Substrate for cell culture vessels, and cell culture vessels

Abstract

To provide a base material for a cell culture vessel to properly hold cells in recesses to allow the cells in the recesses to be appropriately observed, and to provide a cell culture vessel.SOLUTION: A base material of a cell culture vessel of the invention comprises a bottom surface and a surface having a recessed region in which a plurality of recesses is formed. An average depth of the recesses is 200 μm or more. If an angle between a first tangent line, which passes through a connection line between a first curved line corresponding to a first recess and a second curved line corresponding to a second recess and touches the first curved line, and a reference line, which passes through a connection point and is parallel to the bottom surface, is θ1 (deg) and a refractive index of the base material is n, then a following equation is satisfied: θ1<90-θ2+sin-1{sin(θ2)×1.38 / n}. If an angle between a second tangent line, which passes through the connection point and touches the second curved line, and the reference line is θ2(deg), then a following equation is satisfied: θ2<90-θ1+sin-1{sin(θ1)×1.38 / n}.SELECTED DRAWING: Figure 5

Description

Technical Field

[0001] The present invention relates to a base material of a cell culture container and a cell culture container.

Background Art

[0002] In a configuration in which a culture solution is stored on the surface of a base material provided with a plurality of fine recesses, and cells are placed and cultured in each recess, if the depth of the recesses is not sufficiently ensured, the cells in the recesses may pop out during the replacement of the culture solution or the like. In consideration of this point, on the surface of the base material, a plurality of recesses may be formed deeper to such an extent that cell popping out is suppressed (for example, see Non-Patent Document 1).

Prior Art Documents

Non-Patent Documents

[0003]

Non-Patent Document 1

Summary of the Invention

Problems to be Solved by the Invention

[0004] On the other hand, when the depth of each of the plurality of recesses increases, there is a possibility that it may interfere with observing the cells in the recesses with a microscope or the like. Specifically, when light is irradiated from above the base material toward the surface of the base material for cell observation, the light refracted in a certain recess travels to an adjacent recess and is reflected in the adjacent recess. The reflected light appears as a virtual image (reflection) around the adjacent recess, which may become an obstacle when observing the cells in each recess or when performing predetermined image processing on the observation image.

[0005] The present invention has been made in view of the above problems, and an object thereof is to provide a base material of a cell culture container and a cell culture container provided with the base material for appropriately observing the cells in the recesses while appropriately holding the cells in the recesses.

Means for Solving the Problems

[0006] As a result of diligent research into the above-mentioned problems, the present inventors discovered that the desired effects can be obtained by using the substrate for the cell culture vessel of the present invention and the cell culture vessel of the present invention, leading to the present invention.

[0007] In other words, the inventors found that the above problem could be solved by the following configuration. [1] A substrate for a cell culture vessel having a bottom surface and a surface provided on the opposite side of the bottom surface, The above surface has a recess-forming region in which a plurality of recesses are formed, The average depth of the recesses in the above-mentioned multiple recesses is 200 μm or more. The above-mentioned plurality of recesses include a first recess and a second recess that are adjacent to each other, The line of intersection between the cross-section of the substrate passing through the center of the opening of the first recess and the center of the opening of the second recess and the first recess has a first curved line that rises from the bottom position of the first recess and extends towards the second recess while curving. The line of intersection between the above-mentioned cross-section and the above-mentioned second recess has a second curved line that rises from the bottom position of the second recess and extends while curving toward the first recess. The first curved line and the second curved line are connected at the connection point. Let θ1 (deg) be the angle between the first tangent line passing through the connection point and tangent to the first curved line, and the reference line passing through the connection point and parallel to the bottom surface, and let n be the refractive index of the substrate for light with a wavelength of 540 nm. Then the following equation (1) is satisfied: A substrate for a cell culture vessel that satisfies the following equation (2), where θ2 (deg) is the angle between the second tangent line, which passes through the above connection point and is tangent to the above second curved line, and the above reference line. θ1 < 90 - θ2 + sin -1 {sin(θ²) × 1.38 / n} Equation (1) θ² < 90 - θ¹ + sin -1 {sin(θ1)×1.38 / n} Equation (2) [2] The substrate for a cell culture vessel according to [1], wherein the material constituting the above substrate is glass. [3] The diameter of each of the multiple recesses is 250 μm to 700 μm, A substrate for a cell culture vessel according to [1] or [2], wherein the ratio of the depth of the recess to the diameter of the opening in each of the above-mentioned multiple recesses is 0.35 to 0.80. [4] A substrate for a cell culture vessel according to any of [1] to [3], wherein the angle θ1 and the angle θ2 are of the same magnitude. [5] A cell culture vessel substrate as described in any of [1] to [4], A cell culture vessel having a wall member fixed on the substrate at a position surrounding the recess-forming region and partitioning the space above the recess-forming region. [Effects of the Invention]

[0008] According to the present invention, it is possible to provide a cell culture vessel substrate and a cell culture vessel that can appropriately hold cells in the recesses while appropriately observing the cells in the recesses. [Brief explanation of the drawing]

[0009] [Figure 1] This is a diagram showing the disassembled parts of a cell culture vessel according to one embodiment of the present invention. [Figure 2] This figure shows one well in a cell culture vessel. [Figure 3] This is a magnified cross-sectional view of the well. [Figure 4] This is an enlarged plan view showing the recess formation region on the surface of the substrate. [Figure 5] Figure 4 is a cross-sectional view of the substrate when it is cut along plane AA, and is an explanatory diagram of the shapes of the first recess and the second recess. [Figure 6] This is an explanatory diagram illustrating the conditions under which refracted light is reflected. [Figure 7] This is a magnified image of the cross-section of the substrate of the cell culture vessel in Example 1, taken with a scanning electron microscope. [Figure 8]These are the photographed images when observing the concave portion formation region of each example with an inverted microscope.

Embodiments for Carrying Out the Invention

[0010] Hereinafter, one embodiment of the present invention will be described with reference to the drawings. However, the embodiments described below are merely examples given for facilitating the understanding of the present invention and do not limit the present invention. That is, the present invention can be changed or improved from the following embodiments without departing from its gist. In addition, the materials and shapes of each member used for implementing the present invention can be arbitrarily set according to the use of the present invention and the technical level at the time of implementing the present invention. The present invention includes equivalents thereof.

[0011] In this specification, the numerical range represented by "~" means a range including the numerical values described before and after "~" as the lower limit value and the upper limit value. In this specification, "orthogonal", "perpendicular", "vertical", "parallel", and "horizontal" include the range of errors acceptable in the technical field to which the present invention belongs. In this specification, the "cut surface" means the cut surface when observed at a magnification of 100 times with a field emission scanning electron microscope (hereinafter, SEM) manufactured by Hitachi High-Tech Corporation.

[0012] [Configuration Example of Cell Culture Container] [[ID=2,4]]One embodiment of the cell culture container according to the present invention (hereinafter, cell culture container 1) is a rectangular device in plan view, and is composed of a base material 2 and a wall member 3 shown in FIGS. 1 and 2, and includes one or more wells 1A. As shown in FIG. 2, the well 1A is a cylindrical space provided for cell culture, the upper end of the well 1A is open, and the lower end of the well 1A is closed by the base material 2. During the cell culture period, a predetermined amount of a culture solution (most of the components are water) forming a culture medium is stored inside the well 1A and is exchanged at an appropriate frequency.

[0013] In the cell culture vessel 1, as shown in Figures 2 and 3, the base material 2 forms the bottom of the vessel, and the wall member 3 is placed on the surface 2S of the base material 2 to form a wall that partitions the well 1A. An adhesive layer 4, made of adhesive tape or adhesive, is interposed between the surface 2S of the base material 2 and the wall member 3, and the wall member 3 is fixed to the base material 2 by the adhesive layer 4. A specific example of an adhesive is the "SE9140RTV" adhesive manufactured by Toray Dow Corning Silicone Co., Ltd. A specific example of adhesive tape is Nitto Denko's PET-based double-sided tape "5610". The adhesive surfaces of the base material 2 and the wall member 3 may be subjected to corona discharge treatment to improve wettability with the adhesive.

[0014] The base material 2 is composed of a rectangular glass plate in plan view. The lengths of the long and short sides of the base material 2 are not particularly limited. The thickness of the base material 2 is also not particularly limited, but for example, it may be 0.33 mm to 0.43 mm. The base material 2 has two main surfaces that are parallel to each other in the thickness direction. One main surface is the surface 2S located on the side to which the wall member 3 is attached. The other main surface is a flat bottom surface 2B located on the opposite side from surface 2S.

[0015] The surface 2S is provided with the same number of recess-forming regions 5 as the well 1A. The recess-forming regions 5 are areas on the surface 2S where multiple recesses 10 are formed, and are provided at regular intervals in the long and short directions of the substrate 2. As shown in Figure 4, multiple recesses 10 are regularly arranged in the recess-forming regions 5, for example, at equal intervals in a hexagonal or square arrangement. In the recess-forming regions 5, from the viewpoint of maximizing the number of recesses 10, the distance (pitch) between two adjacent recesses 10 is preferably relatively small, for example, 250 μm to 700 μm.

[0016] The recess 10 is a depression formed by eroding or scraping the surface 2S, and as shown in Figures 3 and 4, it has a circular opening 11 at its upper end and a curved surface 12 surrounding the opening 11. The curved surface 12 rises from the deepest position of the recess 10, i.e., the bottom position, and is a hemispherical or other curved surface, specifically an aspherical surface such as a paraboloid, ellipsoid, or hyperboloid.

[0017] Multiple recesses 10 are formed with the same shape and size; in other words, the diameter of the opening 11, the radius of curvature at the bottom of the curved surface 12, and the depth of the recesses 10 are consistent among the recesses 10. The depth of the recess 10 corresponds to the distance from the bottom of the recess to the opening 11.

[0018] Because the shape of the recesses 10 is uniform, the conditions for culturing cells within each recess 10 can be standardized, thereby suppressing variations in the degree of cell growth caused by differences in the shape of the recesses 10.

[0019] Furthermore, the curved surface 12 of each recess 10 is symmetrical with respect to the center of each recess 10, and specifically, the degree of curvature of the curved surface 12 is approximately constant over a 360-degree range around the center. The center of each recess 10 corresponds to the center of the circular opening 11 in plan view.

[0020] Each of the multiple recesses 10 contains cells, more specifically, spheroids, which are aggregates of cells. That is, the spheroids are cultured within the recesses 10, or more precisely, cultured while immersed in the culture medium stored within the recesses 10. It is preferable that the number of spheroids contained in each recess 10 is the same, that is, that the spheroids are evenly distributed across the multiple recesses 10.

[0021] Furthermore, as shown in Figure 3, a coating 6 made of a cell-non-adherent polymer such as MPC (MPC:2-methacryloyloxyethyl phosphorylcholine) polymer is formed on the surface 2S of the substrate 2, covering the curved surface 12 of each recess 10 in the recess-forming region 5. The surface shape of the base material 2, particularly the shape of the recess 10, will be explained in detail in a later section.

[0022] The wall member 3 is an injection-molded plastic product. As shown in Figures 1 and 2, the wall member 3 is constructed by connecting cylindrical partition wall sections 3B, which are located inside the outer frame section 3A, with connecting sections 3C extending from the upper periphery of the partition wall sections 3B. The wall member 3 is provided with the same number of partition wall sections 3B as there are recess-forming regions 5 on the surface 2S of the base material 2. Furthermore, when the wall member 3 is fixed to the base material 2, one partition wall section 3B is positioned to surround one recess-forming region 5 (see Figure 2). As a result, the space above the recess-forming region 5 is partitioned by the partition wall section 3B, forming a well 1A. The height of the partition wall 3B is preferably 8 to 25 mm, and more preferably 10 to 20 mm.

[0023] In well 1A, as can be seen from Figures 2 and 3, the outermost recess 10 (i.e., the side closest to the partition wall 3B) is located somewhat away from the inner wall surface of the partition wall 3B. In other words, the surface 2S of the base material 2 is provided with a peripheral region 7 surrounding the recess-forming region 5, and the partition wall 3B is fixed to the surface 2S of the base material 2 outside the peripheral region 7.

[0024] <Regarding the surface shape of the substrate> The surface shape of substrate 2 will be described in detail. In the recess-forming region 5, as shown in Figure 4, multiple recesses 10 are densely packed together and close to each other. In the configuration example shown in Figure 4, the distance between the centers of two adjacent recesses 10, i.e., the pitch between recesses, is set to approximately 540 μm.

[0025] Each of the multiple recesses 10 has a depth sufficient to accommodate cells. The average depth of the recesses 10 is 200 μm or more, preferably between 200 μm and 550 μm, from the viewpoint of preventing cells (spheroids) from protruding from within the recesses 10. The depth of the recess 10 is the distance from the opening 11 of the recess 10 to the deepest point (bottom) of the recess 10. The average depth of the recesses 10 is the arithmetic mean obtained from the depths of each of the multiple recesses 10.

[0026] Furthermore, it is desirable that the diameter of the opening 11 and the radius of curvature of the curved surface 12 be set within a suitable range. For example, the diameter of the opening 11 is preferably 250 μm to 700 μm, and more preferably 535 μm to 565 μm. The radius of curvature of the curved surface 12 is preferably about 230 μm. In addition, the aspect ratio of each recess 10, that is, the ratio of the depth of the recess 10 to the diameter of the opening 11, is preferably 0.35 to 1.00, and more preferably 0.35 to 0.80. In particular, the effects of the present invention are better when the diameter of the opening 11 is 250 μm to 700 μm and the aspect ratio of the recess 10 is 0.35 to 0.80. Here, the diameter of the opening 11 refers to the distance from the intersection point (corresponding to the connection point 20 in Figure 5) of the line of intersection between the curved surface and the cut surface of one recess and the curved surface and the cut surface of the other recess, when a line is drawn parallel to the bottom surface 2B of the base material 2 (corresponding to the reference line X in Figure 5), passing through the center of the openings of adjacent recesses in the cut surface of the base material (see Figure 5 below), to the point where it intersects with the aforementioned line of intersection in each recess. In Figure 5 below, diameters d1 and d2 correspond to the diameter of the opening 11.

[0027] The multiple recesses 10 include two adjacent recesses 10. Two adjacent recesses 10 are the two recesses 10 that are closest to each other. Here, one of the two adjacent recesses 10 will be called the first recess 13, and the other will be called the second recess 14. The first recess 13 and the second recess 14 are arranged side by side so that their outer edges touch each other in relation to the opening 11.

[0028] When the first recess 13 and the second recess 14 are observed in the cross-section shown in Figure 5, the curved surfaces 12 of the first recess 13 and the second recess 14 appear on the cross-section as intersection lines 16 and 17, respectively, and these intersection lines 16 and 17 become curved lines such as arcs or parabolas, as shown in Figure 5. The above cross-section is the AA surface shown in Figure 4, and is the cross-section of the substrate 2 passing through the center of the opening 11 of the first recess 13 and the center of the opening 11 of the second recess 14.

[0029] If the above intersection lines representing the shape of surface 12 are curved lines other than circular arcs, then their shape can be approximated by the following polynomial.

[0030]

number

[0031] The above polynomial is used to approximate the shape of an aspherical lens, where z represents the sag, h represents the distance from the center, and r represents the radius of curvature. k is the conic coefficient, which is 0 if the surface 12 is spherical, -1 if it is parabolic, greater than 0 if it is ellipsoidal, and less than -1 if it is hyperboloid. A, B, and C represent higher-order aspherical coefficients.

[0032] The intersection line 16 between the above-mentioned cross-section and the first recess 13 has a curved line (hereinafter referred to as the first curved line 18) that rises steeply from the bottom position of the first recess 13 and extends while curving toward the second recess 14. Similarly, the intersection line 17 between the above-mentioned cross-section and the second recess 14 has a curved line (hereinafter referred to as the second curved line 19) that rises steeply from the bottom position of the second recess 14 and extends while curving toward the first recess 13.

[0033] As shown in Figure 5, the first curved line 18 and the second curved line 19 touch each other at their respective upper ends, that is, they are connected at the connection point 20. Furthermore, as shown in Figure 5, the first curved line 18 and the second curved line 19 are symmetrical with respect to a line passing through the connection point 20 and perpendicular to the base surface 2B. Here, the shape of each curved line is represented by its radius if it is a circular arc, and by the polynomial described above if it is a curved line other than a circular arc.

[0034] Furthermore, as shown in Figure 5, the inclination angle of the first tangent line L1 tangent to the first curved line 18 (hereinafter referred to as angle θ1) and the inclination angle of the second tangent line L2 tangent to the second curved line 19 (hereinafter referred to as angle θ2) are of the same magnitude. The first tangent line L1 is a tangent line that passes through the connection point 20 and is tangent to the first curved line 18, and angle θ1 is the magnitude of the acute angle formed between the first tangent line L1 and the reference line X, which passes through the connection point 20 and is parallel to the base surface 2B. The second tangent line L2 is a tangent line that passes through the connection point 20 and is tangent to the second curved line 19, and angle θ2 is the magnitude of the acute angle formed between the reference line X and the second tangent line L2. The units of angles θ1 and θ2 are degrees (°).

[0035] Furthermore, in this invention, from the viewpoint of enabling proper observation of cells within the recess using a microscope, the angles θ1 and θ2 are set to satisfy either equation (1) or (2) below, respectively. θ1 < 90 - θ2 + sin -1 {sin(θ²) × 1.38 / n} Equation (1) θ² < 90 - θ¹ + sin -1 {sin(θ1)×1.38 / n} Equation (2) In equations (1) and (2) above, n is the refractive index of substrate 2 for light with a wavelength of 540 nm, for example, 1.51 to 1.52. Such a value is smaller than the refractive index of polystyrene (= 1.59 to 1.60).

[0036] The above equations (1) and (2) are used to suppress the appearance of a virtual image around the other recess of the first recess 13 and the second recess 14, where the refracted light is located, and are derived from Snell's Law.

[0037] To explain in more detail, for example, in Figure 6, the incidence and refraction of light from the culture medium to the recess 10 (for example, the second recess 14) of the substrate 2 are given by the following relation (3) from Snell's law. ni×sin(90-θa)=n×sin(θb-θa) Equation (3) In equation (3), ni is the refractive index of the culture medium, specifically the refractive index for light with a wavelength of 540 nm. The refractive index of the culture medium is approximately equal to that of water, so ni is approximately 1.38. Also, (90-θa) is an angle equal to the angle of incidence to the curved surface of the recess 10. θa is the acute angle between a reference line that passes through the incident position of the incident light on the curved surface and is parallel to the bottom surface, and the normal to the curved surface of the recess 10 at the incident position. θb is the acute angle between the above reference line and the direction of propagation of the light after refraction.

[0038] By rearranging equation (3) above, we obtain equation (4) below. θb = θa + sin -1 {sin(90-θa)×1.38 / n} Equation (4) Here, at the boundary between the first recess 13 and the second recess 14, i.e., the connection point 20, θa = θ2. Therefore, if θb > θ1, the light refracted in the second recess 14 does not intersect with the curved surface of the first recess 13 (the surface corresponding to the first curvature line 18) toward the first recess 13, and the reflection of the refracted light as a virtual image around the first recess 13 can be suppressed. As described above, equations (1) and (2) are derived.

[0039] Furthermore, when θ1 = θ2, equations (5) and (6) below can be derived according to Snell's Law. 2θ1-sin -1 (sin(θ1)×1.38 / n)<90 Equation (5) 2θ²-sin -1 (sin(θ²) × 1.38 / n) < 90 Equation (6) When the shape of the recesses is uniform, the reflection of refracted light between the recesses 10 can be suppressed by satisfying equations (5) and (6) above. Furthermore, when θ1 = θ2, the shape of the concave areas becomes uniform, which has the advantage of resulting in a more uniform time to spheroid formation. Another advantage is that the size of the spheroids will also be equal.

[0040] θ1 and θ2 are preferably 60 to 90 degrees, and more preferably 65 to 85 degrees, respectively, from the viewpoint of achieving superior effects of the present invention.

[0041] The surface shape of the substrate 2 described above, particularly the shape of each of the multiple recesses 10, allows for proper retention of cells within the recesses 10 and proper observation of the cells within the recesses 10. Specifically, if the depth of the recesses 10 is not sufficiently secured, for example, when changing the culture medium in well 1A, there is a risk that the cells in the recesses 10 will float up, escape from the recesses 10, and invade adjacent recesses 10. In contrast, in the present invention, since the depth of the recesses 10 is 200 μm or more, it is possible to suppress the escape of cells.

[0042] On the other hand, as mentioned above, there is a risk of reflection of refracted light occurring between adjacent recesses 10, and the deeper the recess 10, the more likely reflection of refracted light is to occur. In contrast, in the present invention, each recess 10 is formed to satisfy the above equations (1) and (2), so even if the depth of the recesses 10 is ensured, reflection of refracted light between the recesses 10 can be suppressed.

[0043] In this embodiment, the material constituting the base material 2 is shown to be glass, but resins such as polystyrene, polyester, polycarbonate, and polyolefin may also be used as the material constituting the base material 2. However, from the viewpoint of observability in microscopic observation, it is preferable that the material constituting the base material 2 be glass. This is because glass generally has low fluorescence intensity and high flatness. Specific examples of glass include soda-lime glass, aluminosilicate glass, quartz glass, alkali-free glass, and borosilicate glass. When using a glass plate as the base material, the coefficient of linear thermal expansion of the glass plate is 9 × 10 -7It is preferable that the temperature is below / ℃.

[0044] In this embodiment, an example is shown in which a coating 6 is formed on the surface 2S of the substrate 2, but the embodiment is not limited to this, and a coating 6 may not be formed on the surface 2S of the substrate 2. However, from the viewpoint of forming spheroids of uniform size, it is preferable that a coating 6 is formed on the surface 2S of the substrate 2.

[0045] <Method for manufacturing the base material> Next, the method for manufacturing the substrate 2 of the present invention will be explained using the case where the substrate 2 is a glass plate as an example. First, a glass plate, which will form the substrate 2, is prepared, and a CO2 laser with a wavelength of 9.6 μm is irradiated onto its surface (the side on which the recesses are formed) using a focusing lens with the aperture angle (total angle) set to approximately 20°. As a result, multiple seed holes are formed on the surface of the glass plate by ablation. The depth of the seed hole can be adjusted, for example, by the irradiation time of the CO2 laser.

[0046] Subsequently, wet etching is performed on the surface of the glass plate in which seed holes have been formed, using hydrofluoric acid, hydrochloric acid, or sulfuric acid. This gradually enlarges the diameter of the seed holes, eventually expanding to the diameter of the recesses. As a result, each of the multiple seed holes eventually becomes a recess 10, and the substrate 2 of the cell culture container 1 is completed.

[0047] While the present invention has shown a method combining laser irradiation and wet etching as a method for manufacturing the substrate 2, it is not limited to this method, and a method combining photolithography and wet etching can also be used.

[0048] <Method for manufacturing spheroids> The substrate for the cell culture vessel of the present invention is suitable as a substrate for a cell culture vessel used in the production of spheroids. Spheroids are cell aggregates formed by the three-dimensional aggregation of cells derived from humans, animals, etc. An example of a method for producing spheroids using the cell culture vessel 1 of the present invention is shown below. First, a cell suspension containing cells is added to the inside of well 1A. Once the cells have settled into the recess 10, an amount of culture medium to a height of approximately 2 to 5 mm is added to the inside of well 1A, and the cells placed in the recess 10 are cultured for several hours to several days. As a result, the cells that have proliferated in the recess 10 aggregate three-dimensionally, and spheroids are obtained. It is preferable that the cell culture vessel 1 be sterilized by EOG sterilization (sterilization using ethylene oxide gas at 60°C), autoclave sterilization (sterilization in saturated steam at 121°C for 20 minutes), or other sterilization methods before being used for cell culture. The culture medium may be replenished or replaced during cell culture. [Examples]

[0049] The present invention will be described in detail below with reference to examples. Examples 1 and 2 are examples, and Examples 3 and 4 are comparative examples. However, the present invention is not limited to these examples.

[0050] [Example 1] <Preparation of substrates for cell culture vessels> A glass plate was prepared, and its surface (the side where the recesses are formed) was irradiated with a CO2 laser with a wavelength of 9.6 μm, using a focusing lens with an aperture angle (total angle) set to approximately 20°. As a result, multiple seed holes were formed on the surface of the glass plate by ablation. The glass plate used was "Dragontrail® Pro" (aluminosilicate glass) manufactured by AGC Inc. The refractive index of the glass plate at a wavelength of 540 nm is 1.51. In this process, the CO2 laser irradiation time was appropriately set to ensure that the seed hole opening diameter was 70 μm and the depth was 460 μm. Furthermore, the CO2 laser irradiation conditions were appropriately adjusted to ensure that the seed hole pitch was 540 μm. Subsequently, wet etching was performed on the surface of the glass plate in which seed holes had been formed, using a mixed acid of hydrofluoric acid and hydrochloric acid (hydrofluoric acid concentration of 2 mol / L, hydrochloric acid concentration of 4 mol / L). Wet etching was carried out until the glass plate was thinned by 310 μm. This resulted in obtaining a glass substrate (substrate for cell culture vessels) with multiple recesses formed on its surface.

[0051] The multiple recesses obtained in this way all had an opening diameter of 540 μm and a depth (average depth) of 385 μm. Furthermore, the inclination angles of all the recesses (θ1 and θ2 in Figure 5) were all 70 degrees. At this point, the values ​​on the right-hand sides of equations (1) and (2) are both 79.2 degrees, and it was found that the relationship between equations (1) and (2) is satisfied. Furthermore, the values ​​on the left-hand sides of equations (5) and (6) are both 80.8 degrees, and it was found that the relationship between equations (5) and (6) is satisfied.

[0052] The opening diameters and average depths of the multiple recesses were measured using magnified images at 80x magnification, taken with a Hitachi High-Tech S-4300 electro-emission scanning electron microscope, of which the cross-section of the substrate passing through the center of the opening of the first recess and the center of the opening of the second recess was measured. Figure 7 shows a magnified image of the cross-section of the substrate of the cell culture vessel in Example 1, taken in this manner. In addition, for each of the examples described later, the opening diameter and average depth of the multiple recesses were measured in the same manner as in Example 1.

[0053] <Preparation of cell culture vessels> Toray Dow Corning Silicone Co., Ltd.'s adhesive "SE9140RTV" was applied to the bottom surface of the wall component (an injection-molded polystyrene product; see wall component 3 in Figure 1) using a "JETMASTER" dispenser manufactured by Musashi Engineering Co., Ltd. Subsequently, the side of the wall member to which adhesive had been applied and the side of the cell culture container substrate with the opening formed thereon were overlapped and pressed together, and then air-dried for 12 hours. In this way, a cell culture container with the structure shown in Figures 1 and 2 was obtained.

[0054] <Spheroid Manufacturing> 25 μL of culture medium, consisting of "E-MEM" with 10% by volume of fetal bovine serum "FBS" added, was pipetteed into the wells of the prepared cell culture vessel. Then, to remove bubbles from the depressions, the vessel was centrifuged at 750 × g for 10 minutes. Next, 50 μL of a suspension of human liver cancer-derived cells "HepG2" was pipetteed into the well, and then centrifuged at 200 × g for 1 minute. The suspension contained 22,500 "HepG2" cells. Next, the cells were cultured in a CO2 incubator at 37°C and under 5% CO2 conditions for 4 days to obtain spheroids.

[0055] [Example 2] <Preparation of substrates for cell culture vessels> A photoresist film was formed on one side of a glass plate, "Dragontrail® Pro" manufactured by AGC Inc., by applying a photoresist using a die coater. The photoresist used was "Glibes N-100 PT 2300CP" (product name) manufactured by Tokyo Ohka Kogyo Co., Ltd. Next, using a photomask that formed a pattern with an exposure area of ​​70 μm in diameter and a pitch of 540 μm, the photoresist film was exposed by irradiating it with ultraviolet light at a wavelength of 365 nm. After exposure, a development process was performed to remove the photoresist film from the exposed areas. Next, wet etching was performed using a mixed acid of hydrofluoric acid and hydrochloric acid (hydrofluoric acid concentration 2 mol / L, hydrochloric acid concentration 4 mol / L). As a result, the hydrofluoric acid and hydrochloric acid, which are wet etching solutions, penetrated through the 70 μm diameter hole from which the photoresist film had been removed, creating multiple hemispherical holes (recesses) in the glass, starting from the original hole. Wet etching was continued until the average depth of the multiple recesses reached 260 μm, and finally the photoresist was removed with a stripping solution. This resulted in obtaining a glass substrate (a substrate for cell culture vessels) with multiple recesses formed on its surface.

[0056] The multiple recesses obtained in this way all had an opening diameter of 540 μm and a depth (average depth) of 200 μm. Furthermore, the inclination angles of all the recesses (θ1 and θ2 in Figure 5) were all 75 degrees. At this point, the values ​​on the right-hand sides of equations (1) and (2) are both 77.0 degrees, and it was found that the relationship between equations (1) and (2) is satisfied. Furthermore, the values ​​on the left-hand sides of equations (5) and (6) are both 88.0 degrees, and it was found that the relationship between equations (5) and (6) is satisfied.

[0057] <Preparation of cell culture vessels> The cell culture vessel for Example 2 was obtained in the same manner as the cell culture vessel for Example 1, except that the substrate for the cell culture vessel for Example 2 was used.

[0058] <Spheroid Manufacturing> Spheroids were obtained in the same manner as in Example 1, except that the cell culture vessel of Example 2 was used.

[0059] [Example 3] <Preparation of substrates for cell culture vessels> A glass plate was prepared, and its surface (the side where the recesses are formed) was irradiated with a CO2 laser with a wavelength of 9.6 μm, using a focusing lens with an aperture angle (total angle) set to approximately 20°. As a result, multiple seed holes were formed on the surface of the glass plate by ablation. The glass plate used was "Dragontrail® Star" manufactured by AGC Inc. The refractive index of the glass plate at a wavelength of 540 nm is 1.51. In this process, the CO2 laser irradiation time was appropriately set to ensure that the seed hole opening diameter was 70 μm and the depth was 560 μm. Furthermore, the CO2 laser irradiation conditions were appropriately adjusted to ensure that the seed hole pitch was 520 μm. Subsequently, wet etching was performed on the surface of the glass plate in which seed holes had been formed, using a mixed acid of hydrofluoric acid and hydrochloric acid (hydrofluoric acid concentration of 2 mol / L, hydrochloric acid concentration of 4 mol / L). Wet etching was carried out until the glass plate was thinned by 360 μm. This resulted in obtaining a glass substrate (substrate for cell culture vessels) with multiple recesses formed on its surface.

[0060] The multiple recesses obtained in this way all had an opening diameter of 520 μm and a depth (average depth) of 510 μm. Furthermore, the inclination angles of all the recesses (θ1 and θ2 in Figure 5) were all 80 degrees. At this time, the values ​​on the right-hand sides of equations (1) and (2) were both 74.2 degrees, and the relationship between equations (1) and (2) was not satisfied. Furthermore, the values ​​on the left-hand sides of equations (5) and (6) were both 95.8 degrees, and therefore did not satisfy the relationship between equations (5) and (6).

[0061] <Preparation of cell culture vessels> The cell culture vessel for Example 3 was obtained in the same manner as the cell culture vessel for Example 1, except that the substrate for the cell culture vessel for Example 3 was used.

[0062] <Spheroid Manufacturing> Spheroids were obtained in the same manner as in the production of spheroids in Example 1, except that the cell culture vessel of Example 3 was used.

[0063] [Example 4] <Preparation of substrates for cell culture vessels> A mold having multiple hemispherical protrusions with a diameter of 560 μm and a height of 400 μm was fabricated using a mold processing machine. Using the obtained mold, polystyrene was injection molded to produce a polystyrene substrate (substrate for cell culture vessels) with multiple recesses formed in the inverse shape of the mold. The refractive index of polystyrene at a wavelength of 540 nm is 1.59.

[0064] The multiple recesses obtained in this way all had an opening diameter of 560 μm and a depth (average depth) of 400 μm. Furthermore, the inclination angles of all the recesses (θ1 and θ2 in Figure 5) were all 80 degrees. At this time, the values ​​on the right-hand sides of equations (1) and (2) were both 68.7 degrees, and the relationship between equations (1) and (2) was not satisfied. Furthermore, the values ​​on the left-hand sides of equations (5) and (6) were both 101.3 degrees, and therefore did not satisfy the relationship between equations (5) and (6).

[0065] <Preparation of cell culture vessels> The cell culture vessel for Example 4 was obtained in the same manner as the cell culture vessel for Example 1, except that the substrate for the cell culture vessel for Example 4 was used.

[0066] <Spheroid Manufacturing> Spheroids were obtained in the same manner as in Example 1, except that the cell culture vessel of Example 4 was used.

[0067] [Evaluation Test] <Evaluation of spheroid retention and observability> Using a Zeiss Axio Observer inverted microscope, the recess formation area at the bottom of the wells in each cell culture vessel, and the spheroids within the recesses, were observed at a magnification of 5x. The retention of spheroids within the recesses and the observability of spheroids within the recesses were evaluated according to the following criteria. The evaluation results are shown in Table 1. Figure 8 shows images taken when the recessed areas in each example were observed using an inverted microscope. In Examples 3 and 4, the areas indicated by the arrows are virtual images (reflections).

[0068] (Criteria for evaluating spheroid retention) A: The spheroid is properly held within the recess. B: A spheroid is protruding from within the recess.

[0069] (Criteria for evaluating the observability of spheroids within recesses) A: No virtual images (reflections) are present around the recessed area, making it easy to observe the spheroid. B: A virtual image (reflection) is present around the recess, making it difficult to observe the spheroid.

[0070] [Table 1]

[0071] As shown in Table 1, it was confirmed that using the substrate of the cell culture vessel of the present invention allows for proper retention of cells within the recesses and proper observation of the cells within the recesses (Examples 1 and 2). [Explanation of symbols]

[0072] 1 cell culture vessel 1A well 2 Base material 2B Bottom 2S surface 3 Wall components 3A Outer frame 3B Bulkhead 3C connection part 4 Adhesive layer 5. Recessed area 6 Coating 7 Peripheral region 10 recesses 11 Aperture 12 Curved surface 13. First recess 14. Second recess 16,17 intersection line 18 1st curved line 19 Second curved line 20 connection points L1 1st tangent L2 2nd tangent X reference line d1, d2 diameter

Claims

1. A substrate for a cell culture vessel, comprising a bottom surface and a surface provided on the opposite side of the bottom surface, The surface has a recess-forming region in which a plurality of recesses are formed, The average depth of the recesses in the aforementioned plurality of recesses is 200 to 550 μm. The plurality of recesses include adjacent first recesses and second recesses, The line of intersection between the cross-section of the substrate passing through the center of the opening of the first recess and the center of the opening of the second recess and the first recess has a first curved line that rises from the bottom position of the first recess and extends while curving toward the second recess. The line of intersection between the cut surface and the second recess has a second curved line that rises from the bottom of the second recess and extends while curving toward the first recess. The first curved line and the second curved line are connected at the connection point. When the angle between the first tangent line passing through the connection point and tangent to the first curved line and the reference line passing through the connection point and parallel to the bottom surface is θ1 (deg), and the refractive index of the substrate for light with a wavelength of 540 nm is n, the following equation (1) is satisfied, When the angle between the second tangent line, which passes through the aforementioned connection point and is tangent to the second curved line, and the reference line is θ2 (deg), the following equation (2) is satisfied, A substrate for a cell culture vessel, wherein the material constituting the substrate is glass. θ1 < 90 - θ2 + sin －１ {sin(θ2) × 1.38 / n} Formula (1) θ2 < 90 - θ1 + sin －１ {sin(θ1) × 1.38 / n} Equation (2)

2. The diameter of each of the aforementioned recesses is 250 μm to 700 μm. The substrate for a cell culture vessel according to claim 1, wherein the ratio of the depth of the recess to the diameter of the opening in each of the plurality of recesses is 0.35 to 0.

80.

3. The substrate for a cell culture vessel according to claim 1 or 2, wherein the angle θ1 and the angle θ2 are of the same size.

4. A substrate for a cell culture vessel according to any one of claims 1 to 3, A cell culture vessel having a wall member fixed on the substrate at a position surrounding the recess-forming region and partitioning the space above the recess-forming region.

Images