Cell culture substrate and cell culture method

Abstract

This cell culture substrate is provided with at least one hole having a side surface and a bottom surface, the side surface including a first side section defining a first space and a second side section defining a second space communicating with the first space, the upper end of the first side section being formed in a C-shape having a central angle of 240 degrees or more in plan view, and the lower end of the first side section being formed in a C-shape having a central angle of 240 degrees or more in plan view. The second space extends in a first direction away from the first space from an opening portion of the upper end portion, and an opposing surface of the second side portion opposing the opening portion in the first direction is disposed at a position where a maximum distance between the opening portion and the opposing surface in the first direction is smaller than a diameter of the C-shape of the upper end portion.

Description

Technical Field

[0001] The present invention relates to a cell culture substrate having wells for culturing cells, and a cell culture method using the cell culture substrate. Background Art

[0002] Regarding the cell culture test, for example, a resin substrate called a well plate (or companion plate) provided with a plurality of holes can be used for the test. A cell culture container called an insert is arranged in the well, and cells are cultured in the insert. Cells grow by taking nutrients from the culture medium contained in the well and the insert. In this specification, a substrate provided with at least one hole like a well plate is referred to as a "cell culture substrate".

[0003] The culture medium in the well and the culture medium in the insert can be discharged from the well using a pipette, an aspirator, etc. as needed and replaced with a new culture medium. Usually, the old culture medium in the well is discharged with the insert removed from the well. This is because, when the insert is arranged in the well, the gap between the inner side of the well and the insert is narrow, and it is difficult to insert the front end of the pipette, etc. into the well without contacting the insert. However, each time the culture medium is replaced, it is necessary to take out the insert from each well with great effort.

[0004] For example, Patent Document 1 discloses a cell culture apparatus capable of discharging a culture medium from a well without removing an insert from the well.

[0005] Prior art literature

[0006] Patent Literature

[0007] Patent Document 1: International Publication No. 2018 / 105078 Summary of the invention

[0008] Problems to be solved by the invention

[0009] The cell culture device disclosed in Patent Document 1 is configured to supply and discharge a culture medium via an insertion tube inserted into a well and outside an insert. According to the configuration of Patent Document 1, the culture medium can be replaced with the insert disposed in the well.

[0010] However, as for the structure of patent document 1, there is still room for improvement in terms of holding the insert more stably in the hole. If the insert is not stably held, the insert may shake or tilt during the operation of replacing the culture medium in the hole. It is considered that this may affect the cells in the insert. In addition, if the insert is not stably held, it may be difficult to discharge the culture medium in the insert. It is considered that it is particularly difficult to automatically (using a robot, etc.) discharge the culture medium from the insert.

[0011] An object of the present invention is to solve the above-mentioned problems and to provide a cell culture substrate capable of replacing the culture medium while suppressing the influence on cultured cells.

[0012] Technical means to solve problems

[0013] In order to achieve the above object, the present invention is constructed as follows.

[0014] A cell culture substrate according to one embodiment of the present invention is a cell culture substrate having at least one well, wherein:

[0015] The at least one hole has a side surface and a bottom surface, wherein the side surface includes a first side portion defining a first space and a second side portion defining a second space communicating with the first space,

[0016] When looking down,

[0017] The upper end of the first side portion is formed into a C-shape with a central angle of 240 degrees or more.

[0018] The second space extends from the opening portion of the upper end portion in a first direction away from the first space.

[0019] The opposing surface of the second side portion that is opposed to the opening portion in the first direction is arranged at a position where a maximum distance between the opening portion and the opposing surface in the first direction is smaller than a diameter of the C-shape of the upper end portion.

[0020] Effects of the Invention

[0021] According to the present invention, it is possible to provide a cell culture substrate capable of replacing a culture medium while suppressing the influence on cultured cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 This is a perspective view of a well in a cell culture substrate according to an embodiment of the present invention.

[0023] Figure 2A yes Figure 1 Top view of the hole.

[0024] Figure 2B yes Figure 1 A1-A1 line cross-sectional view.

[0025] Figure 2C yes Figure 1 B1-B1 line cross-sectional view.

[0026] Figure 2D yes Figure 1 B2-B2 line cross-sectional view.

[0027] Figure 3A It is a top view illustrating variations in the shape and arrangement of the grooves.

[0028] Figure 3B It is a top view illustrating variations in the shape and arrangement of the grooves.

[0029] Figure 3C It is a top view illustrating variations in the shape and arrangement of the grooves.

[0030] Figure 3D It is a top view illustrating variations in the shape and arrangement of the grooves.

[0031] Figure 4 It is a perspective view illustrating an insert.

[0032] Figure 5 It is used to describe the settings Figure 1 A three-dimensional view of the support structure in the hole.

[0033] Fig. 6A The anti-rotation ribs of the holes are Figure 4 A top view showing the configuration relationship between the side walls of the insert.

[0034] Figure 6B It is a top view showing a state where another insert is supported in a hole.

[0035] Fig. 7A This is a cross-sectional view for explaining the process of the cell culture method.

[0036] Figure 7B This is a cross-sectional view for explaining the process of the cell culture method.

[0037] Fig. 8A This is a cross-sectional view for explaining the process of the cell culture method.

[0038] Figure 8B This is a cross-sectional view for explaining the process of the cell culture method.

[0039] Fig. 9A This is a cross-sectional view for explaining the process of the cell culture method.

[0040] Fig. 9B This is a cross-sectional view for explaining the process of the cell culture method.

[0041] Fig. 10A This is a cross-sectional view for explaining the process of the cell culture method.

[0042] Fig. 10B This is a cross-sectional view for explaining the process of the cell culture method.

[0043] Fig.11A This is a cross-sectional view for explaining the process of the cell culture method.

[0044] Fig. 11B This is a cross-sectional view for explaining the process of the cell culture method.

[0045] Fig. 12A This is a cross-sectional view for explaining the process of the cell culture method.

[0046] Fig. 12B This is a cross-sectional view for explaining the process of the cell culture method.

[0047] Fig.13 This is a perspective view showing an example of a cell culture substrate.

[0048] Fig.14 yes Fig.13 A top view of the cell culture substrate is shown.

[0049] Fig.15 This is a perspective view illustrating a state where the cell culture substrate is tilted in the X direction.

[0050] Fig.16 Yes means Fig.13 A top view of a modified example of the cell culture substrate.

[0051] Fig.17 This is a graph showing the measurement results of TEER values ​​in cell culture experiment 1.

[0052] Fig.18 (a)~ Fig.18 (e) is a graph showing the results of mRNA expression analysis in cell culture experiment 1.

[0053] Fig.19 (a)~ Fig.19 (d) is a graph showing the results of measuring drug metabolizing enzyme activities in cell culture experiment 1.

[0054] Fig. 20 (a)~ Fig. 20 (c) is a phase contrast microscopic image of the cell surface on day 6 after inoculation in cell culture experiment 2.

[0055] Fig.21 (a)~ Fig.21 (c) is a phase contrast microscopic image of the cell surface on day 7 after inoculation in cell culture experiment 2.

[0056] Fig. 22 (a)~ Fig. 22 (c) is a phase contrast microscopic image of the cell surface on day 8 after inoculation in cell culture experiment 2.

[0057] Fig.23 (a)~ Fig.23 (c) is a phase contrast microscopic image of the cell surface on day 9 after inoculation in cell culture experiment 2.

[0058] Fig.24 (a)~ Fig.24 (c) is a phase contrast microscopic image of the cell surface on the 10th day after inoculation in cell culture experiment 2.

[0059] Fig.25 This is a graph showing the measurement results of TEER values ​​in cell culture experiment 2.

[0060] Fig.26 (a)~ Fig.26 (j) is a graph showing the results of mRNA expression analysis in cell culture experiment 2. DETAILED DESCRIPTION

[0061] (Foundation that forms the basis of the present invention)

[0062] The present inventors have conducted intensive studies on the configuration of a cell culture substrate capable of replacing the culture medium while suppressing the influence on cultured cells, and have obtained the following findings.

[0063] In order to perform the medium replacement work without removing the insert from the well, it is conceivable to ensure a space for inserting a tube such as a pipette in the well in addition to the space for arranging the insert.

[0064] Holes in the shape of hollow cylinders are provided on conventional orifice plates. For example, an insert is disposed approximately at the center of a circular hole when viewed from above. If a space for inserting a pipette or other tube is provided inside a hole of this shape, it becomes difficult to stably hold the insert. For example, in Patent Document 1, an insert having a shape in which a portion of a cylindrical shape is cut off is used in order to ensure space for inserting a tube in the hole. Therefore, the balance of the insert is poor, and when replacing the culture medium in the hole, the insert may be shaken due to the flow of the culture medium, etc. Furthermore, if the hole is tilted in order to efficiently discharge the culture medium, the insert may deviate from a specified position, or the insert may tilt and touch the inner side surface of the hole. This may cause damage to the cells in the insert, such as cells falling off from the bottom surface of the insert. In addition, since the balance of the insert is poor, it is also difficult to replace the culture medium in the insert.

[0065] Therefore, the inventors have repeatedly studied and found that by providing a first space in which an insert can be placed and a second space connected to the first space in the hole, and forming the upper end of the side surface defining the first space into a C-shape when viewed from above, and the second space has a structure extending from the opening of the C-shape, the insert can be stably held in the first space, and the second space can be used as a space for inserting a discharge tube such as a pipette. Based on this new insight, the inventors have completed the following invention.

[0066] A cell culture substrate according to one embodiment of the present invention is a cell culture substrate having at least one hole, wherein the at least one hole has a side surface and a bottom surface, the side surface includes a first side portion defining a first space and a second side portion defining a second space connected to the first space, wherein the upper end portion of the first side portion is formed into a C-shape with a central angle of more than 240 degrees when viewed from above, the second space extends from an opening portion of the upper end portion in a first direction away from the first space, and an opposing surface of the second side portion that is opposite to the opening portion in the first direction is arranged at a position where the maximum distance between the opening portion and the opposing surface in the first direction is less than the diameter of the C-shape of the upper end portion.

[0067] According to this configuration, the insert can be stably maintained in the first space by using the first side portion having a C-shaped upper end portion with a center angle of more than 240 degrees (i.e., an arc shape with a center angle of 240 degrees) when viewed from above. For example, a commercially available ordinary cylindrical insert can be maintained approximately uniformly over its entire circumference. In addition, the second space is a space extending from the opening portion of the C-shaped upper end portion of the first side portion when viewed from above, and even if a pipette or the like is inserted into the second space, it is not easy to affect the cells in the insert. Therefore, while the insert is maintained in the first space of the hole, the second space can be used to discharge and supply the culture medium while suppressing damage to the cultured cells in the insert. Furthermore, since the insert can be stably maintained, the culture medium in the insert can be easily replaced.

[0068] In addition, since the maximum distance in the first direction between the opening portion of the upper end of the first side portion and the opposite surface of the second side portion is smaller than the diameter of the upper end of the first side portion, the increase in the hole size caused by the provision of the second space can be suppressed. Therefore, for example, a plurality of holes can be arranged at the same pitch as in a conventional orifice plate.

[0069] When discharging the culture medium, by tilting the cell culture substrate, the bottom surface of the hole can be tilted from the horizontal direction. Thus, the culture medium in the first space can be easily moved to the second space by gravity, so the culture medium can be efficiently discharged from the second space. It should be noted that, since the insert is stably maintained in the first space, even if the cell culture substrate is tilted, the position (relative position) of the insert relative to the first side portion can be maintained. Therefore, the impact on the cell caused by the insert contacting the first side can be suppressed. In addition, it is possible to suppress the part of the culture medium remaining in the part and not being discharged (liquid residue) due to the local narrowing of the gap between the insert and the first side portion.

[0070] Furthermore, if the above-mentioned cell culture substrate is used, TEER can be easily measured. When TEER is measured, the electrode can be easily inserted by inserting one of a pair of electrodes into the insert in the first space and the other into the second space. In addition, the damage of cells caused by the contact between the electrode and the insert can be suppressed.

[0071] When viewed from above, the opposite surface of the second side portion can be smoothly curved in a manner protruding toward the first direction. According to this configuration, when the culture medium is discharged from the second space, liquid residue is not likely to occur. In addition, since the second side portion has the above configuration, the opening of the discharge tube for discharging the culture medium (e.g., the front end of a pipette) can be inserted, and the second space with controlled volume can be demarcated. Therefore, the increase in the hole size caused by the provision of the second space can be further suppressed.

[0072] The bottom surface includes a first bottom defining the first space and a second bottom defining the second space, and the first bottom and the second bottom can be flush. According to this structure, when the culture medium is discharged, the culture medium can be smoothly moved from the first space to the second space. Therefore, the culture medium can be discharged more effectively. In addition, when the cell culture substrate is tilted when the culture medium is discharged, the tilt angle can be suppressed to be smaller, so the influence on the cultured cells caused by the tilted cell culture substrate can be reduced.

[0073] The bottom surface has at least one groove, and the at least one groove may include a first groove that is continuous from a first portion that contacts the first side portion to a second portion that contacts the second side portion. According to this configuration, liquid in the gap between the insert and the first side portion of the hole can easily move to the second space side through the groove. Therefore, the culture medium in the hole can be more effectively discharged from the second space.

[0074] It should be noted that if the gap between the first side portion and the insert is small, the insert can be more stably held in the first space, but on the other hand, the gap is prone to liquid residue due to capillary phenomenon. In contrast, by providing the first groove on the bottom surface of the hole, the gap between the first side portion and the insert can be suppressed to be small while reducing the amount of culture medium remaining in the gap. The width and depth of the first groove can be designed in a manner that the capillary force of the first groove can be used to suck out liquid from the gap between the first side portion and the insert.

[0075] In the first side portion, a step portion is provided in such a manner that the diameter of the upper end portion is enlarged compared to the diameter of the lower end portion in contact with the bottom surface, and the step portion can extend in the circumferential direction when viewed from above. According to this configuration, the step portion can suppress the rise of the liquid level (the upper surface of the culture medium) in the gap between the insert and the first side portion of the hole. Therefore, when the culture medium is discharged from the hole, the old culture medium is not likely to remain in the gap. In addition, if the liquid level in the gap becomes too high, the liquid level in the second space S2 may sometimes drop by the volume of the culture medium that rises in the gap, and the liquid level in the insert may also drop (i.e., the amount of liquid decreases) due to the siphon phenomenon. By suppressing the rise of the liquid level in the gap by the step portion, the reduction in the amount of liquid (the amount of culture medium) in the insert due to the siphon phenomenon can be suppressed.

[0076] The first side portion may include a support structure in the first space, the support structure supporting the cell culture container (insert) at a distance from the bottom surface, thereby making it possible to more stably hold the insert in the first space.

[0077] The cell culture substrate may be provided with a plurality of holes. For example, when viewed from above, the at least one hole may include a plurality of holes arranged in a row direction and a column direction intersecting the row direction. The first direction in each of the plurality of holes may be a direction intersecting both the row direction and the column direction. According to this configuration, more holes may be arranged in a limited area. The row direction and the column direction may be orthogonal to each other. In this case, the plurality of holes may be arranged in such a way that the first direction of each hole is at an angle of, for example, 45 degrees to the row direction and the column direction when viewed from above.

[0078] The cell culture substrate may also be provided with an outer wall surrounding the plurality of holes when viewed from above. The plurality of holes may include a plurality of outer holes at the end of the row direction or the column direction, and a pool portion for accommodating water may be formed between the sidewall of the side surface forming the plurality of outer holes and the outer wall. By filling the pool portion with water such as purified water, the evaporation of the culture medium in the hole may be suppressed. The pool portion may be divided into more than the same number as the plurality of outer holes. According to this configuration, even if the cell culture substrate is tilted, the water contained in the pool portion is not easy to overflow.

[0079] A cell culture method according to one embodiment of the present invention is a cell culture method for culturing cells in the cell culture container using the cell culture substrate and the cell culture container, wherein the cell culture container is detachably contained in the first space of the at least one hole and is separated from the bottom surface, wherein the cell culture method includes a discharge step for discharging a part or all of the culture medium retained in the at least one hole, and the discharge step includes the following steps: a culture medium moving step for tilting the cell culture substrate in a state where the cell culture container is contained in the first space, so that the culture medium located in the first space is moved to the second space through the opening portion; and a process for inserting a discharge pipe into the second space and discharging the culture medium located in the second space from the at least one hole through the discharge pipe. According to this method, the culture medium can be discharged while suppressing the influence on the cultured cells. This method can be applied to the case where the culture medium is manually discharged from the hole, and can also be applied to the case where the culture medium is automatically discharged (for example, by a robot, etc.). The inventors have verified and confirmed the following. If a conventional cell culture substrate is used, it is difficult to automatically perform the discharge step (i.e., the process of tilting the cell culture substrate to discharge the culture medium). In contrast, if the cell culture substrate of the present invention is used, a pipette or the like can be inserted into the hole while the insert is stably held, so that the above-mentioned discharge process can be automated. In addition, not only the culture medium in the hole can be automatically discharged, but also the culture medium in the insert can be automatically discharged.

[0080] Hereinafter, embodiments of the present invention will be described with reference to the accompanying drawings. It should be noted that the present invention is not limited to the embodiments. In addition, in the accompanying drawings, substantially the same components are denoted by the same reference numerals.

[0081] In addition, for convenience of explanation, in the following, terms indicating directions such as "upper", "lower", and "side" are used assuming a state in normal use, but they do not mean to define the use state of the cell culture substrate according to the present invention.

[0082] Implementation Method

[0083] use Figure 1 and Figure 2A to Figure 2D The overall structure of a cell culture substrate according to an embodiment of the present invention will be described. Figure 1 It is a perspective view of a cell culture substrate according to an embodiment of the present invention. Figure 2A yes Figure 1 Top view of the cell culture substrate. Figure 2B yes Figure 2A A1-A1 line cross-sectional view, Figure 2C yes Figure 2A B1-B1 line cross-sectional view, Figure 2D yes Figure 2A B2-B2 line cross-sectional view.

[0084] like Figure 1 and Figure 2A to Figure 2D As shown, the cell culture substrate according to this embodiment includes at least one well 100 having side surfaces 10 and a bottom surface 20, a side wall 51 constituting the side surface 10 of the well 100, and a bottom wall 52 constituting the bottom surface 20. The cell culture substrate is a resin molded body formed by, for example, injection molding.

[0085] The side surface 10 of the hole 100 includes a first side portion 11 defining a first space S1 and a second side portion 12 defining a second space S2. The second space S2 is connected to the first space S1. The first space S1 is a space for culturing cells. The first space S1 is configured in a manner that an insert (not shown) can be detachably arranged. The second space S2 is a space that can be used for supplying or discharging a culture medium, etc. The second space S2 is configured in a manner that the front end of a pipette, an aspirator, etc., an electrode for TEER measurement, etc. can be inserted without contacting the insert.

[0086] The first side portion 11 has an upper end 11a and a lower end 11b in contact with the bottom surface 20. When viewed from above, the upper end 11a of the first side portion 11 is formed into a C-shaped (arc-shaped) shape with a central angle α of more than 240 degrees. "Viewed from above" refers to a state observed from the upper side of the hole 100 in the thickness direction of the bottom wall 52. According to this structure, the insert can be stably maintained in the first space S1. For example, by providing a plurality of support structures on the upper surface of the first side portion 11 or the side wall 51 constituting the first side portion 11, the insert can be maintained at multiple locations. As long as it is a conventional cylindrical insert, the upper end or outer side surface of the insert can be supported at three locations divided into three equal parts in its circumferential direction. The larger the central angle α, the easier it is to stably maintain the insert. The central angle α is, for example, 300 degrees. It should be noted that if the central angle α becomes too large, the width of the opening portion 11p may be reduced, and it is difficult for the liquid in the first space S1 to move to the second space S2 through the opening portion 11p. The central angle α may be set in such a manner that the opening portion 11 p has a width that enables liquid to easily pass therethrough.

[0087] When viewed from above, the second space S2 extends from the opening portion 11p of the upper end portion 11a in the first direction E1 away from the first space S1. When viewed from above, the second side portion 12 includes an opposing surface 12f that is opposite to the opening portion 11p of the upper end portion 11a in the first direction E1. When viewed from above, the opposing surface 12f of the second side portion 12 can be smoothly curved in a manner that bulges in the first direction E1. According to this structure, when the culture medium is discharged from the second space S2, liquid residue is less likely to occur. In addition, by providing the second side portion 12 with the opposing surface 12f as described above, the front end of a pipette for discharging the culture medium can be easily inserted, and the second space S2 with a suppressed volume can be demarcated. When viewed from above, the opposing surface 12f can be, for example, in the shape of an arc (here, a semicircle). As Figure 2B As shown, the facing surface 12f may be inclined so that the distance from the opening portion 11p increases as it goes upward.

[0088] The maximum distance L1 between the opening portion 11p and the opposite surface 12f of the second side portion 12 in the first direction E1 (i.e., the maximum length of the second space S2 in the first direction E1) is smaller than the diameter Da of the C-shaped portion of the upper end portion 11a. According to this configuration, the increase in the size of the hole 100 can be suppressed. In addition, it becomes possible to arrange the holes 100 at a standard pitch. The diameter Da of the arc of the upper end portion 11a is, for example, 16.0 mm, and the maximum distance L1 is, for example, 10.5 mm. When viewed from above, the maximum width L2 of the second space S2 in the second direction E2 orthogonal to the first direction E1 can be, for example, less than the width of the opening portion 11p in the second direction E2. The maximum width L2 of the second space S2 is, for example, 7.7 mm.

[0089] The first direction E1 may be the radial direction of the C-shaped upper end 11a. According to this configuration, when the cell culture container is tilted to discharge the culture medium, it becomes easy to move the liquid in the first space S1 to the second space S2, and the amount of culture medium remaining in the first space S1 can be reduced. In addition, in the TEER measurement, a pair of electrodes can be easily inserted into the vicinity of the center of the insert arranged in the first space S1 and the second space S2, respectively. The planar shape of each hole 100 may be linearly symmetrical with the line connecting the center of the upper end 11a and the center of the opening 11p as the axis.

[0090] The bottom surface 20 includes a first bottom 21 defining a first space S1 and a second bottom 22 defining a second space S2. The first bottom 21 includes an observation surface 21a for microscopic observation or the like. The observation surface 21a is, for example, a circle with a diameter of 9.5 mm. The observation surface 21a can be arranged at a distance from the periphery of the first bottom 21. When an insert is configured, the observation surface 21a at least partially overlaps with the bottom surface of the insert when viewed from above. The first bottom 21 and the second bottom 22 can be flush. According to this configuration, since the liquid in the first space S1 becomes easy to move to the second space S2, the culture medium can be discharged more efficiently. When viewed from above, the area of ​​the bottom surface 20 is, for example, 187 mm 2 , which is the same as the bottom area of ​​the wells in commercially available well plates.

[0091] When discharging the culture medium, for example, the cell culture substrate can be tilted in such a manner that the first bottom 21 of the hole 100 is relatively lower than the second bottom 22. The cell culture substrate can be tilted, for example, in the first direction E1. The direction in which the cell culture substrate is tilted (the tilted direction) is not defined in the first direction E1, as long as it is a direction having a component in the first direction E1, that is, a direction less than ±90 degrees relative to the first direction E1. The tilted direction is preferably a direction that is at an angle of less than ±45 degrees to the first direction E1. According to this configuration, the culture medium in the first space S1 can be easily moved to the second space S2 by gravity. In this state, the front end of a pipette or the like can be arranged near the lowest point in the second bottom 22, which is the lowest position, to discharge the culture medium. When the cell culture substrate is tilted in the directions F1 and F2 that are at an angle of ±45 degrees to the first direction E1, points Q1 and Q2 in the second bottom 22 are the lowest points, respectively.

[0092] <Stairs>

[0093] A step portion may also be provided in the first side portion 11 so that the diameter Da of the upper end portion 11a of the first side portion 11 is enlarged compared to the diameter Db of the lower end portion 11b of the first side portion 11. The diameter Da is, for example, 16.0 mm, and the diameter Db is, for example, 12.5 mm. The step portion includes an upward step surface 11c located between the upper end portion 11a and the lower end portion 11b. When viewed from above, the step portion extends in the circumferential direction. According to this structure, the liquid level in the gap between the insert and the first side portion 11 can be suppressed from rising. Therefore, the reduction in the amount of liquid (the amount of culture medium) in the insert due to the siphon phenomenon can be suppressed. In addition, when the culture medium is discharged from the hole 100, the old culture medium is not easy to remain in the above-mentioned gap, so that liquid residue can be suppressed. The step portion may extend along the entire first side portion 11. That is, when viewed from above, the step portion may extend in the circumferential direction from one end of the C-shaped to the other end.

[0094] The height (level) of the step surface 11c depends on the amount of culture medium supplied to the hole 100, and can be set to the same level as the height of the liquid level in the insert. Thus, the height of the liquid level in the gap between the insert and the first side portion 11 can be made consistent with the height of the liquid level in the insert and in the second space S2, so that the amount of culture medium in the insert is not easily reduced due to the siphon phenomenon. The distance between the observation surface 21a and the step surface 11c in the thickness direction of the bottom wall 52 is, for example, 5.6 mm.

[0095] The width of the step surface 11c (radial width of the upper end 11a) can be set to a width that can suppress the rise of the liquid level in the above-mentioned gap by utilizing the Coanda effect. On the other hand, if the width of the step surface 11c becomes too large, the diameter Da may increase relative to the diameter of the insert, and it may become difficult to stably hold the insert. Therefore, the width of the step surface 11c can be set in a manner that can suppress the rise of the liquid level while suppressing the increase of the diameter Da. The width of the step surface 11c is, for example, 0.8 mm to 1.3 mm.

[0096] At least a portion of the second side portion 12 defining the second space S2 may not be provided with a step surface having the same height as the step surface 11c of the first side portion 11. Thus, the shaking of the liquid surface caused by the step surface 11c can be absorbed. In the illustrated example, such a step portion is not provided on the entire second side portion 12.

[0097] <slot>

[0098] One or more grooves including the first groove 31 may be provided on the bottom surface 20. The grooves are arranged in the area other than the observation surface 21a of the bottom surface 20. The bottom of the groove is located below the observation surface 21a.

[0099] The first groove 31 only needs to be continuous from the first portion in contact with the first side portion 11 to the second portion in contact with the second side portion 12. According to this configuration, the liquid in the gap between the insert and the first side portion 11 and the first bottom portion 21 of the well 100 can be easily moved to the second space S2 side through the first groove 31. Therefore, by arranging the tip of a pipette or the like close to the second portion of the first groove 31, the culture medium in the well 100 can be more effectively discharged from the second space S2.

[0100] The first groove 31 may extend from the first portion to the second portion along the side surface 10. In the illustrated example, the first groove 31 is continuous from the first portion between the first side portion 11 and the observation surface 21a to the second portion in contact with the opposite surface 12f of the second side portion 12. The first groove 31 may extend along the entire periphery of the bottom surface 20 when viewed from above.

[0101] The region without grooves in the bottom surface 20 may be at the same height (level) as the observation surface 21a. For example, the region without grooves in the first bottom 21 (including the observation surface 21a) may be flush with the region without grooves in the second bottom 22. In the illustrated example, the first groove 31 is provided only on the periphery of the bottom surface 20, and the portion of the bottom surface 20 located inside the first groove 31 is flush. According to this configuration, liquid residue caused by the unevenness of the bottom surface 20 can be reduced.

[0102] The width r of the first groove 31 may be smaller than the width w of the gap between the insert and the first side portion 11 when the insert is supported at a predetermined position of the first space S1 (see Fig. 7A By reducing the width r of the first groove 31, when the culture medium is discharged, the culture medium remaining in the gap between the insert and the first side portion 11 and the first bottom portion 21 can be sucked into the first groove 31 by capillary action, and moved to the second space S2 side in the first groove 31, so that the liquid residue can be further reduced. On the other hand, if the width of the first groove 31 is too small, the capillary force may be reduced. Therefore, the width r of the first groove 31 may be greater than the minimum thickness of the side wall 51. In addition, from the viewpoint of ensuring the strength of the mold for forming the cell culture substrate, the width r of the first groove 31 may be, for example, greater than 0.6 mm. The width r of the first groove 31 is, for example, 0.9 mm.

[0103] The depth (maximum depth) d of the first groove 31 may be, for example, greater than the minimum thickness of the side wall 51. The depth d of the first groove 31 may be set in such a way that the capillary force of the first groove 31 is higher than the capillary force generated in the gap between the insert and the first side portion 11 and the first bottom portion 21. On the other hand, the depth d of the first groove 31 may be less than the maximum height H of the liquid (here, the culture medium) rising in the gap between the insert and the first side portion 11. The depth d of the first groove 31 is, for example, 0.5 mm. It should be noted that the "maximum height H" can be calculated using the following formula (1) based on the width w of the gap between the insert and the first side portion 11, the surface tension σ of the culture medium, the contact angle θ of the culture medium relative to the resin material of the insert and the hole (for example, 90 degrees), the density ρ of the culture medium, and the gravitational acceleration g.

[0104]

[0105] In addition to the first groove 31, other grooves may be provided on the bottom surface 20. The other grooves may be connected to the first groove 31, or may be separated from the first groove 31. The width and depth of the other grooves may be the same as those of the first groove 31, or may be different.

[0106] Figure 3A to Figure 3DEach of them is a diagram illustrating variations in the shape and arrangement of the grooves, and is a plan view showing the bottom surface 20 of the hole 100. The periphery of the bottom surface 20 is defined by the lower ends of the first side portion 11 and the second side portion 12.

[0107] like Figure 3A As shown, a second groove 32 extending from the vicinity of the opening portion 11p of the first side portion 11 to a portion of the first groove 31 that contacts the opposite surface 12f of the second side portion 12 may be further provided on the bottom surface 20 of the hole 100. The end of the second groove 32 on the opposite surface 12f side is connected to the first groove 31. Thus, the liquid remaining in the gap between the insert and the first side portion 11 and the first bottom portion 21 can be more effectively guided to the opposite surface 12f side via the second groove 32. Therefore, the time required to discharge the culture medium can be shortened.

[0108] like Figure 3B As shown in FIG. 1 , a third groove 33 may be further provided on the opening portion 11p of the first side portion 11 in the bottom surface 20 of the hole 100. In a plan view, the third groove 33 is arranged so as to be connected to both ends of the C-shaped portion of the first groove 31 along the first side portion 11. Thus, the first groove 31 and the third groove 33 can form an annular groove surrounding the bottom surface of the insert. Therefore, the liquid remaining in the gap between the insert and the first side portion 11 and the first bottom portion 21 can be efficiently sucked out into the annular groove, thereby shortening the time required to discharge the culture medium.

[0109] It should be noted that, when only the first groove 31 is provided, or when Figure 3A and Figure 3B In the example shown, most of the bottom surface of the well 100 is flat (flush with the observation surface). Therefore, liquid residue caused by the unevenness of the bottom surface of the well 100 (undulations caused by providing the grooves) can be reduced.

[0110] exist Figure 3C In the example shown, except Figure 3B In addition to the first groove 31 and the third groove 33 shown in the figure, a plurality of second grooves 32 may be further provided. In a plan view, one end of each second groove 32 is connected to the third groove 33, and the other end is connected to the portion of the first groove 31 that contacts the opposite surface 12f of the second side portion 12. Figure 3A and Figure 3B Compared with the example shown, the time required to discharge the culture medium can be shortened.

[0111] like Figure 3DAs shown, a plurality of protrusions 34 may be provided on the second bottom portion 22. The plurality of protrusions 34 are arranged in a manner to define a groove between them that can serve as a flow path for the culture medium. An area 35 in the second bottom portion 22 where the plurality of protrusions 34 are not formed may be flush with the bottom of the first groove 31. The height (level) of the upper surface of each protrusion 34 may be the same as the height of the observation surface. The liquid remaining in the gap between the insert and the first side portion 11 and the first bottom portion 21 may move to the opposite surface 12f side through the third groove 33 and the area 35, thereby shortening the time required to discharge the culture medium.

[0112] <Supporting structure>

[0113] Refer again Figure 1 as well as Figure 2A to Figure 2D . The first side portion 11 may have a support structure 40 in the first space S1, and the support structure 40 is used to support the insert at a distance from the bottom surface 20. Thereby, the insert can be more stably maintained in the first space S1. The insert is preferably arranged in a manner that the center of the bottom surface of the insert is roughly consistent with the center of the upper end portion 11a of the first side portion 11 when viewed from above. That is, the insert is preferably arranged in the first space S1 without eccentricity. In addition, the insert is preferably arranged in a manner that the central axis of the insert is parallel to the thickness direction of the bottom wall 52 of the hole 100 when viewed in section. Thereby, the narrowing of the gap between the first side portion 11 and the insert can be suppressed.

[0114] A part or the whole of the support structure 40 may be arranged on the step surface 11c or between the step surface 11c and the upper end portion 11a. Thus, the support structure 40 can be formed by utilizing the space expanded in the radial direction by the step surface 11c. In addition, since the support structure 40 can be arranged above the liquid level of the culture medium in the gap between the insert and the first side portion 11, the influence of the support structure 40 on the culture medium replacement operation (for example, it becomes difficult to discharge the culture medium, etc.) can be suppressed.

[0115] The support structure 40 can be constructed in a manner that can support one or more commercially available inserts. The support structure 40 includes a portion that supports a portion of the insert from below. The support structure 40 is preferably constructed in a manner that the insert can be loaded and unloaded by simply moving the insert in the up and down directions (along the thickness direction of the bottom wall 52) relative to the support structure 40. In addition, the support structure 40 and the portion of the insert supported by the support structure 40 preferably have a size relationship relative to the hole that allows easy loading and unloading of the insert. Thereby, damage to cells caused by loading and unloading of the insert, such as the peeling off of cells seeded on the bottom surface of the insert, can be suppressed.

[0116] Figure 4: is a perspective view showing an example of an insert. The insert 60 has a cylindrical side wall and a bottom wall formed by a porous membrane filter. A plurality of (here two) protrusions 62 are provided on the outer side surface 61 of the side wall of the insert 60. The insert 60 is, for example, an insert manufactured by Falcon.

[0117] One side reference Figure 5 An example of the support structure 40 for supporting the insert 60 will be described while referring to the perspective view shown in FIG.

[0118] The cell culture substrate has a plurality of ribs as a support structure 40 arranged between the upper end 11a and the lower end 11b of the first side portion 11. The plurality of ribs include a plurality of support ribs 41, an anti-misinsertion rib 42 arranged between two adjacent support ribs among the plurality of support ribs 41, and a plurality of anti-rotation ribs 43. The lower surfaces of these ribs 41 to 43 are in contact with the step surface 11c. Since the upper surfaces of the ribs 41 to 43 are located further below the upper end 11a of the first side portion 11, a support structure for supporting other types of inserts can be additionally provided at the upper end 11a of the first side portion 11. The ribs 41 to 43 can be arranged in a manner that does not hinder the insertion of other types of inserts.

[0119] Each of the plurality of (here, two) support ribs 41 has a recess 41a on the upper surface thereof for receiving one of the protrusions 62 of the insert 60. That is, the width of the recess 41a is greater than the width of each protrusion 62 of the insert 60.

[0120] The anti-misinsertion rib 42 is arranged at a spacing t between two adjacent support ribs 41. The spacing t is smaller than the width of each protrusion 62 of the insert 60. The anti-misinsertion rib 42 can be divided into a plurality of parts. The divided parts can be arranged at a spacing smaller than the width of each protrusion 62. By providing the anti-misinsertion rib 42, it is possible to prevent the insert 60 from being mistakenly inserted below a specified height. Therefore, it is possible to suppress the siphon phenomenon caused by the liquid level in the insert 60 becoming lower than the liquid level of the liquid (culture medium) filled in the hole 100 due to mistaken insertion.

[0121] A plurality of (four in this case) anti-rotation ribs 43 may be arranged at predetermined intervals along the circumference of the first side portion 11. When viewed from above, each anti-rotation rib 43 extends inward from the first side portion 11. The width of each anti-rotation rib 43 decreases as it moves away from the first side portion 11. In the example shown in the figure, each anti-rotation rib 43 is in the shape of a triangular prism. Fig. 6A As shown, in a plan view, the innermost portion of each anti-rotation rib 43 may contact the outer side surface of the insert 60. Thus, the insert 60 can be prevented from rotating.

[0122] The structure of the insert and the supporting structure 40 of the hole 100 are not limited to the above example. A plurality of (here, three) cutouts 44 may be formed on the upper surface 51a of the side wall 51 of the hole 100. Figure 6B As shown in FIG. 1 , when an insert 60a having a plurality of (three in this case) protrusions 63 extending from one end of a cylindrical shape in a flange shape is used, the protrusions 63 of the insert 60a can be supported by the cutout 44. The insert 60a is, for example, an insert made by FUJIFILM, MILLIPORE, VITRIGEL, or GREINER. The three protrusions 63 of the insert 60a are respectively arranged at positions that divide the upper end of the side wall of the insert 60a into three equal parts in the circumferential direction. Each cutout 44 is configured to receive one protrusion 63.

[0123] In addition, the upper surface 51a of the side wall 51 of the hole 100 may not be provided with any concavo-convex part other than the cutout part 44. Thus, for example, when a cylindrical insert having a flange at one end (for example, manufactured by CORNING) is used, the flange of the insert can be supported by the flat area other than the cutout part 44 on the upper surface 51a of the side wall 51.

[0124] exist Figure 5 In the example shown, the support structure 40 is configured to support a variety of inserts. It should be noted that the support structure 40 only needs to be configured to support any type of insert.

[0125] (Cell Culture Method)

[0126] An example of a method for culturing cells using the cell culture substrate of the present embodiment will be described. Figures 7A to 12B Each of them is a cross-sectional view for explaining the process of the cell culture method. FIG. 7A to FIG. 12A Representation and Figure 2A The cross section corresponding to the A1-A1 line shown is Figure 7B to Figure 12B Respectively represent Figure 2A The cross section corresponding to the line B1-B1 shown.

[0127] like Fig. 7A and Figure 7B As shown, an insert 60 is arranged in the first space S1 of the hole 100 of the cell culture substrate, and cells 70 are inoculated in the insert 60 using a known method. For example, a suspension containing cells 70 and a culture medium 71 can be prepared, and a specified amount of the suspension can be supplied to the insert 60. As the insert 60, there is no particular limitation, and a known insert can be used. The insert has a porous membrane filter on the bottom surface. The porous membrane filter is constructed in a manner that allows small molecules (chemical substances, etc.) dissolved or suspended in a liquid to pass through, but does not allow cells to pass through. Here, as the insert 60, Figure 4Insert 60 is shown.

[0128] Next, if Fig. 8A and Figure 8B As shown, a culture medium 72 is supplied into the hole 100. Here, for example, the culture medium 72 is injected into the second space S2 of the hole 100 using a pipette 80. At this time, if the width w of the gap Sg between the insert 60 and the first side portion 11 of the hole 100 is small, the liquid level of the culture medium 72 in the gap Sg sometimes rises due to the capillary phenomenon. As a result, the liquid level of the culture medium 72 in the gap Sg becomes higher than the liquid level of the culture medium in the second space S2 by dh (>0). In this specification, the width w of the gap Sg refers to the distance between the lower end of the outer side surface of the insert 60 and the first side portion 11 of the hole 100 in the radial direction of the insert 60 when viewed from above. The width w of the gap Sg is, for example, about 1 mm to 2 mm.

[0129] like Fig. 9A and Fig. 9B As shown, the culture medium 72 is further injected into the hole 100. If the liquid level of the culture medium 72 in the gap Sg reaches the height of the step surface 11c, the rise of the liquid level can be suppressed by the Coanda effect. For example, the height of the liquid level in the gap Sg becomes approximately the same as the height of the step surface 11c. Thus, the increase of the height difference dh of the liquid level can be suppressed. In this way, since the rise of the liquid level in the gap Sg can be suppressed by providing the step surface 11c, the reduction of the liquid amount in the insert 60 caused by the siphon phenomenon can be suppressed. In addition, by suppressing the rise of the liquid level in the gap Sg, when the culture medium 72 is discharged, it is less likely that liquid residue will be generated in the gap Sg between the insert 60 and the first side portion 11.

[0130] It should be noted that the step surface 11c may cause fluctuations and oscillations in the culture medium 72. In this embodiment, since no step surface having the same height as the step surface 11c is provided on the second side portion 12, the oscillations in the culture medium 72 caused by fluctuations can be absorbed by the second space S2.

[0131] When the supply of the culture medium 72 is completed, the amount of the culture medium supplied to the insert 60 and the hole 100 can be adjusted so that the liquid level of the culture medium 72 in the second space S2, the liquid level in the gap Sg limited by the height of the step surface 11c, and the liquid level of the culture medium 71 in the insert 60 are approximately the same.

[0132] After that, the wells 100 are covered with a cap, and the cell culture substrate is placed in a humidified CO2 incubator at a predetermined temperature (eg, 37°C) to culture the cells. The cells take in necessary nutrients from the culture medium 72 and grow.

[0133] As the number of cells increases, the medium is replaced as needed. The medium replacement includes a discharge step of discharging at least a portion of the old medium with reduced nutrients from the wells, and a supply step of supplying new medium to the wells after the old medium is discharged.

[0134] In the discharge process, Fig. 10A and Fig. 10B As shown, first, the cell culture substrate is tilted in a state where the first space S1 contains the insert, so that the second bottom of the second space S2 is relatively lower than the first bottom of the first space S1. Thus, part of the culture medium 72 in the first space S1 is moved to the second space S2 through the opening of the first side portion 11 (culture medium moving step). Fig. 10A and Fig. 10B Indicated in Figure 2A The cell culture substrate is tilted in the direction F2 shown in the figure. As shown in the figure, due to the capillary phenomenon, the liquid level in the gap Sg between the insert 60 and the first side portion 11 is located above the liquid level in the second space S2.

[0135] Next, the pipette 80 is inserted into the second space S2. The tip of the pipette 80 may be disposed at the lowest point Q2 in the second bottom 22 (see Figure 2A ) nearby.

[0136] Next, if Fig.11A and Fig. 11B As shown, the culture medium 72 in the second space S2 is discharged from the well 100 via the pipette 80. The portion of the culture medium 72 in the gap Sg is more strongly affected by the capillary phenomenon than by gravity, and thus remains in the gap Sg.

[0137] like Fig. 12A and Fig. 12B As shown, if most of the culture medium 72 in the second space S2 is discharged, the culture medium 72 remaining in the gap Sg can be sucked out by the capillary force of the first groove 31. The sucked out culture medium 72 moves to the second space S2 side in the first groove 31 and can be discharged from the hole 100 by the pipette 80. In this way, by utilizing the capillary force of the first groove 31, the amount of old culture medium 72 remaining in the gap Sg can be reduced.

[0138] After the old culture medium is discharged, a new culture medium is supplied to the well 100 (supply step). Method and reference for supplying a new culture medium Figures 8A to 9B The same method as above.

[0139] In the above, the cell culture method using the well 100 having the step portion and the first groove 31 is described, but even when the well 100 does not have the step portion, the first groove 31, or both, the cell culture can be performed in the same method as described above. Even when there is no step portion and the first groove 31, the liquid residue can be reduced by reducing the discharge speed of the culture medium or dividing the discharge operation of the culture medium into multiple times.

[0140] (Other Examples of Cell Culture Substrates)

[0141] Fig.13 It is a perspective view showing an example of the cell culture substrate 200 . Fig.14 2 is a top view of a cell culture substrate 200. The cell culture substrate 200 is a well plate having 24 wells 100 arranged in a two-dimensional manner. Fig.13 and Fig.14 In the figure, the height direction of the cell culture substrate 200 is set as the Z direction. When viewed from the Z direction, the cell culture substrate 200 is, for example, a rectangle. The extending directions of two mutually orthogonal sides (here, the long side and the short side) in the rectangle are respectively set as the X direction and the Y direction.

[0142] The cell culture substrate 200 includes a plurality of wells 100 and an outer wall 53 surrounding the plurality of wells 100 in a plan view. Figure 1 to Figure 6B In this example, the number of holes is 24, but it is not limited to 24, and can also be, for example, 6, 48, 96, etc.

[0143] The outer wall 53 extends in a rectangular shape when viewed from above. When viewed from above, the outer wall 53 has two first outer wall portions 53x extending in the X direction and two second outer wall portions 53y extending in the Y direction. The length of the first outer wall portion 53x is, for example, 127.5 mm, and the length of the second outer wall portion 53y is, for example, 185.5 mm. The height of the outer wall 53 is, for example, 21.1 mm.

[0144] In a plan view, the holes 100 are arranged, for example, in a row direction and a column direction intersecting (e.g., orthogonal to) the row direction. The holes 100 are arranged so that the first direction E1 of each hole 100 intersects both the row direction and the column direction, which are the arrangement directions of the holes 100. According to this configuration, more holes 100 can be arranged within a limited area.

[0145] exist Fig.14In the example shown, the row direction of the holes 100 is the X direction, and the column direction is the Y direction. The first direction E1 of each hole 100 is, for example, at an angle of 45 degrees to the X direction and the Y direction respectively. That is, the second space S2 of each hole 100 extends along the X direction and the Y direction of the first space S1. Preferably, the plurality of holes 100 are arranged in the same direction (for example, so that the first direction E1 is at an angle of 45 degrees to the X direction and the Y direction). Thus, when the cell culture substrate 200 is tilted, the culture medium in the first space S1 of the plurality of holes 100 can be moved to the second space S2 at the same time.

[0146] The arrangement pitch Px of the holes 100 in the X direction and the arrangement pitch Py of the holes 100 in the Y direction can be the same. In this example, the arrangement pitches Px and Py are both 19 mm, which is the same as the arrangement pitch of a commercially available orifice plate. Thus, an observation microscope or a pipette corresponding to a commercially available orifice plate can be applied to the cell culture substrate 200.

[0147] exist Fig.14 In the example shown, by tilting the cell culture substrate 200 in the X direction, in the Y direction, or in both directions, the culture medium in each well 100 can be moved to the second space S2. "Tilting in the X direction" means tilting in such a way that one of the two second outer wall portions 53y, which is located on the second space S2 side of each well 100, is lower than the other side. "Tilting in the Y direction" means tilting in such a way that one of the two first outer wall portions 53x, which is located on the second space S2 side of each well 100, is lower than the other side. When tilting in the X direction, for example, a pipette or the like can be arranged near the lowest point Q2 in the second bottom 22 of each well 100 to suck the culture medium. When tilting in the Y direction, for example, a pipette or the like can be arranged near the lowest point Q1 in the second bottom 22 of each well 100 to suck the culture medium. It should be noted that the tilting direction of the cell culture substrate 200 can be any direction as long as it forms an angle of ±45 degrees or less with the first direction E1. In addition, the tilt direction of the cell culture substrate 200 may be changed stepwise when the culture medium is discharged.

[0148] Fig.15 It is a stereoscopic diagram illustrating a state in which the cell culture substrate 200 is tilted in the X direction. The tilt angle β of the bottom surface of each hole 100 (the surface including the observation surface of the hole) relative to the horizontal plane only needs to be greater than 0°. The tilt angle β is preferably greater than 5°. Thus, the movement speed of the culture medium to the second space S2 can be increased. On the other hand, the tilt angle β can be less than 20°. Thus, the influence on the cells caused by tilting the cell culture substrate 200 or resetting can be reduced. The tilt angle β is, for example, about 10°. The operation of tilting the cell culture substrate 200 can be performed manually or automatically.

[0149] Refer again Fig.13 and Fig.14 The cell culture substrate 200 may further include a pool portion 90 between the outer wall 53 and the plurality of wells 100. By filling the pool portion 90 with water such as purified water, evaporation of the culture medium in the wells 100 can be suppressed.

[0150] The plurality of holes 100 include a plurality of outer holes 101 located at the ends of the row direction or the column direction and an inner hole 102 located inside the outer holes 101. The pool portion 90 is arranged between the side wall 51 and the outer wall 53 of the plurality of outer holes 101. The pool portion 90 is divided into the same number or more than the outer holes 101. By dividing the pool portion 90 into the same number or more than the outer holes 101, even if the cell culture substrate 200 is tilted, the water contained in the pool portion 90 is not easy to overflow. In this example, the number of the outer holes 101 is 16, and the pool portion 90 is divided into 19.

[0151] Fig.16 1 is a top view showing a modified example of the cell culture substrate of the present embodiment. In the modified example of the cell culture substrate 200a, the second space S2 has a region near the opposing surface 12f having a width greater than the opening portion 11p of the upper end of the C-shaped first side portion. The planar shape of each hole 100 may not be axisymmetric.

[0152] The inventors studied Fig.13 and Fig.14 The usefulness of the cell culture substrate (well plate) 200 shown in the figure was tested, and a cell culture experiment was performed using the well plate 200 to confirm the operability of the well plate 200 and evaluate the function of the cultured cells. In addition, the same experiment was performed using a commercially available well plate and compared with the case of using the well plate 200. The method and results of the cell culture experiment are described below.

[0153] <Cell culture experiment 1>

[0154] (Example 1)

[0155] In Example 1, prepare Fig.13 and Figure 4 The well plate 200 is shown. Next, inserts manufactured by Falcon are placed in the 24 wells in the well plate 200, respectively.

[0156] Next, intestinal stem cells differentiated from hiPS cells are inoculated into each insert and induced to differentiate into intestinal epithelial cells under feeder-free conditions. The differentiation induction protocol is in accordance with the method described in Japanese Patent Gazette No. 2022-019411. The entire disclosure of Japanese Patent Gazette No. 2022-019411 is incorporated herein by reference. As needed, the culture medium is replaced by the following method, for example, at a frequency of 24 hours to 2 days. The culture experiment was terminated on the 30th day after cell inoculation. After termination, the differentiated cells were evaluated by mRNA expression analysis and drug metabolizing enzyme activity assay.

[0157] ·Methods for changing culture medium

[0158] Reference for the discharge and supply of culture medium when changing culture medium Figures 7A to 12B The method is used to perform the above-mentioned operation manually. Specifically, the well plate is tilted while the insert is held in the first space of each well, and the medium is discharged from the second space of each well using a pipette. After the discharge, the well plate is restored to a horizontal state, and new medium is supplied to the second space of each well.

[0159] Determination of TEER value

[0160] The transepithelial electrical resistance (TEER) value of the cells was measured from day 18 after cell inoculation, and the change of TEER value over time was studied. The measurement was performed using Millicell ERS-2.

[0161] mRNA expression analysis

[0162] For epithelial cells differentiated by the above method, the expression levels of Villin1, a marker of absorptive epithelial cells, MDR1 and PEPT1, drug transporters, and CYP3A4 and CYP2C9, drug metabolizing enzymes, were measured by RT-qPCR, and the relative expression levels to adult small intestine (ASI) were determined. The measurement was performed using the KAPA SYBR Fast qPCR Kit.

[0163] Determination of drug metabolizing enzyme activity

[0164] For epithelial cells differentiated by the above method, the metabolic activities of CYP3A4 / 5, CYP2C9, CYP2C19 and CYP2D6 were studied using midazolam, diclofenac, mephenytoin and bufuralol as substrates. Here, the activities of CYP3A4 / 5, CYP2C9, CYP2C19 and CYP2D6 were calculated by measuring the production of 1'-hydroxymidazolam, 4'-hydroxydiclofenac, 4'-hydroxymephenytoin and 1'-hydroxybufuralol.

[0165] (Example 2)

[0166] Differentiation induction into intestinal epithelial cells and evaluation of epithelial cells were performed in the same manner as in Example 1, except that an insert manufactured by Greiner was used as the insert.

[0167] (Comparative Example 1)

[0168] Differentiation induction into intestinal epithelial cells and evaluation of epithelial cells were performed in the same manner as in Example 1, except that a commercially available Falcon well plate having 24 wells and a Falcon insert were used and the medium was replaced by the following method.

[0169] ·Methods for changing culture medium

[0170] In Comparative Example 1, the insert was removed from each well with tweezers, and the culture medium was drained from the well using a pipette. Then, the insert was returned to the well and the culture medium was supplied to the well. These operations were all performed manually.

[0171] (Comparative Example 2)

[0172] Differentiation induction into intestinal epithelial cells and evaluation of epithelial cells were performed in the same manner as in Comparative Example 1, except that a commercially available Greiner well plate having 24 wells and a Greiner insert were used.

[0173] (TEER value measurement results)

[0174] Fig.17 1 is a graph showing the results of measuring TEER values ​​on the 18th to 30th day after inoculation in Examples 1 and 2 and Comparative Examples 1 and 2. Fig.17 As shown, the TEER values ​​of the cells of Examples 1 and 2 are equal to or higher than the TEER values ​​of the cells of Comparative Examples 1 and 2, respectively. It can be confirmed that even when the well plate 200 of this embodiment is used, cells having a barrier function equal to or higher than that when a commercially available well plate is used can be cultured.

[0175] In addition, especially after the 24th day, the TEER values ​​of the cells of Examples 1 and 2 were higher than the TEER values ​​of the cells of Comparative Examples 1 and 2, respectively, and it was found that the use of the well plate 200 of this embodiment can improve the barrier function of epithelial cells. The reason is presumed to be as follows.

[0176] In Comparative Examples 1 and 2, since the insert is lifted from the hole with forceps each time the culture medium is discharged, the cells in the insert are stimulated by the vibration of the insert or the operation using forceps, and the result of repeated stimulation may lead to a decrease in barrier function. In contrast, in Examples 1 and 2, since the culture medium in the hole is discharged without removing the insert, it is speculated that the influence on the cells caused by the replacement of the culture medium is suppressed, and the reduction of the barrier function is suppressed. In addition, since the insert is properly and stably maintained in each hole, it can be considered that even if the well plate is tilted when the culture medium is discharged, the cells in the insert will hardly be affected by the tilt of the well plate (even if affected, the influence is extremely small compared to the operation using forceps). Furthermore, in Examples 1 and 2, the culture medium replacement operation is smooth and the time required for replacement is shortened. It is speculated that by utilizing the second space of the hole to supply the culture medium and measure the TEER value, the influence on the cells caused by these operations is suppressed compared to Comparative Examples 1 and 2.

[0177] (Evaluation results)

[0178] Fig.18 (a)~ Fig.18 (e) is a graph showing the results of mRNA expression analysis in epithelial cells differentiated in Examples 1 and 2 and Comparative Examples 1 and 2. Fig.18 (a)~ Fig.18 As shown in (e), the mRNA expression level of Example 1 is at the same level as that of Comparative Example 1, and the mRNA expression level of Example 2 is at the same level as that of Comparative Example 2.

[0179] Fig.19 (a)~ Fig.19 (d) is a graph showing the results of measuring the activity of drug metabolizing enzymes in differentiated epithelial cells in Examples 1 and 2 and Comparative Examples 1 and 2. Fig.19 (a)~ Fig.19 As shown in (d), the metabolic activity of each marker in Example 1 is at the same level as that in Comparative Example 1, and the metabolic activity of each marker in Example 2 is at the same level as that in Comparative Example 2.

[0180] These evaluation results confirmed that, when the well plate 200 of the present embodiment is used, it is possible to culture cells having functions equivalent to or higher than those when a commercially available well plate is used, while greatly improving operability.

[0181] <Cell culture experiment 2>

[0182] (Example 3)

[0183] In Example 3, prepare Fig.13 and Fig.14 The cell culture substrate (well plate) 200 shown. Next, inserts manufactured by Greiner were placed in the 24 wells of the well plate 200, respectively.

[0184] Next, the three-dimensional intestinal organoids differentiated from hiPS cells were unicellularized and inoculated into each insert, and differentiation into two-dimensional intestinal organoids was induced under feeder-free conditions. The differentiation induction protocol is in accordance with the method described in International Publication No. 2022 / 091020 by the inventors. The entire disclosure of International Publication No. 2022 / 091020 is cited in this specification as a reference. The amount of culture medium on the basal side is set to 800 μL. As needed, the culture medium is replaced at a frequency of, for example, once every 24 hours to 3 days. In addition, the TEER value is measured from the fourth day after inoculation. The culture medium replacement and TEER value determination are performed by the same method as in Examples 1 and 2 above. Furthermore, the cell surface was observed using a phase contrast microscope, and the changes in the structure (villous structure) of the cell surface associated with differentiation were studied. The culture experiment was terminated on the 10th day after inoculation. After termination, the differentiated cells were evaluated by mRNA expression analysis.

[0185] mRNA expression analysis

[0186] For the intestinal organoids differentiated by the above method, the expression levels of LGR5 (intestinal stem cell marker), Villin1 (absorptive epithelial cell marker), MUC2 (goblet cell marker), Lysozyme (intestinal Paneth cell marker), Ki67 (proliferative cell marker), CYP3A4 (drug metabolizing enzyme), MDR1 (drug transporter), ALB (mature hepatocyte differentiation marker), OLFM4 (intestinal stem cell marker), and CDX2 (transcription factor involved in the proliferation and differentiation of intestinal epithelial cells) were measured by RT-qPCR. The measurement was performed using the KAPA SYBR Fast qPCR Kit.

[0187] (Example 4)

[0188] Differentiation induction into intestinal two-dimensional organoids and evaluation of differentiated cells were performed in the same manner as in Example 3, except that the amount of the culture medium on the basal side was changed to 1200 μL.

[0189] (Comparative Example 3)

[0190] Differentiation induction into intestinal two-dimensional organoids and evaluation of differentiated cells were performed in the same manner as in Example 3, except that a commercially available Greiner well plate having 24 wells and a Greiner insert were used, and the medium replacement method was different. In Comparative Example 3, the medium replacement was performed in the same manner as in Comparative Example 1.

[0191] (Cell Observation Results)

[0192] Fig. 20 (a)~ Fig. 20 (c) are phase contrast microscopic images of the cell surface of Comparative Example 3, Example 3 and Example 4 on the 6th day after inoculation. Fig.21 (a)~ Fig.21 (c)~ Fig.24 (a)~ Fig.24 (c) are phase contrast microscope images of the cell surfaces of Comparative Example 3, Example 3, and Example 4 at 7 to 10 days after inoculation.

[0193] The maturity of the cells can be determined based on the density of the villi in the villus-like structure on the cell surface. If the results on the same day are compared, it can be seen that the villus density of the cells of Examples 3 and 4 is higher than that of the cells of Comparative Example 3 (the villi are denser), and the maturation of the cells of Examples 3 and 4 progresses faster. In addition, for example, the villus density of Comparative Example 3 on the 10th day after inoculation is the same as the villus density of Examples 3 and 4 on the 8th to 9th day after inoculation. It can be seen from this that the cells of Examples 3 and 4 matured about 1 to 2 days earlier than the cells of Comparative Example 3.

[0194] Therefore, it was confirmed that the use of the well plate of the present embodiment can promote cell maturation more than the use of a commercially available well plate. The reason for promoting maturation is presumed to be as follows.

[0195] In Comparative Example 3, since the insert was lifted from the hole with forceps each time the culture medium was discharged, the cells in the insert were stimulated by the vibration of the insert or the operation using the forceps, and it is thought that the maturation of the cells may be delayed. In contrast, in Examples 3 and 4, the culture medium in the hole was discharged without removing the insert, so that the culture medium replacement was smooth and the time required for replacement was shortened, and the second space of the hole was used to supply the culture medium and measure the TEER value, so it can be inferred that the influence on the cell maturation rate caused by each operation of the cell test was suppressed. In addition, it is believed that the insert is appropriately and stably maintained in each hole, and the influence on the maturation rate of the cells caused by tilting the well plate is almost not observed.

[0196] (TEER value measurement results)

[0197] Fig.25: is a graph showing the measurement results of TEER values ​​from the 4th day to the 10th day after inoculation of Examples 3 and 4 and Comparative Example 3. Fig.25 As shown, the TEER values ​​of the cells of Examples 3 and 4 are comparable to the TEER value of the cells of Comparative Example 3.

[0198] It should be noted that, in detail, in Examples 3 and 4, the TEER values ​​are slightly lower than those in Comparative Example 3, but this does not mean that the barrier function is reduced. In the intestinal two-dimensional organoids, it has been confirmed that the TEER value decreases if the crypt villus structure is formed. Since the TEER value in the body is 100 Ω·cm -2 Therefore, it is considered that in Examples 3 and 4, the intestinal two-dimensional organoids are in a state closer to that of a living organism.

[0199] (Evaluation results)

[0200] Fig.26 (a)~ Fig.26 (i) is a graph showing the results of mRNA expression analysis of each marker for differentiated cells in Examples 3 and 4 and Comparative Example 3. The expression level of ASI is also shown in each graph. Fig.26 (a)~ Fig.26 As shown in (i), the mRNA expression levels of Examples 3 and 4 were comparable to that of Comparative Example 3.

[0201] The evaluation results of the cell culture experiment 2 confirmed that the use of the well plate 200 of the present embodiment can significantly improve the operability and allow cells to mature at a rate equivalent to or faster than that of a commercially available well plate.

[0202] In the above-mentioned Examples 1 to 4, the culture medium was replaced manually, but the inventors have confirmed through other cell culture experiments that the use of an automated device to automatically replace the culture medium can further improve operability. As the main factors that can be automated, it can be cited that if the well plate 200 of the present embodiment is used, the culture medium can be discharged from the well without removing the insert from the well; even if the well plate is tilted, the insert will be properly maintained in the well. Furthermore, the inventors have also confirmed that even if the culture medium replacement is automated, the function of the cultured cells can be made equal to or higher than that of the cells cultured using the conventional cell culture substrate.

[0203] The cell culture substrate and cell culture method disclosed herein can also be expressed, for example, as follows.

[0204] [Project 1]

[0205] A cell culture substrate is a cell culture substrate having at least one hole, wherein the at least one hole has a side surface and a bottom surface, wherein:

[0206] The side surface includes a first side portion defining a first space and a second side portion defining a second space connected to the first space.

[0207] When looking down,

[0208] The upper end of the first side portion is formed into a C-shape with a central angle of 240 degrees or more.

[0209] The second space extends from the opening portion of the upper end portion in a first direction away from the first space.

[0210] The opposing surface of the second side portion that is opposed to the opening portion in the first direction is arranged at a position where a maximum distance between the opening portion and the opposing surface in the first direction is smaller than a diameter of the C-shape of the upper end portion.

[0211] [Project 2]

[0212] The cell culture substrate according to item 1, wherein the opposing surface of the second side portion is smoothly curved so as to be convex in the first direction in a plan view.

[0213] [Item 3]

[0214] The cell culture substrate according to item 1 or 2, wherein the bottom surface includes a first bottom defining the first space and a second bottom defining the second space, and the first bottom is flush with the second bottom.

[0215] [Item 4]

[0216] The cell culture substrate according to any one of items 1 to 3, wherein the bottom surface has at least one groove.

[0217] The at least one groove includes a first groove continuous from a first portion in contact with the first side portion to a second portion in contact with the second side portion.

[0218] [Item 5]

[0219] The cell culture substrate according to any one of items 1 to 4, wherein a step portion is provided on the first side portion so that the diameter of the upper end portion is larger than the diameter of the lower end portion in contact with the bottom surface, and the step portion extends in the circumferential direction when viewed from above.

[0220] [Item 6]

[0221] The cell culture substrate according to any one of items 1 to 5, wherein the first side portion includes a support structure in the first space, the support structure being used to support the cell culture container at a distance from the bottom surface.

[0222] [Item 7]

[0223] The cell culture substrate according to any one of items 1 to 6, wherein, in a plan view,

[0224] The at least one hole includes a plurality of holes arranged in a row direction and a column direction intersecting the row direction, and

[0225] The first direction in each of the plurality of holes is a direction intersecting both the row direction and the column direction.

[0226] [Item 8]

[0227] The cell culture substrate according to item 7, further comprising an outer wall surrounding the plurality of holes in a plan view,

[0228] The plurality of holes include a plurality of outer holes located at ends in the row direction or the column direction,

[0229] A pool portion for storing water is formed between the side wall of the side surface forming the plurality of outer holes and the outer wall, and the pool portion is divided into the same number or more than the number of the plurality of outer holes.

[0230] [Item 9]

[0231] A cell culture method, comprising: using the cell culture substrate and the cell culture container as described in any one of items 1 to 8 to culture cells in the cell culture container, wherein the cell culture container is detachably accommodated in the first space of the at least one hole and spaced apart from the bottom surface, wherein:

[0232] The cell culture method includes a discharge step of discharging a part or all of the culture medium held in the at least one hole, and the discharge step includes the following steps:

[0233] A step of moving the culture medium by tilting the cell culture substrate while the cell culture container is contained in the first space, so that the culture medium in the first space moves to the second space through the opening; and

[0234] A step of inserting a discharge tube into the second space and discharging the culture medium in the second space from the at least one hole via the discharge tube.

[0235] It should be noted that the present invention is not limited to the above-mentioned embodiment, and can be implemented with other various schemes. In the cell culture substrate with multiple holes, as long as at least a portion of the holes have the above-mentioned structure. The cell culture substrate of the present invention can be applied in any form of an open system and a closed system. In addition, it can not only be used in the case of manually performing operations such as culture medium replacement, but also can be used in an automatic cell culture device that automatically replaces culture medium.

[0236] Industrial Applicability

[0237] The cell culture substrate according to the present invention can replace or discharge the culture medium while suppressing the influence on the cultured cells, and is therefore useful in applications such as the pharmaceutical industry, the cosmetics industry, and the food industry.

[0238] Explanation of symbols

[0239] 10 Side

[0240] 11 First side

[0241] 11a Upper end portion of the first side portion

[0242] 11b Lower end of the first side portion

[0243] 11c Step surface

[0244] 11p Opening at the top

[0245] 12 Second side

[0246] 12f Opposite side

[0247] 20 Bottom

[0248] 21 First Bottom

[0249] 21a Observation surface

[0250] 22 Second Bottom

[0251] Slots 31, 32, 33

[0252] 40 Support structure

[0253] 41 Support ribs

[0254] 42 Anti-misinsertion rib

[0255] 43 Anti-rotation rib

[0256] 44 incision

[0257] 51 Sidewall

[0258] 52 bottom wall

[0259] 53 outer wall

[0260] 60, 60a Insert

[0261] 70 cells

[0262] 71, 72 Culture medium

[0263] 80 Pipette

[0264] 90 Ikebe

[0265] 100 holes

[0266] 101 Outer hole

[0267] 200, 200a Cell culture substrate

[0268] Da Diameter of the upper end of the first side portion

[0269] E1 First direction

[0270] E2 Second direction

[0271] Sg Gap between insert and first side

[0272] S1 First Space

[0273] S2 Second Space

Claims

1. A cell culture substrate, comprising: a cell culture substrate having at least one hole, wherein the at least one hole has a side surface and a bottom surface; in, The side surface includes a first side portion defining a first space and a second side portion defining a second space communicating with the first space, When looking down, The upper end of the first side portion is formed into a C-shape with a central angle of more than 240 degrees. The second space extends from the opening portion of the upper end portion in a first direction away from the first space, The surface of the second side portion that faces the opening in the first direction is arranged at a position where the maximum distance between the opening and the surface in the first direction is smaller than the diameter of the C-letter of the upper end portion.

2. The cell culture substrate according to claim 1, in, The opposing surface of the second side portion is smoothly curved so as to be convex toward the first direction in a plan view.

3. The cell culture substrate according to claim 1 or 2, in, The bottom surface includes a first bottom defining the first space and a second bottom defining the second space, and the first bottom is flush with the second bottom.

4. The cell culture substrate according to claim 1 or 2, in, The bottom surface has at least one groove, The at least one groove includes a first groove continuous from a first portion in contact with the first side portion to a second portion in contact with the second side portion.

5. The cell culture substrate according to claim 1 or 2, in, The first side portion is provided with a step portion such that the diameter of the upper end portion is larger than the diameter of the lower end portion in contact with the bottom surface, and the step portion extends in the circumferential direction in a plan view.

6. The cell culture substrate according to claim 1 or 2, in, The first side portion includes a support structure in the first space, and the support structure is used to support the cell culture container at a distance from the bottom surface.

7. The cell culture substrate according to claim 1 or 2, in, When looking down, The at least one hole includes a plurality of holes arranged in a row direction and a column direction intersecting the row direction, The first direction in each of the plurality of holes is a direction intersecting both the row direction and the column direction.

8. The cell culture substrate according to claim 7, in, The cell culture substrate further comprises an outer wall surrounding the plurality of holes in a plan view. The plurality of holes include a plurality of outer holes located at ends in the row direction or the column direction, A pool portion for accommodating water is formed between the side wall of the side surface forming the plurality of outer holes and the outer wall, and the pool portion is divided into the same number or more than the number of the plurality of outer holes.

9. A cell culture method, comprising: using the cell culture substrate and the cell culture container according to claim 1 or 2 to culture cells in the cell culture container, wherein the cell culture container is detachably accommodated in the first space of the at least one hole and is spaced apart from the bottom surface, in, The cell culture method includes a discharge step of discharging a part or all of the culture medium held in the at least one hole, The discharging process includes the following steps: A step of moving the culture medium by tilting the cell culture substrate in a state where the cell culture container is accommodated in the first space, thereby moving the culture medium in the first space to the second space through the opening; as well as A step of inserting a discharge tube into the second space and discharging the culture medium in the second space from the at least one hole via the discharge tube.

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