Cell culture materials

Abstract

To provide a cell culture substrate that is excellent in oxygen permeability and three-dimensional cell aggregation, and is suitable for culturing cell aggregates. [Solution] A cell culture substrate 10 having a polyorganosiloxane layer 11, wherein part or all of one surface 11a of the polyorganosiloxane layer 11 is designated as a culture area, and the culture area has two or more recesses 11c that open to the one surface 11a.

Description

[Technical Field]

[0001] This invention relates to a cell culture substrate. [Background technology]

[0002] In the field of regenerative medicine, cell aggregates such as spheroids (hereinafter also referred to as "cell aggregates"), which are masses of cells cultured in three dimensions, are attracting attention for applications such as transplantation therapy, organ regeneration research, and drug screening. One method for forming cell aggregates is the adherent culture method, in which cells are grown on a substrate for culturing cells (hereinafter also referred to as "cell culture substrate"). In the adherent culture method, polystyrene cell culture substrates, which are inert to cells, are usually used. However, because polystyrene cell culture substrates have low oxygen permeability, oxygen does not reach the culture medium sufficiently, leading to oxygen deficiency. This makes the cells inside the cell aggregate more susceptible to death due to oxygen deprivation during culture, thus limiting the size of the cell aggregate that can be cultured. In addition, depending on the type of cell, the cells may not aggregate easily, making it difficult to form cell aggregates.

[0003] As a cell culture substrate with improved oxygen permeability, for example, Patent Document 1 discloses a cell culture vessel comprising a thin film sheet containing silicone, having a thickness of 5 μm or less and being oxygen permeable, and a cell culture plate having one recess in the center, wherein multiple through holes are formed at the bottom of the recess and penetrate the cell culture plate, and the thin film sheet covers the lower side of the cell culture plate so as to block these through holes, thereby forming cell culture wells. [Prior art documents] [Patent Documents]

[0004] [Patent Document 1] International Publication No. 2023 / 248620 [Overview of the project] [Problems that the invention aims to solve]

[0005] However, with the cell culture vessel described in Patent Document 1, depending on the type of cell, cells may not aggregate easily during the culture process, resulting in cases where cell aggregates cannot be obtained, or even if initial cell aggregates are formed, they may not grow well, indicating room for improvement. The present invention aims to provide a cell culture substrate that is suitable for culturing cell aggregates, exhibiting excellent oxygen permeability and three-dimensional cell aggregation. [Means for solving the problem]

[0006] The present invention has the following aspects. [1] Having a polyorganosiloxane layer, A portion or all of one side of the polyorganosiloxane layer is designated as the culture area. The cell culture substrate has two or more recesses that open to one of its surfaces in the culture area. [2] The storage modulus of the polyorganosiloxane constituting the polyorganosiloxane layer, as measured in accordance with JIS K 7224-10, is 5 × 10⁻¹⁰. 3 ~2×10 6 The cell culture substrate described in [1] above, wherein the cell culture substrate is Pa. [3] The cell culture substrate according to [1] or [2], wherein the depth of the recess is 0.1 to 5 μm. [4] The cell culture substrate according to any one of [1] to [3], wherein the thickness of the polyorganosiloxane layer is 20 to 500 μm. [5] A cell culture substrate according to any one of [1] to [4], wherein the distance between adjacent recesses is 1 to 10 μm. [6] The cell culture substrate according to any one of [1] to [5], wherein the opening diameter of the recess is 1 to 10 μm. [7] The cell culture substrate according to any one of [1] to [6], wherein the recess is a through-hole penetrating the polyorganosiloxane layer. [8] The cell culture substrate according to any one of [1] to [6], wherein the recess is a bottomed pore having a bottom within the polyorganosiloxane layer. [9] The cell culture substrate according to any one of [1] to [6], wherein the concave portion is in a groove shape.

[10] The cell culture substrate according to any one of [1] to [6], wherein the concave portion is formed by a plurality of intersecting groove portions.

[11] Further comprising a reinforcing layer, The cell culture substrate according to any one of [1] to

[10] , wherein the reinforcing layer is provided on the other surface of the polyorganosiloxane layer.

[12] The cell culture substrate according to

[11] , wherein the reinforcing layer is a non-woven fabric.

[13] The cell culture substrate according to

[11] , wherein the reinforcing layer is a layer of polyorganosiloxane having a storage elastic modulus larger than that of the polyorganosiloxane constituting the polyorganosiloxane layer. [Effect of the Invention]

[0007] According to the present invention, it is possible to provide a cell culture substrate that is excellent in oxygen permeability and three-dimensional aggregation of cells and is suitable for culturing cell masses. [Brief Description of the Drawings]

[0008] [Figure 1] It is a cross-sectional view schematically showing an example of the cell culture substrate of the present invention. [Figure 2] It is a perspective view showing an example of the concave portion. [Figure 3] It is a perspective view showing another example of the concave portion. [Figure 4] It is a perspective view showing another example of the concave portion. [Figure 5] It is a view for explaining an example of a method for producing a cell culture substrate, (a) is a plan view and (b) is a cross-sectional view. [Figure 6] It is a cross-sectional view schematically showing another example of the cell culture substrate of the present invention. [Figure 7] It is an observation image of a fluorescence microscope showing the results of culturing cells using the cell culture substrates obtained in Examples 1 to 10. [Figure 8]These are fluorescence microscope images showing the results of culturing cells using the cell culture substrates obtained in Comparative Examples 1-6. [Modes for carrying out the invention]

[0009] The following description will refer to one embodiment of the cell culture substrate according to the present invention, with appropriate reference to Figure 1. However, the following description is merely one example of an embodiment of the present invention, and the present invention is not limited to the following description unless it exceeds the gist of the invention. In this specification, the "~" symbol indicating a numerical range means that the numbers before and after it are included as the lower and upper limits, respectively. The numerical ranges of content, various physical properties, and characteristic values ​​disclosed herein can be modified by arbitrarily combining their lower and upper limits to create new numerical ranges. In the following description, the drawings may be enlarged to clearly show their features, and the dimensional ratios of the components may differ from those of the actual components. The materials and dimensions exemplified in the following description are examples only, and the release sheets according to the present invention are not limited to these examples. They can be modified as appropriate without departing from the spirit of the invention. Also, in Figures 2 to 6, the same reference numerals are used for components that are the same as those in Figure 1, and their descriptions may be omitted.

[0010] [Cell culture substrate] Figure 1 is a schematic cross-sectional view showing an example of the cell culture substrate of the present invention. The cell culture substrate 10 shown in Figure 1 consists only of a polyorganosiloxane layer 11 and is in the form of a sheet or film.

[0011] <Polyorganosiloxane layer 11> The polyorganosiloxane layer 11 is a layer composed of polyorganosiloxane. Since polyorganosiloxane is inert to cells and has excellent oxygen permeability, the cell culture substrate 10 having the polyorganosiloxane layer 11 has excellent oxygen permeability and compatibility with cells. In addition, depending on the cell type, it is easier to form initial cell aggregates compared to conventional styrene cell culture substrates. Furthermore, because it has low temperature dependence, stable oxygen permeability can be obtained over a wide temperature range. Moreover, because it has excellent heat resistance, chemical resistance, and moldability, it is easy to form the recessed portion 11c, described later, into the desired shape. A portion or all of one surface 11a of the polyorganosiloxane layer 11 is designated as a culture area, and the culture area has two or more recesses 11c that open to the surface 11a.

[0012] The polyorganosiloxane layer 11 in the illustrated example is composed of a porous layer 111 and a non-porous layer 112. The surface of the porous layer 111 opposite to the non-porous layer 112 is one surface 11a of the polyorganosiloxane layer 11. The surface of the non-porous layer 112 opposite to the porous layer 111 is the other surface 11b of the polyorganosiloxane layer 11.

[0013] (Polyorganosiloxane) The polyorganosiloxane (hereinafter also referred to as "polyorganosiloxane (A)") constituting the polyorganosiloxane layer 11 is a compound having siloxane bonds (Si-O-Si). One or more organic groups may be bonded to the silicon atom in the siloxane bond. The organic group is not particularly limited, but examples include alkyl groups such as methyl, ethyl, and propyl groups; cycloalkyl groups such as cyclopentyl and cyclohexyl groups; alkenyl groups such as vinyl and allyl groups; aryl groups such as phenyl and tolyl groups; thiol and alkylthiol groups; aralkyl groups such as benzyl and phenylethyl groups; or halogenated hydrocarbons in which one or more hydrogen atoms of these organic groups are substituted with halogen atoms such as chlorine or fluorine atoms.

[0014] As the polyorganosiloxane (A), addition reaction type and condensation type polyorganosiloxane compositions can be used, and the curing mechanism may be any of the following: thermal curing, room temperature curing, moisture curing, or energy ray curing (e.g., ultraviolet (UV) curing). The polyorganosiloxane (A) before curing may be liquid or solid at room temperature, but it is preferable that it be liquid from the viewpoint of easily forming recesses 11c of the desired shape.

[0015] Specific examples of polyorganosiloxane (A) include polydimethylsiloxane, polydiethylsiloxane, polydiphenylsiloxane, and polymethylphenylsiloxane. Copolymers composed of polysiloxane units with different structures can also be used. These copolymers may be random copolymers or block copolymers. Among these, polydimethylsiloxane is preferred from the viewpoint of versatility and oxygen permeability. Furthermore, from the viewpoint of affinity with culture medium, polyorganosiloxane modified with hydrophilic groups can also be used.

[0016] The storage modulus of polyorganosiloxane (A) (25°C, 1Hz) is 5 × 10⁻⁶ 3 ~2×10 6 Pa is preferred, 1 × 10 4 ~2×10 6 Pa is more preferred, 1 × 10 5 ~2×10 6Pa is even more preferable. For polyorganosiloxanes (A) with the same polysiloxane chain structure, a higher storage modulus generally correlates to a higher crosslink density, and a higher crosslink density tends to decrease oxygen permeability. Furthermore, a higher storage modulus of polyorganosiloxane (A) leads to better shape retention, which is advantageous in terms of handling. Therefore, if the storage modulus of polyorganosiloxane (A) is within the above range, it exhibits excellent oxygen permeability, making cell culture easier and providing an excellent balance between cell culture efficiency and the handling of the cell culture substrate 10. For polyorganosiloxanes (A) with the same structure, setting the storage modulus to the lower limit is advantageous for increasing oxygen permeability, while setting it to the upper limit is advantageous for increasing handling. Therefore, a storage modulus is selected according to the balance between oxygen permeability and handling. Additionally, oxygen permeability may be adjusted by selecting the structure of the polysiloxane chain constituting the polyorganosiloxane (A).

[0017] The storage modulus of polyorganosiloxane (A) is measured in accordance with JIS K 7224-10, "Plastics - Test methods for dynamic mechanical properties - Part 10: Complex shear viscosity using a parallel plate vibrating rheometer." Specifically, first, uncured polyorganosiloxane (A) is molded to a size of 100 mm x 100 mm x 2 mm thick, and then cured to obtain a 2 mm thick sheet-like cured material. The obtained sheet-like cured material is cut out into a disc shape with a diameter of 8 mm to obtain a disc-shaped test specimen with a diameter of 8 mm x a thickness of 2 mm. The storage modulus of the obtained test specimen is measured using a dynamic viscoelasticity measuring device under conditions of 25°C and 1 Hz, and the measured value is taken as the storage modulus of polyorganosiloxane (A).

[0018] (Porous layer) The porous layer 111 is a region in the thickness T1 direction of the polyorganosiloxane layer 11 where recesses 11c exist, and is composed of a plurality of recesses 11c and regions 11d between adjacent recesses 11c (hereinafter also referred to as "non-recessed areas" or "protrusions"). The non-recessed portion 11d of the porous layer 111 is composed of polyorganosiloxane (A).

[0019] The thickness of the porous layer 111, that is, the thickness of the non-recessed portion 11d, is the depth T of the recessed portion 11c 11 and is the same. When there are recessed portions 11c with different depths, the thickness of the porous layer 111 is the same as the depth of the deepest recessed portion 11c. The depth of the recessed portion 11c will be described later.

[0020] (Non-porous layer) The non-porous layer 112 is a region in the thickness direction of the polyorganosiloxane layer 11 where there are no recessed portions 11c, and is also referred to as a solid layer. When there are recessed portions 11c with different depths in the culture area, in the region in the thickness direction of the polyorganosiloxane layer 11, the region where the deepest recessed portion 11c does not exist is defined as the non-porous layer 112. The non-porous layer 112 is composed of polyorganosiloxane.

[0021] The thickness T of the non-porous layer 112 12 is not particularly limited, but for example, 50 to 680 μm is preferable, 100 to 600 μm is more preferable, and 200 to 500 μm is even more preferable. The thickness T of the non-porous layer 112 12 If it is above the above lower limit value, the handling property when peeling the cells after culture will be good. The thickness T of the non-porous layer 112 12 If it is below the above upper limit value, the oxygen permeability can be maintained well, and a sufficient amount of oxygen for culture can permeate. The thickness of the non-porous layer 112 is determined by cutting the cell culture substrate 10 in the thickness direction, slicing the cross-section, and observing the sliced cross-section with a scanning electron microscope (SEM).

[0022] (Recessed portion) There are two or more recessed portions 11c in the culture area, and they are formed so as to open to one surface 11a of the polyorganosiloxane layer 11. The presence of two or more recesses 11c in the culture area facilitates cell adhesion to the non-recessed surface 11d, as well as easier three-dimensional aggregation of cells, thus promoting growth into cell aggregates. In particular, vertical growth of cell aggregates is facilitated. In particular, the width of the non-recessed portion 11d in the planar direction (the spacing P described later) and the opening dimensions of the recessed portion 11c (the opening diameter R and groove width described later) are preferably both smaller than the size of the seed cell. This is because partially supporting the cells rather than supporting the entire surface of the cell culture substrate 10 makes it easier for the cells to aggregate three-dimensionally and increases the contact area with the culture medium, thus promoting growth into a cell aggregate. Here, the size of the cell refers to the length in the longitudinal direction in the case of fibrous cells.

[0023] Examples of the shape of the recess 11c include a hole-like shape as shown in Figure 2, and a groove-like shape as shown in Figures 3 and 4. Among these, the hole-like shape is preferred from the viewpoint of cell aggregate formation.

[0024] If the recess 11c is in the shape of a hole, the shape of the opening of the recess 11c when viewed from the front is not particularly limited, and may be circular as shown in Figure 2, or it may be elliptical or polygonal. If the recess 11c is hole-shaped, the recess 11c may be a through-hole penetrating the polyorganosiloxane layer 11, or a bottomed hole having a bottom within the polyorganosiloxane layer 11, but a bottomed hole is preferred. As shown in Figure 1, if the polyorganosiloxane layer 11 is composed of a porous layer 111 and a non-porous layer 112, the hole-shaped recess 11c is a bottomed hole.

[0025] If the recess 11c is groove-shaped, the recess 11c may be formed of multiple grooves 11m extending in one direction, as shown in Figure 3, or it may be formed of multiple intersecting grooves 11m, as shown in Figure 4. If the recess 11c is groove-shaped, the recess 11c may be a through groove penetrating the polyorganosiloxane layer 11, or a bottomed groove having a bottom within the polyorganosiloxane layer 11, but a bottomed groove is preferred. As shown in Figure 1, if the polyorganosiloxane layer 11 is composed of a porous layer 111 and a non-porous layer 112, the groove-shaped recess 11c is a bottomed groove.

[0026] Depth T of recess 11c 11 That is, the thickness of the porous layer 111 can be set according to the size of the cell, but for example, 0.05 to 20 μm is preferred, 0.08 to 15 μm is more preferred, 0.1 to 15 μm is even more preferred, 0.1 to 10 μm is even more preferred, 0.1 to 5 μm is particularly preferred, and 1 to 5 μm is most preferred. Depth T of the recess 11 If the depth T of the recess is within the above range, cells will be more likely to aggregate three-dimensionally, and growth into a cell mass will be further promoted. 11 If the value is below the above upper limit, it becomes easier to shape the silicone. Also, the depth T of the recess 11 If the value is above the lower limit mentioned above, the surface of the cell culture substrate will not become as flat as it should be, thus maintaining good three-dimensional aggregation of cells and further improving cell aggregate formation. Depth T of recess 11c 11 The thickness T of the nonporous layer 112 is 12 It can be obtained using the same method.

[0027] Note that the depth T of the recess 11c 11 When the thickness T1 of the polyorganosiloxane layer 11 matches, it means that the polyorganosiloxane layer 11 is composed solely of the porous layer 111.

[0028] The spacing P between adjacent recesses 11c should be set according to the size of the cells to be cultured, such that it is smaller than the size of the cells. For example, 0.5 to 50 μm is preferred, 1 to 50 μm is more preferred, 1 to 25 μm is even more preferred, 1 to 10 μm is even more preferred, 3 to 10 μm is particularly preferred, and 5 to 10 μm is most preferred. If the spacing P between adjacent recesses 11c is within the above range, the cells will be able to aggregate three-dimensionally more easily, and the growth into a cell aggregate will be further promoted. In particular, if the spacing P between the recesses 11c is below the upper limit, the area in contact with the non-recessed surface 11d formed between two adjacent recesses 11c will not increase, the three-dimensional aggregation of cells will be well maintained, and the cell aggregate formation will be further improved. Also, if the spacing P between the recesses 11c is above the lower limit, the cells will be more easily supported by the non-recessed surface 11d, the three-dimensional aggregation of cells will be well maintained, and the cell aggregate formation will be further improved. The distance P between adjacent recesses 11c is the distance from any recess 11c to the recess 11c adjacent to that recess 11c, and is determined by SEM observation from a front view.

[0029] The opening diameter R of the recess 11c should be set according to the size of the cells to be cultured, so as to be smaller than the size of the cells. For example, 0.5 to 50 μm is preferred, 1 to 50 μm is more preferred, 1 to 25 μm is even more preferred, 1 to 10 μm is even more preferred, 3 to 10 μm is particularly preferred, and 5 to 10 μm is most preferred. If the opening diameter R of the recess 11c is within the above range, the cells will be able to aggregate three-dimensionally more easily, and the growth into a cell aggregate will be further promoted. In particular, if the opening diameter R of the recess 11c is below the upper limit, the cells to be cultured will be less likely to reach the bottom surface of the recess 11c, the area in contact with the bottom surface will be suppressed, the three-dimensional aggregation of cells will be well maintained, and the cell aggregate formation will be further improved. Also, if the opening diameter R of the recess 11c is above the lower limit, the surface of the culture area of ​​the cell culture substrate will be less likely to become flat, so the three-dimensional aggregation of cells will be well maintained, and the cell aggregate formation will be further improved. The opening diameter R of the recess 11c is determined by the same method as the distance P between adjacent recesses 11c.

[0030] Furthermore, if the shape of the recess 11c is, for example, a hole as shown in Figure 2, and the shape of the opening of the recess 11c when viewed from the front is circular, then the opening diameter R of the recess 11c is the diameter of the circle. If the shape of the opening of the recess 11c when viewed from the front is elliptical, then the opening diameter R of the recess 11c is the major axis of the ellipse. If the shape of the opening of the recess 11c when viewed from the front is polygonal, then the opening diameter R of the recess 11c is the diameter of the circumscribed circle of the opening of the recess 11c. If the recess 11c is formed by a plurality of grooves 11m extending in one direction, as shown in Figure 3, for example, the opening diameter R of the recess 11c is the width of the grooves 11m. If the recess 11c is formed by multiple intersecting grooves 11m, as shown in Figure 4, for example, the opening diameter R of the recess 11c is the width of one of the two intersecting grooves 11m. If the widths of the two intersecting grooves 11m are different, the narrower width is used as the opening diameter R of the recess 11c.

[0031] (thickness) The thickness T1 of the polyorganosiloxane layer 11, i.e., the depth T of the recess 11c. 11 and the thickness T of the nonporous layer 112 12 The total thickness is preferably 20 to 700 μm, more preferably 100 to 600 μm, and even more preferably 200 to 500 μm. If the thickness T1 of the polyorganosiloxane layer 11 is greater than or equal to the lower limit above, the cell culture substrate 10 can be well maintained in a sheet or film form. It also has excellent handling properties. If the thickness T1 of the polyorganosiloxane layer 11 is less than or equal to the upper limit above, good oxygen permeability can be maintained. The thickness T1 of the polyorganosiloxane layer 11 is determined by the same method as the thickness of the nonporous layer 112.

[0032] <Manufacturing method> The cell culture substrate 10 can be manufactured by methods such as nanoimprinting, 3D printing, electrospinning, foaming, sphere filling, and hydrogenation molding. Among these methods, a recess 11c can be easily formed in a desired region on one surface 11a of the polyorganosiloxane layer 11, and the depth T of the recess 11c can be easily formed.11 From the viewpoint of easily controlling the aperture diameter R, shape, and the spacing P between adjacent recesses 11c, the nanoimprint method is preferred.

[0033] Nanoimprinting is a method of transferring the surface structure of a porous layer to a transfer target by pressing a mold, which has a reversed structure of the porous layer structure, i.e., a convex portion on its surface with a shape opposite to that of a concave portion 11c, onto the transfer target, and applying heat, pressure, or ultraviolet (UV) light. For example, as shown in Figures 5(a) and (b), uncured polyorganosiloxane S is poured onto a mold 20 having a plurality of protrusions 21 on its surface, and then pressure-molded with a glass plate 40 via a spacer 30 to a predetermined thickness. Subsequently, the uncured polyorganosiloxane S is crosslinked by heat treatment to obtain a cell culture substrate consisting of a polyorganosiloxane layer having a surface structure that has the surface structure of the mold 20 transferred to it. In the cases of Figures 5(a) and (b), the shape of the protrusion 21 of the mold 20 is transferred to one surface of the polyorganosiloxane layer, forming a recess with the shape shown in Figure 3. The height of the protrusion 21 of the mold 20 is roughly equal to the depth of the recess in the polyorganosiloxane layer, i.e., the thickness of the porous layer. The height of the spacer 30 is roughly equal to the thickness of the polyorganosiloxane layer. The distance from the protrusion 21 of the mold 20 to the glass plate 40 is roughly equal to the thickness of the non-porous layer.

[0034] The method for forming the surface structure of type 20 is not particularly limited, but for example, fine pattern formation methods such as photolithography, electron beam exposure, and X-ray exposure can be employed.

[0035] <Effects and Effects> The cell culture substrate of this embodiment, as described above, has a polyorganosiloxane layer and therefore exhibits excellent oxygen permeability. As a result, oxygen is sufficiently distributed during cell culture, making it less likely for oxygen deficiency to occur. Furthermore, because the culture area of ​​the polyorganosiloxane layer has two or more depressions, cells tend to aggregate three-dimensionally, making it easier for cell aggregates to grow. Thus, the cell culture substrate of this embodiment is suitable for culturing cell aggregates because it has excellent oxygen permeability and three-dimensional cell aggregation properties.

[0036] <Other Embodiments> The cell culture substrate of the present invention is not limited to those described above. For example, the cell culture substrate may have a curved surface on one side of the polyorganosiloxane layer, particularly the surface of the culture area. In a culture area, the depths of two or more recesses may be the same or different. The opening diameters and pore shapes of each recess may be the same or different. The spacing between adjacent recesses may be the same or different. The opening diameters of the recesses and the spacing between adjacent recesses may be the same or different. Furthermore, multiple groups of recesses formed from two or more recesses may be formed, and at least one of the following may be different for each group of recesses: the diameter and depth of the recess opening, the shape of the recess opening, the spacing between adjacent recesses, and the arrangement pattern of the recesses. Also, the spacing between adjacent groups of recesses may be the same or different. For example, a combination of a group of recesses formed from hole-shaped recesses as shown in Figure 2 and a group of recesses formed from groove-shaped recesses as shown in Figures 3 and 4 may be used.

[0037] Furthermore, the shape of the top surface of the non-recessed area formed between two adjacent recesses 11c may be a flat surface or a convex curved surface. In particular, by using a convex curved surface, the contact area with the cells can be smaller than in the case of a flat surface, allowing for easier movement of cells in the culture area, which further enhances the three-dimensional aggregation of cells and promotes the formation of cell aggregates. In addition, the shape of the non-recessed area may be the same for all of them, or it may be a configuration in which non-recessed areas of different shapes are mixed together.

[0038] The polyorganosiloxane layer may consist solely of porous layers. If the polyorganosiloxane layer consists solely of porous layers, the depressions present in the culture area of ​​the polyorganosiloxane layer are, for example, through-holes or through-grooves. A polyorganosiloxane layer consisting solely of porous layers may be formed directly on a culture container such as a petri dish or a reinforcing layer, as described later.

[0039] The cell culture substrate 10 may further include, for example, a reinforcing layer 12 provided on the other surface 11b of the polyorganosiloxane layer 11, in addition to the polyorganosiloxane layer 11, as shown in Figure 6. The inclusion of the reinforcing layer 12 improves handling.

[0040] The thickness T2 of the reinforcing layer 12 is preferably 15 to 495 μm, more preferably 30 to 200 μm, and even more preferably 50 to 100 μm. If the thickness T2 of the reinforcing layer 12 is above the lower limit, handling performance is further improved. If the thickness T2 of the reinforcing layer 12 is below the upper limit, good oxygen permeability can be maintained. From the viewpoint of improving handling performance, a thicker reinforcing layer 12 (thickness T2) is advantageous, but from the viewpoint of oxygen permeability, a thinner reinforcing layer 12 is preferable. Therefore, it is preferable to increase the hardness (storage modulus) of the reinforcing layer 12 to reduce its thickness T2. The thickness T2 of the reinforcing layer 12 is determined by the same method as the thickness of the non-porous layer 112.

[0041] The reinforcing layer 12 is preferably one that has oxygen permeability, and examples include nonwoven fabrics and polyorganosiloxane layers. When the reinforcing layer 12 is a polyorganosiloxane layer, the polyorganosiloxane layer 11 having a culture area, i.e., having a recess 11c on its surface, is also called the "first polyorganosiloxane layer," and the polyorganosiloxane layer that is the reinforcing layer 12 is also called the "second polyorganosiloxane layer."

[0042] The polyorganosiloxane constituting the second polyorganosiloxane layer (hereinafter also referred to as "polyorganosiloxane (B)") preferably has a higher storage modulus than the polyorganosiloxane (A) constituting the first polyorganosiloxane layer. Specifically, the storage modulus of polyorganosiloxane (B) is preferably 10 times or more than the storage modulus of polyorganosiloxane (A), more preferably 10 to 10,000 times, and even more preferably 100 to 1,000 times. The storage modulus of polyorganosiloxane (B) is greater than that of polyorganosiloxane (A), which improves the handling of the cell culture substrate.

[0043] The storage modulus of polyorganosiloxane (B) is 5 × 10⁻⁶. 3 ~1 × 10 7 Pa is preferred, 1 × 10 5 ~1 × 10 7 Pa is more preferred, 1 × 10 6 ~1 × 10 7 Pa is even more preferable. If the storage modulus of polyorganosiloxane (B) is within the above range, handling properties are improved while maintaining sufficient oxygen permeability of the cell culture substrate. For polyorganosiloxanes (B) with the same polysiloxane chain structure, the crosslinking density tends to increase as the storage modulus increases, and oxygen permeability tends to decrease. Therefore, it is preferable that the storage modulus of polyorganosiloxane (B) be below the above upper limit. The storage modulus of polyorganosiloxane (B) can be determined using the same method as that used for the storage modulus of polyorganosiloxane (A).

[0044] <Application> The cell culture substrate of the present invention is suitable as a scaffold material when culturing cells. In the cell culture method using the cell culture substrate of the present invention, cells are cultured using the cell culture substrate to form cell aggregates, and then the cultured cell aggregates are detached from the cell culture substrate and collected. In the cell culture method of this embodiment, the cell culture substrate is placed in a culture vessel such as a petri dish or a regular dish, with the side containing the porous layer of the polyorganosiloxane layer (the culture side surface) exposed. Cells are then seeded and attached to the culture area of ​​the cell culture substrate, specifically on the surface of at least the porous layer of the polyorganosiloxane layer (the area where the depressions exist). In this case, it is preferable to provide a collagen layer on the surface of the porous layer of the polyorganosiloxane layer and seed and attach the cells via this collagen layer, i.e., to the surface of the collagen layer. Providing a collagen layer makes it easier to retain the seed cells after implantation.

[0045] The collagen layer only needs to be provided on the surface of at least the porous layer of the polyorganosiloxane layer (in the area where the depressions exist), and the collagen layer may be provided over the entire surface of the culture side. Furthermore, the collagen layer may be a continuous membrane or a discontinuous membrane (for example, composed of multiple membranes dispersed in an island-like manner). The collagen layer is formed by applying collagen, which forms the collagen layer, to at least the surface of the porous layer on the culture side of the polyorganosiloxane layer, or to the entire surface on the culture side, using a known method. The thickness of the collagen layer is preferably 0.1 to 1000 nm, more preferably 0.5 to 100 nm, and even more preferably 1 to 10 nm. If the thickness of the collagen layer is above the lower limit, seed cells will be more likely to implant. If the thickness of the collagen layer is below the upper limit, it will have less influence on the shape of the recesses in the porous layer, making it easier for cells to aggregate three-dimensionally and promoting growth into cell aggregates.

[0046] In this specification, a cell culture substrate with a collagen layer, wherein the collagen layer is provided on the culture-side surface (one surface 11a in Figure 1) of the polyorganosiloxane layer, is also referred to as a "composite substrate." That is, the composite substrate comprises the cell culture substrate of the present invention and a collagen layer, wherein the collagen layer is provided on at least the surface of the porous layer on the culture-side surface of the polyorganosiloxane layer.

[0047] The cells to be cultured are not particularly limited, but examples include fibroblasts, osteoblasts, cardiomyocytes, nerve cells, hepatocytes, chondrocytes, etc. The animal species of the cells are not particularly limited, but examples include mammals such as humans, rats, mice, pigs, and dogs. The culture conditions, such as those for the culture medium, are not particularly limited and can be set appropriately depending on the type of cell and the intended use of the cultured cells.

[0048] In the cell culture method of this embodiment, cells are cultured using the cell culture substrate of the present invention as described above. As a result, oxygen is sufficiently distributed during culture, making it less likely for oxygen deficiency to occur. Furthermore, cells can easily aggregate three-dimensionally on the culture area of ​​the cell culture substrate to form cell aggregates, and these cell aggregates can grow easily. [Examples]

[0049] The present invention will be described in more detail below with reference to examples. The following examples illustrate specific examples of the present invention and do not limit the scope of the invention.

[0050] [Example 1] <Manufacturing of cell culture substrates> A 1 mm thick silicon substrate was used as a mold by photolithography to form a hole-shaped recess 11c on its surface, as shown in Figure 2, with a protrusion having the opposite shape to the circular hole-shaped recess 11c.

[0051] An addition-reaction type thermosetting liquid polydimethylsiloxane (manufactured by Shin-Etsu Chemical Co., Ltd., trade name "SIM-360") and a curing agent (manufactured by Shin-Etsu Chemical Co., Ltd., trade name "CAT-360") were mixed in a mass ratio of 10:1. The mixture was stirred and degassed using a stirrer (manufactured by Shin-Kee Co., Ltd., product name "Awatori Rentaro"), and then degassed under reduced pressure for 10 minutes to prepare uncured polyorganosiloxane (A). Using uncured polyorganosiloxane (A), the specimens were molded and cured as follows, and the storage modulus of polyorganosiloxane (A) was measured in accordance with JIS K 7224-10, yielding 6.8 × 10⁻⁶.5 It was Pa. First, uncured polyorganosiloxane (A) was molded to a size of 100 mm x 100 mm x 2 mm thick. It was then preheated at 70°C for 1 hour in a hot air oven (Tokyo Rikakikai Co., Ltd., product name "WFO-520W"), followed by heat treatment at 100°C for 3 hours to cure it, obtaining a 2 mm thick sheet-like cured material. The obtained sheet-like cured material was then cut into an 8 mm diameter disc shape to obtain a disc-shaped test specimen with a diameter of 8 mm and a thickness of 2 mm. The storage modulus of the obtained test specimen was measured using a dynamic viscoelasticity measuring device (TA Instruments, product name "ARES-G2") at 25°C and 1 Hz.

[0052] Uncured polyorganosiloxane (A) was poured onto the surface of the previously fabricated mold where the protrusions were formed, and pressure-molded with a glass plate via a spacer to the desired thickness. Then, it was heat-treated at 150°C for 30 minutes to crosslink the uncured polyorganosiloxane (A). After cooling to room temperature, it was removed from the mold to obtain a cell culture substrate measuring 30 mm in length, 30 mm in width, and 500 μm in thickness. This substrate consisted of a polyorganosiloxane layer composed of a porous layer and a non-porous layer, with recesses 11c, as shown in Figure 2, formed on one side. The porous layer with multiple recesses was formed in the central region (20 mm × 20 mm) of one side of the polyorganosiloxane layer, and the region with the recesses (20 mm × 20 mm) was used as the culture area. The depth of the recesses, i.e., the thickness of the porous layer, was 5 μm, and the thickness of the non-porous layer was 495 μm. Furthermore, the spacing between adjacent recesses and the opening diameter of each recess were both set to 5 μm.

[0053] <Pretreatment of culture devices> The previously obtained cell culture substrate was placed on a polystyrene culture dish measuring 60 mm x 60 mm. A polymethyl methacrylate ring with an outer diameter of 22 mm, an inner diameter of 16 mm, and a height of 8 mm was then placed on the culture area of ​​the cell culture substrate to form a culture device. The culture device was disinfected by immersing it in 70% by mass ethanol for 10 minutes. After disinfection, the culture device was washed by immersing it in sterile water for 2 minutes twice. A collagen solution (manufactured by Nitta Gelatin Co., Ltd., product name "Cell Matrix (registered trademark) Type IP") at a concentration of 0.3 mg / mL was placed in the culture area (2 cm) inside the ring. 2 A 3 nm thick collagen layer was formed by applying 0.4 mL per unit area of ​​the culture area twice. Next, the culture device containing the collagen layer was sterilized using a UV lamp with ultraviolet light at a wavelength of 254 nm, and then left to stand for 1 hour to dry. The UV sterilization was performed inside a clean bench. Next, the culture device, after UV sterilization, was washed twice with phosphate-buffered saline (PBS solution), then UV sterilization was performed under the same conditions as before, and it was left to stand for 1 hour to dry. After UV sterilization, the culture devices were stored in a 37°C incubator until seeding.

[0054] <Cell Culture> As cells, we used NIH3T3 cells (mouse fibroblasts, with a longitudinal length of approximately 10-20 μm) obtained from the Human Science Research Resource Bank. Cells are placed on a collagen layer formed on the culture area of ​​the cell culture device, with 0.4 mL (2.2 × 10) of cells per culture device. 4 Seeds were sown in a 60mm petri dish and left to stand for 3 hours. After 3 hours, 8 mL each of DMEM (Dulbecc's Modified Eagle Medium, Thermo Fisher Scientific) and 10% by mass FBS (Fetal Bovine Serum, Thermo Fisher Scientific) were added to each culture device, and the cells were incubated for 5 days. No culture medium was changed during the incubation period.

[0055] <Rating> (Evaluation of cell aggregate culture suitability) Rhodamine Phalloidin X-bonded (Fujifilm Wako Pure Chemical Corporation) was used as the staining solution, and staining and observation were performed according to the following procedure. (1) Washing: The cultured cells (hereinafter referred to as "sample") were washed three times with PBS solution. (2) Immobilization: The sample was immobilized with a 3.7% by mass formaldehyde solution and allowed to stand for 10 minutes. (3) Washing: The sample was washed three times with PBS solution. (4) Clearing: The sample was cleared with a PBS solution containing 0.1% by mass of Triton X-100 and allowed to stand for 3-5 minutes. (5) Washing: The sample was washed three times with PBS solution. (6) Pre-culture: Samples were pre-cultured in a PBS solution containing 1% by mass BSA (bovine serum albumin) and allowed to stand for 20-30 minutes. (7) Staining: The sample was stained with staining solution (5 units / mL) and allowed to stand for 20 minutes. (8) Washing: The sample was washed three times with PBS solution. (9) Embedding: The sample was embedded in a solution of PBS and glycerol mixed in a 1:1 mass ratio. (10) Observation: Observation images were acquired using a fluorescence microscope (Keyence Corporation, product name "All-in-One Fluorescence Microscope BZ-X800", excitation wavelength (Ex): 568 nm, fluorescence wavelength (Em): 585 nm).

[0056] Figure 7 shows the observation images obtained using a fluorescence microscope. The gray areas in the figure represent cultured cells. The image display scale is 1 mm wide x 0.75 mm high. Furthermore, cell culture suitability was evaluated based on the following evaluation criteria. The results are shown in Table 1. Note that superior cell aggregate culture suitability indicates superior oxygen permeability and cell aggregate formation. ◎: The area ratio of cell aggregates measuring 100-500 μm is 20% or more of the area of ​​the observed image. ○: The area ratio of cell aggregates measuring 100-500 μm is between 5% and 20% of the area of ​​the observed image. ×: The area ratio of cell aggregates measuring 100-500 μm is less than 5% of the area of ​​the observed image.

[0057] [Example 2] In Example 1, a cell culture substrate was prepared in the same manner as in Example 1, except that the spacing between adjacent recesses in the porous layer and the opening diameter of the recesses were both set to 1 μm. Cells were then cultured and evaluated using the obtained cell culture substrate. The results are shown in Figure 7 and Table 1.

[0058] [Example 3] In Example 1, a cell culture substrate was prepared in the same manner as in Example 1, except that the spacing between adjacent recesses in the porous layer and the opening diameter of the recesses were both set to 10 μm. Cells were then cultured and evaluated using the obtained cell culture substrate. The results are shown in Figure 7 and Table 1.

[0059] [Example 4] In Example 1, a cell culture substrate was prepared in the same manner as in Example 1, except that the spacing between adjacent recesses in the porous layer and the opening diameter of the recesses were both set to 50 μm. Cells were then cultured and evaluated using the obtained cell culture substrate. The results are shown in Figure 7 and Table 1.

[0060] [Example 5] Except for using a mold obtained by photolithography to form groove-shaped recesses 11c with multiple grooves 11m extending in one direction, as shown in Figure 3, and convex portions with the opposite shape, a cell culture substrate was manufactured in the same manner as in Example 1, and cells were cultured and evaluated using the obtained cell culture substrate. The results are shown in Figure 7 and Table 2.

[0061] [Example 6] In Example 5, a cell culture substrate was prepared in the same manner as in Example 5, except that the spacing between adjacent recesses in the porous layer and the opening diameter of the recesses were both set to 1 μm. Cells were then cultured and evaluated using the obtained cell culture substrate. The results are shown in Figure 7 and Table 2.

[0062] [Example 7] In Example 5, a cell culture substrate was prepared in the same manner as in Example 5, except that the spacing between adjacent recesses in the porous layer and the opening diameter of the recesses were both set to 10 μm. Cells were then cultured and evaluated using the obtained cell culture substrate. The results are shown in Figure 7 and Table 2.

[0063] [Example 8] A cell culture substrate was manufactured in the same manner as in Example 1, except that a mold was used in which a groove-shaped recess 11c, composed of multiple intersecting grooves 11m as shown in Figure 4, was formed by photolithography, creating a protrusion with the opposite shape to the groove-shaped recess 11c. Cells were cultured using the obtained cell culture substrate and evaluated. Structural suitability was also evaluated. These results are shown in Figure 7 and Table 2.

[0064] [Example 9] In Example 8, a cell culture substrate was prepared in the same manner as in Example 8, except that the spacing between adjacent recesses in the porous layer and the opening diameter of the recesses were both set to 1 μm. Cells were then cultured and evaluated using the obtained cell culture substrate. The results are shown in Figure 7 and Table 2.

[0065] [Example 10] In Example 8, a cell culture substrate was prepared in the same manner as in Example 8, except that the spacing between adjacent recesses in the porous layer and the opening diameter of the recesses were both set to 10 μm. Cells were then cultured and evaluated using the obtained cell culture substrate. The results are shown in Figure 7 and Table 2.

[0066] [Comparative Example 1] In Example 1, a cell culture substrate was prepared in the same manner as in Example 1, except that a recess was not formed. Cells were then cultured and evaluated using the obtained cell culture substrate. The results are shown in Figure 8 and Table 3.

[0067] [Comparative Example 2] A cell culture substrate was prepared in the same manner as in Example 1, except that polystyrene was used instead of uncured polyorganosiloxane (A). Cells were cultured and evaluated using the obtained cell culture substrate. The results are shown in Figure 8 and Table 3.

[0068] [Comparative Example 3] Cell culture substrates were prepared in the same manner as in Example 2, except that polystyrene was used instead of uncured polyorganosiloxane (A). Cells were cultured and evaluated using the obtained cell culture substrates. The results are shown in Figure 8 and Table 3.

[0069] [Comparative Example 4] A cell culture substrate was prepared in the same manner as in Example 3, except that polystyrene was used instead of uncured polyorganosiloxane (A). Cells were cultured and evaluated using the obtained cell culture substrate. The results are shown in Figure 8 and Table 3.

[0070] [Comparative Example 5] A cell culture substrate was prepared in the same manner as in Example 1, except that a mold was obtained by photolithography, using a groove-shaped recess 11c formed by multiple grooves 11m extending in one direction as shown in Figure 3, and a convex portion with the opposite shape was formed, and polystyrene was used instead of uncured polyorganosiloxane (A). Cells were cultured and evaluated using the obtained cell culture substrate. The results are shown in Figure 8 and Table 4.

[0071] [Comparative Example 6] A cell culture substrate was prepared in the same manner as in Example 1, except that a mold was used to form a groove-shaped recess 11c composed of multiple intersecting grooves 11m as shown in Figure 4, by creating a convex portion with the opposite shape to the groove-shaped recess 11c using photolithography, and polystyrene was used instead of uncured polyorganosiloxane (A). Cells were cultured and evaluated using the obtained cell culture substrate. The results are shown in Figure 8 and Table 4.

[0072] [Table 1]

[0073] [Table 2]

[0074] [Table 3]

[0075] [Table 4]

[0076] The abbreviations used in Tables 1-4 are as follows: • PDMS: Polyorganosiloxane (A) • PS: Polystyrene

[0077] As is clear from the results in Tables 1 and 2 and Figure 7, when cells were cultured using the cell culture substrate obtained in Examples 1 to 10, which consisted of a polyorganosiloxane layer having a porous layer with multiple recesses formed on the culture surface, the cells grew into cell aggregates as multiple three-dimensional aggregates, demonstrating excellent suitability for cell culture. Therefore, the cell culture substrate of the present invention was shown to have excellent oxygen permeability and three-dimensional cell aggregation properties, as well as excellent cell aggregate formation and growth. In contrast, as is clear from the results in Figure 8, the cell culture substrate consisting of a polyorganosiloxane layer without recesses formed on the culture surface obtained in Comparative Example 1 showed cell aggregate formation, but the degree of growth was smaller compared to Examples 1-10, indicating inferior suitability for cell aggregate culture. Furthermore, although not mentioned in the results, the cell culture substrates obtained in Examples 1-6 also exhibited good handling characteristics during operations such as placing them on culture dishes.

[0078] Furthermore, as is clear from Table 1, the results from Examples 1 to 3 show that the cell culture substrate made of polyorganosiloxane layers exhibits excellent cell aggregate formation and growth even when the spacing between adjacent recesses and the opening diameter of the recesses in the porous layer are changed to 1 μm and 10 μm, respectively, which are smaller than the size of the cells. Furthermore, a comparison of the results of Examples 1-3 and Example 4 in Table 1 revealed that the cell culture substrate made of polyorganosiloxane layers exhibits superior cell aggregate culture suitability when the spacing between adjacent recesses and the opening diameter of recesses in the porous layer are smaller than the cell size, compared to when they are larger than the cell size (50 μm).

[0079] Furthermore, the results from Examples 1-3 in Table 1 and Examples 5-10 in Table 2 show that cell culture substrates made of polyorganosiloxane layers exhibit excellent cell aggregate formation and growth even when the shape of the recesses in the porous layer differs. It was also found that the substrates remained excellent for cell aggregate formation and growth even when the recesses were changed to 1 μm and 10 μm, respectively, which are smaller than the size of the cells.

[0080] Although not shown in Tables 1 and 2, in Examples 2, 6, and 9, where the spacing P between the recesses of the polyorganosiloxane layer was 1 μm, the cell-supporting surface of the porous layer was significantly more prone to deformation compared to Examples 1, 5, and 8, where the spacing P was 5 μm. Therefore, from the viewpoint of more stably supporting cells and further promoting cell aggregate formation, it was found that the spacing P between the recesses is preferable to be greater than 1 μm, and more preferably 5 μm or greater.

[0081] In contrast, as is clear from the results in Figure 8, when cells were cultured using the polystyrene cell culture substrates obtained in Comparative Examples 2 to 6, the cells spread out and grew evenly on the culture surface, making it difficult to form cell aggregates, and the cell culture suitability was inferior to that of Examples 1 to 10. [Explanation of symbols]

[0082] 10 Cell culture substrate 11 Polyorganosiloxane layer 11a One side 11b The other side 11c recess 11d Non-recessed 11m groove 111 Porous layer 112 Non-porous layer 12 Reinforcement layer 20-inch 21 Convex part 30 Spacers 40 glass plates P: Distance between adjacent recesses R recess opening diameter T 11 Depth of the recess T 12 Thickness of the nonporous layer Thickness of the T1 polyorganosiloxane layer Thickness of T2 reinforcement layer

Claims

1. It has a polyorganosiloxane layer, A portion or all of one side of the polyorganosiloxane layer is designated as the culture area. The cell culture substrate has two or more recesses that open to one of its surfaces in the culture area.

2. The storage modulus of the polyorganosiloxane constituting the polyorganosiloxane layer, as measured in accordance with JIS K 7224-10, is 5 × 10⁻¹⁰. ３ ~2 x 10 ６ The cell culture substrate according to claim 1, wherein the material is Pa.

3. The cell culture substrate according to claim 1 or 2, wherein the depth of the recess is 0.1 to 5 μm.

4. The cell culture substrate according to claim 1 or 2, wherein the thickness of the polyorganosiloxane layer is 20 to 500 μm.

5. The cell culture substrate according to claim 1 or 2, wherein the distance between adjacent recesses is 1 to 10 μm.

6. The cell culture substrate according to claim 1 or 2, wherein the opening diameter of the recess is 1 to 10 μm.

7. The cell culture substrate according to claim 1 or 2, wherein the recess is a through-hole penetrating the polyorganosiloxane layer.

8. The cell culture substrate according to claim 1 or 2, wherein the recess is a bottomed pore having a bottom within the polyorganosiloxane layer.

9. The cell culture substrate according to claim 1 or 2, wherein the recess is groove-shaped.

10. The cell culture substrate according to claim 1 or 2, wherein the recess is formed by a plurality of intersecting grooves.

11. With additional reinforcement layers, The cell culture substrate according to claim 1 or 2, wherein the reinforcing layer is provided on the other surface of the polyorganosiloxane layer.

12. The cell culture substrate according to claim 11, wherein the reinforcing layer is a nonwoven fabric.

13. The cell culture substrate according to claim 11, wherein the reinforcing layer is a layer of polyorganosiloxane having a larger storage modulus than the polyorganosiloxane constituting the polyorganosiloxane layer.

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