner surface 52a of the recess 52 in the container 50 is subjected to a non-specific cell adsorption suppression treatment. This non-specific cell adsorption suppression treatment is a treatment that suppresses the non-specific adsorption of cells. This non-specific cell adsorption suppression treatment can be performed, for example, by hydrophilization treatment. More specifically, the non-specific cell adsorption suppression treatment can be performed by a treatment that includes at least one group selected from the group consisting of groups represented by the following formulas (1), (2), (3), and (4) on the inner surface 52a of the recess 52.

[0058] [Chemical formula]

[0059] Here, in formula (1), R 31 , , 13 , , 11 ,

[0063] , , 11 ,

[0061] , , , , 32 , 21 , , 12 , , , , , ,

[0060] ,

[0062] is NH or an oxygen atom. m is an integer from 0 to 4. R 13 is a hydrogen atom, a hydroxyl group, or a methoxy group. In formula (3), R 32 is a hydrogen atom or a methyl group. n is an integer from 2 to 100.

[0060] Also, the non-specific adsorption suppression treatment of cells can be performed by forming a coating layer mainly composed of a polymer containing a specific hydrophilic constituent unit in the concave portion 52. Here, the hydrophilic constituent unit in the polymer that is the main component of the coating layer can include at least one constituent unit selected from the group consisting of the constituent units represented by the following formula (5), formula (6), formula (7), and formula (8).

[0061] [Chemical formula]

[0062] Here, * represents a bond. In formula (5), R 11 is a hydrogen atom or a methyl group. R 12 is NH or an oxygen atom. m is an integer from 0 to 4. R 13 is a hydrogen atom, a hydroxyl group, or a methoxy group. In formula (6), R 21 is a hydrogen atom or a methyl group. In formula (7), R 31 is a hydrogen atom or a methyl group. R 32 is a hydrogen atom or a methyl group. n is an integer from 2 to 100.

[0063] As an example, regarding the constituent unit represented by formula (5), R 11When R is a hydrogen atom, examples of monomers that serve as raw materials for hydrophilic constituent units include N-(2-hydroxyethyl)acrylamide (HEAA). 11 Examples of monomers that serve as raw materials for hydrophilic structural units when the group is a methyl group include 2-hydroxyethyl methacrylate (HEMA).

[0064] Furthermore, the hydrophilic constituent units in the polymer that forms the main component of the coating layer may include constituent units represented by the following formula (9).

[0065] [ka]

[0066] Here, * represents conjugation. In equation (9), R 41 is an alkyl group having a carbonyl group and an amino group. p is an integer from 1 to 1000. q is an integer from 40 to 4995. r is an integer from 0 to 4000. s is an integer from 1 to 3.

[0067] The polymer that forms the main component of the coating layer may further contain hydrophobic structural units. In this case, the hydrophobic structural units may include at least one structural unit selected from the group consisting of structural units represented by the following formulas (10) and (11).

[0068] [ka]

[0069] Here, * represents conjugation. In equation (10), R 51 R is a hydrogen atom or a methyl group. 52 R is a linear or branched alkyl group having 1 to 10 carbon atoms, an alicyclic alkyl group having 3 to 8 carbon atoms, or a combination thereof. In formula (11), R 61 This is either a hydrogen atom or a methyl group.

[0070] Examples of linear or branched alkyl groups having 1 to 10 carbon atoms include methyl, ethyl, propyl, methylethyl, butyl, 1,2-dimethylethyl, pentyl, 1-methylbutyl, 2-methylbutyl, and hexyl groups. Examples of alicyclic alkyl groups having 3 to 8 carbon atoms include cyclopropyl, cyclobutyl, cyclopentyl, and cyclohexyl groups.

[0071] The total content of hydrophilic constituent units in the polymer that forms the main body of the coating layer is preferably 10 mol% or more. The total content of hydrophilic constituent units is preferably 20 mol% to 80 mol%, and more preferably 30 mol% to 70 mol%. Furthermore, the total content of hydrophobic constituent units in the polymer is preferably 90 mol% or less. The total content of hydrophobic constituent units is preferably 40 mol% to 80 mol%, and more preferably 50 mol% to 70 mol%.

[0072] The polymer that forms the main component of the coating layer may further contain crosslinkable structural units in addition to the hydrophilic and hydrophobic structural units described above. The content of crosslinkable structural units may be, for example, 0.05 mol% to 20 mol%, and preferably 0.5 mol% to 10 mol%.

[0073] By providing such a coating layer, the contact angle of the inner surface 52a of the recess 52 with respect to pure water at 25°C (room temperature) is set to be between 3° and 90°. By making the inner surface 52a of the recess 52 hydrophilic, the nonspecific adsorption of compounds onto the inner surface 52a of the recess 52 can be suppressed. However, setting the contact angle to less than 3° may be undesirable from a cost perspective due to the extensive processing required. On the other hand, if the contact angle exceeds 90°, the hydrophilicity decreases, which may reduce the ability to suppress the nonspecific adsorption of compounds. By setting the contact angle to be between 3° and 90°, the nonspecific adsorption of compounds onto the inner surface 52a of the recess 52 can be suppressed at a relatively low cost.

[0074] The contact angle of the inner surface 52a of the recess 52 with respect to pure water is preferably 5° or more, and more preferably 8° or more. Furthermore, the contact angle of the inner surface 52a of the recess 52 with respect to pure water is preferably 80° or less, and more preferably 70° or less.

[0075] 2. Second Embodiment In the following description, the cell characteristic detection set 100 according to the second embodiment will be explained with reference to Figures 9 and 10. Figure 9 is a perspective view of the contact body 80 of the second embodiment, and Figure 10 is a cross-sectional view illustrating the cell characteristic detection set 100 of the second embodiment, showing the procedure for attaching the contact body 80 to the substrate 20.

[0076] The cell characteristic detection set 100 according to the second embodiment differs from the cell characteristic detection set 100 of the first embodiment in that the cell culture device 10 is equipped with a contact body 80 instead of a contact body 70. The following description will focus on the differences from the first embodiment. Points that are not specifically described are the same as in the first embodiment.

[0077] As shown in Figure 9, the contact body 80 comprises a base 81 and a plate-shaped body 82 suspended from the base 81. The plate-shaped body 82 has a pair of overhanging portions 83 that extend toward the pair of support members 30 from the side walls 82A on both sides of the plate-shaped body 82. The pair of contact portions that abut against the pair of support members 30 are the pair of overhanging portions 83. The contact surfaces 83A of the pair of overhanging portions 83 that abut against the pair of support members 30 are flat, as in the first embodiment. The plate-shaped body 82 has a semicircular notch 84 in the side wall 82A between the base 81 and the overhanging portions 83.

[0078] Next, an example of the procedure for attaching the contact body 80 to the opening 22 of the substrate 20 will be described with reference to Figure 10. Since the contact body 80 has the configuration described above, the outermost width W3 of the pair of protruding portions 83 of the contact body 80 is greater than the distance L3 between the two opposing positions 22a in the opening 22.

[0079] As shown in S1 of Figure 10, first, the protruding portion 83 on the notched side of the pair of protruding portions 83 of the contact body 80 is inserted into the opening 22 of the substrate 20 (S1). At this time, the notch 84 serves as a relief portion for the plate-shaped body 82 when inserting the contact body 80 into the opening 22. Next, as shown by the arrow in Figure S1, the notch 84 of the plate-shaped body 82 of the contact body 80 is inserted while rotating along the edge of the opening 22 of the substrate 20, and the other protruding portion 83 is also inserted into the opening 22. Then, the contact body 80 is positioned so that the upper surface of the base portion 81 of the contact body 80 and the upper surface of the substrate 20 are parallel (S2). Next, as shown by the arrow in Figure S2, the contact body 80 is inserted downwards, and the base portion 81 of the contact body 80 is locked into the opening 22 of the substrate 20 (S3). Thus, the contact body 80 according to this embodiment can be inserted while rotating with the notch 84 along the edge of the opening 22 of the substrate 20, making it easy to attach between the pair of support bodies 30.

[0080] In this embodiment, although not limited thereto, for example, the distance L3 between two opposing positions 22a of the opening 22 in the substrate 20 is the same as in the first embodiment, 5 to 10 mm (e.g., 7 mm), and the shortest distance L8 between a pair of protruding portions 83 at the contact position with the support 30 coincides with the outermost width W3 of the pair of protruding portions 83 of the contact body 80, and is 6 to 9 mm (e.g., 8 mm). The dimensions of each part of the contact body 80 in the second embodiment shown in Figure 9 are, although not limited thereto, for example, the length W4 in the direction along the direction X1 of the base portion 81 is 6 to 8 mm (e.g., 7 mm), and the height H3 from the lower surface of the base portion 81 to the lower surface of the plate-shaped body 82 is 10 to 14 mm (e.g., 12 mm). The height H4 of the contact surface in the protruding portion 83 is 0.5 to 3 mm (e.g., 1 mm), and the width B3 of the contact surface is 2 to 3 mm (e.g., 2 mm). Furthermore, the diameter of notch 84 is 6-9 mm (for example, 7 mm).

[0081] 3. Other Embodiments (1) In the first embodiment described above, the pair of contact portions of the contact body 70 was described as a pair of elastic pieces 72 suspended from the base 71, and the pair of elastic pieces 72 each had a pair of projections 73 extending toward a pair of support bodies 30. However, in the present invention, the pair of elastic pieces 72 of the contact body 70 does not necessarily have a pair of projections 73. For example, the pair of tip portions or the pair of intermediate portions of the pair of elastic pieces 72 extending from the base 71 may be configured to spread toward a pair of support bodies 30, and the tip portions or intermediate portions may contact the pair of support bodies 30. Also, when the pair of elastic pieces 72 have a pair of projections 73, the configuration is not limited to having the pair of projections 73 at the tip portions of the pair of elastic pieces 72, but the pair of projections 73 may be provided at each intermediate position of the pair of elastic pieces 72. Furthermore, the pair of elastic pieces 72 may have multiple pairs of projections 73 by having two or more projections 73 for each elastic piece 72.

[0082] (2) In the second embodiment described above, the pair of contact portions of the contact body 80 are a pair of overhanging portions 83 that extend toward a pair of support bodies 30 from the side walls 82A on both sides of the plate-shaped body 82 that is suspended from the base 81, and the plate-shaped body 82 has a notch 84 in the side wall 82A between the base 81 and the overhanging portions 83. However, the pair of overhanging portions 83 are not limited to the shape described in the second embodiment, and the contact body 80 may have one or more pairs of overhanging portions 83 in the side wall 82A between the base 81 and the lower end of the plate-shaped body 82. Also, the notch 84 in the plate-shaped body 82 only needs to be a relief portion of the plate-shaped body 82 when the contact body 80 is inserted into the opening 22 of the substrate 20, and is not limited to a semicircular shape, but may be a polygonal shape such as a triangle or square, or an ellipse, etc.

[0083] (3) In the first and second embodiments described above, a configuration in which the contact body has a contact surface and makes surface contact with the pair of support bodies 30 was described as an example. However, the configuration of the contact body is not limited as long as it has a base and a pair of contact portions that extend from the base and are inserted into the opening 22 to contact each of the pair of support bodies 30, and may be configured to make contact with the pair of support bodies by line contact or point contact. Furthermore, the contact body may have a configuration other than that of the first and second embodiments.

[0084] (4) In the first embodiment described above (see Figures 2 and 3), a configuration was described as one in which the displacement amount ΔW of the support 30 is measured from below the transparent or translucent container 50 using an optical microscope 60. However, the invention is not limited to such an example, and for example, the optical microscope 60 may measure the displacement amount ΔW from the side of the transparent container 50. Also, for example, the measuring instrument for detecting the displacement amount ΔW may be a measuring instrument other than an optical microscope. Also, for example, the displacement amount ΔW of the support 30 may be detectable from the outside by connecting a strain gauge to the support 30. Furthermore, the container 50 does not have to be light-transmitting, and the cell culture device 10 side may be light-transmitting, and the cell characteristic detection set 100 may be capable of detecting the displacement amount ΔW from the outside.

[0085] (5) In the first embodiment described above (see Figure 1), the pair of support bodies 30 were described as a pair of film bodies arranged opposite each other. However, the configuration is not limited to such an example, and for example, the shape of the support bodies 30 may be cylindrical or prismatic. Also, although the configuration in which the pair of support bodies 30 are made of resin was described as an example, the support bodies 30 may be made of metal, a soft material (silicone or elastomer), etc.

[0086] (6) In the first and second embodiments described above, a support 30 comprising a support body portion 32 and a bent end portion 34 was described (Figure 4). However, the support 30 does not have to have a bent end portion 34, and may consist only of the support body portion 32. Furthermore, as shown in Figure 11, the support 30 may have a recess 36 in the support body portion 32. By providing this recess 36, the cell aggregate 15 held by the pair of support 30 can be aggregated as a whole. As a result, the thickness of the cell aggregate 15 near the recess 36 increases, making it less likely for stress to concentrate on a part of the cell aggregate 15 when the cell aggregate 15 pulsates, and as a result the cell aggregate 15 becomes less likely to be damaged.

[0087] (7) The support 30 may also have a support body portion 32 formed on a flat surface and a notch portion 37 on the widthwise side surface of the support body portion 32. With this configuration, the portion of the support body portion 32 closer to the tip than the notch portion 37 is easier to bend, and the bent portion corresponding to the bent end portion 34 described in the first and second embodiments above can be easily formed afterward. Here, it is preferable that the notch portion 37 is formed at a position of 0.5 mm to 2.0 mm from the tip of the support body portion 32. The support 30 illustrated in Figure 12 includes a recess 36 and a notch portion 37. The recess 36 shown in Figure 12 is formed on both sides in the widthwise direction of the support body portion 32, similar to the recess 36 shown in Figure 11. On the other hand, the longitudinal length of the recess 36 shown in Figure 12 is shorter than the longitudinal length of the recess 36 shown in Figure 11. The notches 37 are formed by cutting out triangular shapes on both sides of the width direction of the support body 32, towards the tip of the recess 36. The position, size, and shape of the recess 36 and the notches 37 may be determined as appropriate depending on the type of cell aggregate 15.

[0088] (8) In the first and second embodiments described above, examples were given in which a pair of bent ends 34 are formed by bending them toward each other. However, the bent ends 34 may also be formed by bending the lower ends of the support body portion 32 toward each other.

[0089] (9) In the first embodiment described above (see Figure 1), the cell aggregate 15 was described as a skeletal muscle cell aggregate, cardiomyocyte aggregate, or smooth muscle cell aggregate produced by differentiation induction from induced pluripotent stem cells (iPS cells). However, the invention is not limited to such examples, and for example, the cell aggregate 15 may be a muscle cell aggregate, adipocyte aggregate, osteocyte aggregate, nerve cell aggregate, epithelial cell aggregate, chondrocyte aggregate, or tendon tissue differentiated from embryonic stem cells (ES cells), nuclear transfer embryonic stem cells (ntES cells), somatic stem cells, umbilical cord blood stem cells, etc. Furthermore, for example, the cell aggregate 15 may be produced by a method other than differentiation induction.

[0090] (10) The configurations disclosed in each of the above embodiments can be applied in combination with configurations disclosed in other embodiments, as long as no inconsistencies arise. With regard to other configurations, the embodiments disclosed herein are merely illustrative in all respects. Therefore, various modifications can be made as appropriate without departing from the spirit of this disclosure. [Industrial applicability]

[0091] The technology described herein can be used, for example, in a cell characteristic detection device for detecting the contractile characteristics of cardiomyocyte aggregates. [Explanation of Symbols]

[0092] 5: Cell culture set 10: Cell culture devices 15:Cell aggregate 20: Circuit board 30:Support 32: Support main body part 34: Bent end 50: Container 52a: Inner surface 70, 80: Contact object 75: Other contact bodies 100: Cell characteristic detection set ΔW: Displacement

Claims

1. A substrate having at least one aperture, A pair of supports for supporting cell aggregates, each of which is suspended from two opposing positions of the opening and is elastically deformable, The contact body comprises a base portion that engages with the opening, and a pair of contact portions that extend from the base and are inserted into the opening to abut against each of the pair of support members, A cell culture device in which, when the contact body is inserted into a pair of supports, the distance between the pair of contact portions at the contact positions with the supports is greater than the distance between the two positions of the opening.

2. The pair of contact portions are a pair of elastic pieces suspended from the base, The cell culture device according to claim 1, wherein the pair of elastic pieces each have a pair of protruding portions extending toward the pair of supports.

3. The pair of abutment portions are a pair of protruding parts that extend toward the pair of support members from the side walls on both sides of the plate-shaped main body that is suspended from the base, The cell culture device according to claim 1, wherein the plate-shaped body has a notch in the side wall between the base and the protruding portion.

4. The contact body is detachably attached to the opening of the substrate and replaceable with another contact body. The cell culture device according to claim 1, wherein the shortest distance between the locking surface to the substrate at the base and the straight line connecting the two contact positions of the pair of contact parts is different from that of the other contact body.

5. The cell culture device according to claim 1, wherein the pair of supports has a support body portion extending downward from the substrate and a bent end portion formed by bending the lower end of the support body portion inward of the pair of supports.

6. The cell culture device according to claim 1, wherein the pair of supports are a pair of film bodies arranged opposite each other.

7. The cell culture device according to claim 1, wherein the cell aggregate is a skeletal muscle cell aggregate, a cardiomyocyte aggregate, or a smooth muscle cell aggregate.

8. A cell culture device according to any one of claims 1 to 7, A cell culture set comprising a container capable of accommodating at least a portion of a pair of supports for the cell culture device along with a culture medium.

9. A cell characteristic detection set using the cell culture set described in claim 8, A pair of supports holding cell aggregates are housed in the container into which the culture medium has been introduced. A cell characteristic detection set that enables the detection of the characteristics of the cell aggregate by detecting the amount of displacement of the support from outside the cell culture set.

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