Surface structure for cell culture substrate and cell culture chip

Abstract

To provide a surface structure for a cell culture substrate that exhibits high cell adhesiveness, and a cell culture chip.SOLUTION: A surface structure for a cell culture substrate is a surface on which cells are cultured, and the surface includes, at least in regions where the cells come into contact, recesses that allow biomolecules involved in cell adhesion to penetrate. The surface structure of the cell culture substrate is also a surface on which cells are cultured, and the surface includes, at least in regions where the cells come into contact, a plurality of protrusions that define valleys between the protrusions, the valleys being capable of receiving biomolecules involved in adhesion between the cells and the surface.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] The present invention relates to a surface structure of a cell culture substrate and a cell culture chip. [Background technology]

[0002] In cell culture, adherent cells function after adhering to the surface of a culture vessel. However, most resins that make up culture vessels do not have cell adhesive properties without some kind of surface treatment.

[0003] Therefore, in Patent Document 1 listed below, a hydrophobic polymer surface is formed directly on the surface of a hydrophobic resin or by using a material with a hydrophobic polymer skeleton, and then polymer chains containing a hydrophilic skeleton are grafted onto the surface, thereby obtaining a surface with improved wettability. When L6 cells were seeded and cultured on a 24-well cell culture dish with this surface, it was found that the cell adhesiveness was improved.

[0004] The degree of surface wettability that maximizes cell adhesion varies depending on the type and density of functional groups and the type of cell, but is generally said to be a water contact angle of 40 to 70 degrees. Increasing the surface wettability of cell culture substrates has generally been shown to be a guideline for improving cell adhesion.

[0005] In order to obtain such a moderately hydrophilic surface with excellent cell adhesiveness, in addition to grafting the above-mentioned hydrophilic materials, methods have also been reported in which hydrophilic functional groups are introduced onto the surface by corona discharge treatment (Patent Document 2), plasma treatment (Patent Document 3), VUV treatment (Non-Patent Document 1), etc. [Prior art documents] [Patent documents]

[0006] [Patent Document 1] Japanese Patent Application Laid-Open No. 2009-017809 [Patent Document 2] Japanese Patent Application Publication No. 06-098756 [Patent Document 3] Special Publication No. 2012-144624 [Non-patent literature]

[0007] [Non-Patent Document 1] Applied Surface Science, (2009), 3648-3654, 255(6) [Non-patent document 2] Biosensors, (2020), 10(11),182 [Non-patent document 3] Chemical Engineering Journal 300, (2016), 394-403 Summary of the Invention [Problem to be solved by the invention]

[0008] However, methods such as grafting hydrophilic materials onto resin surfaces directly, as described in Patent Document 1, can impart surface properties that are applicable to a wide range of applications to resins, but the manufacturing process is complicated and requires expensive equipment.

[0009] On the other hand, surface modification methods using high-energy irradiation of surfaces in the atmosphere, such as VUV treatment and plasma treatment, are the primary methods for hydrophilic modification because they are simple to manufacture and require minimal labor. However, physical methods using corona discharge treatment, VUV treatment, and plasma treatment are known to cause hydrophilic surfaces to revert to the original hydrophobic nature of the substrate when left at room temperature (see Non-Patent Documents 2 and 3). Therefore, even if a surface is modified to be hydrophilic, it will revert to a surface with poor cell adhesion if left unattended, making it unsuitable as a substrate for cell culture. Furthermore, when microfluidic devices are fabricated using substrates whose wettability changes over time, it becomes difficult to accurately control the flow of liquid within the device.

[0010] In view of the above problems, an object of the present invention is to provide a surface structure of a cell culture substrate that exhibits high cell adhesiveness and a cell culture chip. [Means for solving the problem]

[0011] The surface structure of the cell culture substrate according to the present invention is a surface structure of the cell culture substrate on which cells are cultured, The surface has recesses, at least in the region where the cells come into contact, that can accommodate biomolecules for cell adhesion to the surface.

[0012] According to this configuration, biomolecules that allow cells to adhere to the surface penetrate into the recesses and adhere to the bottom and side surfaces of the recesses, thereby exhibiting high cell adhesiveness.

[0013] In the surface structure of the cell culture substrate, the recesses preferably have openings with a width of 30 nm or more.

[0014] This configuration allows biomolecules to easily enter the recesses.

[0015] In the surface structure of the cell culture substrate, the recesses preferably have a depth of 2 nm or more.

[0016] This configuration stabilizes the biomolecules within the recesses.

[0017] In the surface structure of the cell culture substrate, the recess preferably has a first groove extending along a first direction.

[0018] Furthermore, in the surface structure of the cell culture substrate, it is preferable that the recessed portion comprises a plurality of the first grooves and a plurality of second grooves extending along a second direction different from the first direction and intersecting with the first grooves.

[0019] These structures can be fabricated using existing methods that are known to be capable of fabricating structures of several nanometers or tens of nanometers in size, so they can be fabricated without searching for a method that can fabricate the structures. Furthermore, by disclosing the present structures, it is possible to select materials that can be used and processes that can fabricate the disclosed structures using the materials. Therefore, it is possible to select simple processes or processes that do not use expensive equipment, which not only allows for fabrication feasibility but also allows for efficient planning of costs, resources, scheduling, and other factors required for mass production.

[0020] The surface structure of the cell culture substrate according to the present invention is a surface structure of the cell culture substrate on which cells are cultured, the surface comprises a plurality of protrusions at least in the region where the cells contact; The plurality of protrusions form valleys between the protrusions that can be penetrated by biomolecules to allow cells to adhere to the surface.

[0021] According to this configuration, biomolecules that allow cells to adhere to the surface penetrate into the valleys between the protrusions and adhere to the side surfaces of the protrusions, thereby exhibiting high cell adhesiveness.

[0022] In the surface structure of the cell culture substrate, the adjacent protrusions are preferably arranged so that the distance between the apexes is 30 nm or more.

[0023] This configuration allows biomolecules to easily enter the recesses between the protrusions.

[0024] In the surface structure of the cell culture substrate, the protrusions preferably have a height of 2 nm or more.

[0025] With this configuration, the biomolecules are stabilized in the recesses between the protrusions.

[0026] In the surface structure of the cell culture substrate, the skewness (Ssk) of the surface is preferably 0.2 or more.

[0027] In addition, in the surface structure of the cell culture substrate, the Spd (Density of peaks) of the surface is 1300 / μm 2 It is preferable that the following configuration is true:

[0028] This method is applicable to processes that can form random patterns on a surface and primarily produce protruding surface structures. Because many of these processes can be performed on relatively large areas, high throughput can be achieved.

[0029] A cell culture chip according to the present invention includes any one of the surface structures of the cell culture substrates described above.

[0030] According to this configuration, biomolecules that allow cells to adhere to the surface penetrate into the recesses or valleys and adhere to the bottom or side surfaces of the recesses or valleys, thereby exhibiting high cell adhesiveness. [Brief explanation of the drawings]

[0031] [Figure 1] Schematic diagram showing the configuration of the surface structure of the cell culture substrate according to the first embodiment. [Figure 2] Schematic diagram showing cells attached to the surface of a cell culture substrate. [Figure 3] Schematic diagram showing the configuration of the surface structure of a cell culture substrate according to a second embodiment. [Figure 4] Schematic diagram showing the configuration of the surface structure of a cell culture substrate according to a third embodiment. [Figure 5] Schematic diagram showing the configuration of the surface structure of a cell culture substrate according to a fourth embodiment. [Figure 6] Schematic diagram showing cells attached to the surface of a cell culture substrate. [Figure 7A] AFM image of the surface without VUV irradiation treatment (untreated) [Figure 7B] AFM image of the surface immediately after VUV irradiation treatment [Figure 8] Graph showing the difference in the number of cells attached to the surface due to differences in VUV irradiation treatment [Figure 9]Graph showing the difference in water contact angle on the surface due to differences in VUV irradiation treatment [Figure 10A] AFM image of the surface without VUV irradiation treatment (untreated) [Figure 10B] AFM image of the surface after 1 minute of VUV irradiation treatment [Figure 10C] AFM image of the surface after 16 minutes of VUV irradiation [Figure 11] Graph showing the change in the water contact angle and cell adhesion number on the surface depending on the VUV irradiation time [Figure 12A] AFM image of the surface without VUV irradiation treatment (untreated) [Figure 12B] AFM image of the surface immediately after VUV irradiation treatment [Figure 13] Graph showing the difference in the number of cells attached to the surface due to differences in VUV irradiation treatment [Figure 14] Graph showing the difference in water contact angle on the surface due to differences in VUV irradiation treatment DETAILED DESCRIPTION OF THE INVENTION

[0032] Embodiments of the surface structure of a cell culture substrate according to the present invention will be described with reference to the drawings. Note that the following drawings are schematic illustrations, and the dimensional ratios in the drawings do not necessarily correspond to the actual dimensional ratios, and the dimensional ratios between the drawings do not necessarily correspond to each other.

[0033] [Surface structure of cell culture substrate] First Embodiment 1 is a schematic diagram showing the configuration of the surface structure of a cell culture substrate 1 according to the first embodiment. The cell culture substrate 1 can be applied to substrates used for cell culture, such as culture vessels, beads, plates, and petri dishes.

[0034] The cell culture substrate 1 is made of, for example, cycloolefin polymer, polyethylene, ultra-high molecular weight polyethylene, polypropylene, polystyrene, polyvinyl chloride, polyvinyl alcohol, acrylic resin, polyethylene terephthalate, polyacetal, polycarbonate, polyamide, polyimide resin, phenol resin, amino resin, epoxy resin, polyester, and acrylonitrile-butadiene-styrene copolymer synthetic resin (ABS resin).

[0035] Cells are cultured on a surface 2 of a cell culture substrate 1. The surface 2 has recesses 3, at least in the region where the cells come into contact, into which biomolecules can enter to allow cells to adhere to the surface 2. A plurality of recesses 3 may be provided.

[0036] Here, the biomolecules that allow cells to adhere to the surface 2 of the cell culture substrate 1 are, for example, biomolecules such as fibronectin and vitronectin, which are extracellular matrices (ECMs) that function for adhesion.

[0037] FIG. 2 is a schematic diagram showing a state in which cells 9 are adhered to the surface 2 of the cell culture substrate 1. The cells 9 have a cell nucleus and pseudopodia. ECM 9a invades the recesses 3 and adheres to the bottom and sides of the recesses 3. The cells 9 seeded on the surface 2 are connected to the ECM 9a that has fallen into the recesses 3 via integrins 9b. This allows the cells 9 to adhere to the surface 2.

[0038] The recess 3 has an opening 3a that opens to the surface 2. The opening 3a is not particularly limited, but may be square. The width A of the opening 3a is preferably 30 nm or more, and more preferably 40 nm or more. If the width A of the opening 3a is 30 nm or more, the ECM 9a can easily penetrate into the recess 3.

[0039] Furthermore, the width A of the opening 3a is preferably 1 μm or less, and more preferably 100 nm or less. When the width A of the opening 3a is 1 μm or less, the recess 3 is easily filled with ECM 9a, and cell adhesiveness is improved.

[0040] The depth B of the recesses 3 is preferably 2 nm or more, and more preferably 2.5 nm or more. When the depth B of the recesses 3 is 2 nm or more, the ECM 9a is stabilized in the recesses 3.

[0041] Second Embodiment The second embodiment has the same configuration as the first embodiment except for the configuration described below, so the commonalities will be omitted and differences will be mainly described. Note that in the second embodiment, elements having substantially the same configuration or substantially the same function (action) as the parts described in the first embodiment will be shown, and their description will not be repeated.

[0042] 3 is a schematic diagram showing the configuration of the surface structure of a cell culture substrate 1 according to the second embodiment. As shown in FIG. 3, the recess 3 may have a first groove 31 extending along a first direction (X direction). The cross section of the first groove 31 is not particularly limited, but is generally triangular. The cross section of the first groove 31 may also be generally rectangular, U-shaped, or the like.

[0043] The recess 3 may have a plurality of first grooves 31 arranged in the Y direction. The number of first grooves 31 is not particularly limited, but the number of cells 9 that adhere depends on the density at which the first grooves 31 are arranged in the Y direction.

[0044] The width A of the opening 3a of the recess 3 is preferably 30 nm or more, and more preferably 40 nm or more. Here, the width A of the opening 3a is the opening width of the first groove 31. The width A of the opening 3a is preferably 1 μm or less, and more preferably 100 nm or less.

[0045] The depth B of the recess 3 (first groove 31) is preferably 2 nm or more, and more preferably 2.5 nm or more.

[0046] <Third embodiment> The third embodiment has the same configuration as the first embodiment except for the configuration described below, so the commonalities will be omitted and differences will be mainly described. Note that in the third embodiment, elements having substantially the same configuration or substantially the same function (action) as the parts described in the first embodiment will be shown, and their description will not be repeated.

[0047] 4 is a schematic diagram showing the configuration of the surface structure of a cell culture substrate 1 according to a third embodiment. As shown in FIG. 4, the recess 3 may include a plurality of first grooves 31 extending along a first direction (X direction) and a plurality of second grooves 32 extending along a direction different from the first direction (Y direction) and intersecting with the first grooves 31. The cross sections of the first grooves 31 and the second grooves 32 are not particularly limited, but are generally triangular. The cross sections of the first grooves 31 and the second grooves 32 may also be generally rectangular, U-shaped, or the like. The cross-sectional shapes of the first grooves 31 and the second grooves 32 may be the same or different.

[0048] The width A of the opening 3a of the recess 3 is preferably 30 nm or more, and more preferably 40 nm or more. Here, the width A of the opening 3a is the opening width of the first groove 31 and the second groove 32. The width A of the opening 3a is preferably 1 μm or less, and more preferably 100 nm or less.

[0049] The depth B of the recess 3 (first groove 31 and second groove 32) is preferably 2 nm or more, and more preferably 2.5 nm or more.

[0050] The surface 2 has a land portion 33 surrounded by a first groove 31 and a second groove 32. The land portion 33 has a substantially quadrangular truncated pyramid shape, although this is not particularly limited. The upper surface of the land portion 33 may be a triangle or a circle, in addition to a rectangle.

[0051] <Fourth embodiment> The fourth embodiment has the same configuration as the first embodiment except for the configuration described below, so the commonalities will be omitted and differences will be mainly described. Note that in the fourth embodiment, elements having substantially the same configuration or substantially the same function (action) as the parts described in the first embodiment will be shown, and their description will not be repeated.

[0052] 5 is a schematic diagram showing the configuration of the surface structure of a cell culture substrate 1 according to the fourth embodiment. The surface 2 has a plurality of protrusions 4 at least in the region that comes into contact with the cells 9. The plurality of protrusions 4 have valleys 5 between the protrusions 4 into which ECM 9a can penetrate to allow the cells 9 to adhere to the surface 2. In other words, it can be said that the protrusions 4 are surrounded by the valleys 5.

[0053] FIG. 6 is a schematic diagram showing a state in which cells 9 have adhered to the surface 2 of the cell culture substrate 1. ECM 9a penetrates into the valleys 5 and adheres to the bottom and side surfaces of the valleys 5 (the sides of the protrusions 4). The cells 9 seeded on the surface 2 are connected to the ECM 9a that has fallen into the valleys 5 via integrins 9b. This allows the cells 9 to adhere to the surface 2.

[0054] The protrusions 4 are not particularly limited, but are conical in shape. The protrusions 4 may have a pointed shape, such as a quadrangular pyramid or a triangular pyramid. The slope of the protrusions 4 from the base to the apex may not be linear, but may be curved.

[0055] The number of protrusions 4 may be one or more, but it is preferable that a plurality of protrusions 4 are gathered together. When a plurality of protrusions 4 are gathered together, the protrusions 4 must be spaced apart to a certain extent. Adjacent protrusions 4 are preferably arranged so that the distance A' between the apexes 4a is 30 nm or more, and more preferably 40 nm or more. If the distance A' between the apexes 4a of the protrusions 4 is 30 nm or more, the ECM 9a can penetrate into the valleys 5.

[0056] Furthermore, the distance A' between the apexes 4a of the protrusions 4 is preferably 1 μm or less, and more preferably 100 nm or less. If the distance A' between the apexes 4a is 1 μm or less, the valleys 5 are easily filled with ECM 9a, thereby increasing cell adhesiveness.

[0057] The height B' of the protrusions 4 is preferably 2 nm or more, and more preferably 2.5 nm or more. If the height B' of the protrusions 4 is 2 nm or more, the ECM 9a is stabilized in the valleys 5.

[0058] Furthermore, the skewness (Ssk) of the surface 2 is preferably 0.2 or more. The skewness (Ssk) is a parameter that indicates the symmetry of the height distribution, and is defined in ISO 25178.

[0059] In addition, the Spd (Density of peaks) of surface 2 is 1300 / μm 2 It is preferable that:

[0060] [Surface structure fabrication method] <Nanoimprint> Nanoimprinting is called thermal nanoimprinting when it uses thermosetting or thermoplastic resins and involves a heat cycle, while photoimprinting when it uses photocurable resins. Photoimprinting often uses ultraviolet light for curing, and is therefore also called UV nanoimprinting.

[0061] In this method, a mold with the negative shape shown in Figures 1 and 3 is used, and the mold is pressed against the material applied to the substrate. In the case of thermal nanoimprinting, the temperature is maintained above the curing temperature Tg of the thermosetting or thermoplastic resin for a certain period of time, and then the mold is slowly cooled while still pressed against the substrate, and the mold is then peeled off, thereby creating a surface structure with cell adhesive properties. This method is advantageous for creating the regular structures shown in Figures 1 and 3.

[0062] <Lithography technology> It is a technology for transferring the circuit pattern of a semiconductor device drawn on a mask master to a resist on a silicon wafer through an exposure apparatus. Currently, lithography with a line width of 10 nm or less has been realized. A resist with a thickness of 2 nm is applied on a substrate having a smooth surface, and the resist at the site where the light of the mask master passes is cured by double patterning of immersion of EUV light or ArF light (193 nm) in the resist, and the uncured resist is removed. By depositing or applying a material to the removed site and removing the resist material, a cell-adhesive surface structure can be produced. This method is also an advantageous method for producing a regular structure shown in FIG. 1, FIG. 3, etc., similar to nanoimprint.

[0063] <VUV irradiation> By performing VUV irradiation at an integrated irradiation dose of about 300 mJ / cm 2 a cell-adhesive surface structure can be produced. This method is an advantageous method for producing the random protrusion structure shown in FIG. 5.

[0064] [Examples] Examples are shown below.

[0065] Sample: The sample used was a cycloolefin polymer (COP) (ZEON INC, ZEONEX 690R) with a size of 1.8 cm × 1.8 cm.

[0066] VUV irradiation treatment: Each sample was subjected to VUV (172 nm) irradiation treatment at 24 °C in an air atmosphere using a VUV irradiation apparatus (Ushio INC, Excimer mini). The VUV irradiation was performed at a distance of 2 mm from the excimer lamp (sample surface illuminance 5.2 mW / cm 2 ) and was carried out so that the integrated irradiation dose became about 300 mJ / cm 2 .

[0067] The surface structure of the sample was measured by AFM. AFM characterization was performed using a scanning probe microscope (Hitachi High-Tech Corporation, L-trace II) in tapping mode under ambient conditions. The cantilever used was an SI-DF40 with aluminum backing (Hitachi High-Tech Corporation). The scan speed was ~1 Hz, the resolution was 512 samples per line, and the measurement area was 5 x 5 μm^2 (512 x 512 pixels). Figures 7A and 7B show AFM images of the surface. The AFM images in Figures 7A and 7B are 0.5 μm x 0.5 μm. Figure 7A shows an AFM image of the surface without VUV irradiation (untreated). Figure 7B shows an AFM image of the surface immediately after VUV irradiation. As shown in Figure 7B, VUV irradiation resulted in the formation of multiple random protrusions on the surface.

[0068] Next, cells (BALB / c 3T3) were seeded on each of the surfaces shown in Figure 7A and Figure 7B, and the number of cells adhering to each surface was investigated. The results are shown in Figure 8. As is clear from the results in Figure 8, the surface on which multiple protrusions were formed by VUV irradiation treatment exhibited higher cell adhesiveness than the untreated surface.

[0069] Furthermore, since increasing the wettability of the surface of a cell culture substrate has generally been shown to be a guideline for improving cell adhesion, we investigated the contact angle of water on surfaces prepared by VUV irradiation treatment. The results are shown in Figure 9. The contact angle of water on surfaces treated with VUV irradiation was approximately 40°, a result consistent with previous guidelines. As described in Non-Patent Documents 2 and 3, for example, it is known that surfaces rendered hydrophilic by VUV irradiation return to hydrophobicity over time. The degree of hydrophobicity recovery varies depending on the storage environment of the sample. The water contact angle of the surface decreases to approximately 40° (water contact angle 24 hours after irradiation) as shown on the right side of Figure 9 upon VUV irradiation. However, after leaving the surface in the atmosphere at a predetermined temperature for a predetermined time, it returns to approximately 90°. Observation of the surface with restored hydrophobicity revealed a structure similar to that of the surface immediately after VUV irradiation, as shown in Figure 7B. The high cell adhesiveness of the surface with restored hydrophobicity was also similar to that of the surface immediately after VUV irradiation. This indicates that the high cell adhesiveness is attributable to the surface structure.

[0070] Here, we explain the change in surface structure with VUV irradiation time. The above sample was irradiated with VUV (172 nm) at 24°C in an air atmosphere using a VUV irradiation device (Ushio INC, Excimer mini). VUV irradiation was performed at a distance of 2 mm from the excimer lamp (sample surface illuminance: 5.2 mW / cm2). Figures 10A to 10C show the change in surface structure with VUV irradiation time. Figure 10A is an AFM image of a surface that was not irradiated with VUV (untreated). Figure 10B is an AFM image of a surface that was irradiated with VUV for 1 minute. Figure 10C is an AFM image of a surface that was irradiated with VUV for 16 minutes. The AFM images in Figures 10A to 10C are 0.5 μm × 0.5 μm.

[0071] As shown in Figure 10B, after 1 minute of VUV irradiation, multiple random protrusions were formed on the surface. However, as shown in Figure 10C, after 16 minutes of VUV irradiation, the protrusions disappeared, resulting in a structure similar to that of an untreated surface. This is presumably because the cell culture substrate 1 is made of a polymer resin, and there are areas on the surface that are prone to evaporation (e.g., areas with many single-bond molecular chains) and areas that are difficult to evaporate (e.g., areas with many double-bond molecular chains). Specifically, VUV irradiation initially evaporates the areas that are prone to evaporation, forming protrusions in the areas that are difficult to evaporate. However, as VUV irradiation continues, the areas that are difficult to evaporate also evaporate, resulting in the disappearance of the protrusions. Therefore, to form a surface structure with multiple protrusions as shown in Figure 10B, VUV irradiation must be performed for an appropriately set short period of time.

[0072] Figure 11 shows the change in the water contact angle and the number of attached cells on the surface as a function of VUV irradiation time. As shown in Figure 11, the water contact angle decreases as the VUV irradiation time increases. Therefore, VUV irradiation has been performed for a long time (16 minutes in this example) to improve the surface wettability. However, when VUV irradiation is performed for a long time, the surface protrusions disappear, as shown in Figure 10C, and it is presumed that cell adhesion decreases.

[0073] Below, an example will be shown in which polystyrene (PS) is used as a sample. Sample: The sample used was polystyrene (PS) measuring 1.8 cm x 1.8 cm.

[0074] VUV irradiation treatment: Each sample was irradiated with VUV (172 nm) at 24°C in an air atmosphere using a VUV irradiation device (Ushio INC, Excimer mini). VUV irradiation was performed at a distance of 2 mm from the excimer lamp (sample surface illuminance 5.2 mW / cm). 2 ) with an accumulated irradiation dose of approximately 300 mJ / cm 2 I went there to make it so.

[0075] AFM images of the obtained surface are shown in Figures 12A and 12B. The AFM images in Figures 12A and 12B are 0.5 μm × 0.5 μm. Figure 12A is an AFM image of the surface that was not subjected to VUV irradiation treatment (untreated). Figure 12B is an AFM image of the surface immediately after VUV irradiation treatment. As shown in Figure 12B, multiple random protrusions were formed on the surface by VUV irradiation treatment.

[0076] Like Figure 8, Figure 13 shows the difference in the number of cells adhering to the surface due to different VUV irradiation treatments. Like Figure 9, Figure 14 shows the difference in the water contact angle of the surface due to different VUV irradiation treatments. As can be seen from Figure 14, VUV irradiation treatment reduces the water contact angle of the surface to approximately 20°. Furthermore, although the water contact angle of the surface decreases to approximately 20° due to VUV irradiation, it returns to approximately 50° when left in the air at a predetermined temperature for a predetermined time. When the surface with restored hydrophobicity was observed, it had a structure similar to the surface immediately after VUV irradiation treatment shown in Figure 12B.

[0077] On the other hand, as can be seen from Figure 13, the surface treated with VUV irradiation exhibited higher cell adhesion than the untreated surface. Furthermore, the level of cell adhesion of the surface whose hydrophobicity was restored was almost the same as that of the surface immediately after VUV irradiation. This indicates that the high cell adhesion of polystyrene (PS) is also attributable to the surface structure. Thus, the same tendency was observed for polystyrene (PS) as for cycloolefin polymer (COP). Therefore, it is presumed that, similar to cycloolefin polymer (COP) and polystyrene (PS), multiple random protrusions can be formed on the surface of the aforementioned other polymer resins by irradiating them with VUV for an appropriately set short period of time, resulting in high cell adhesion.

[0078] Although the embodiments of the present invention have been described above with reference to the drawings, the specific configurations should not be considered to be limited to these embodiments. The scope of the present invention is defined not only by the description of the above embodiments but also by the claims, and further includes all modifications within the meaning and scope of the claims.

[0079] The structures employed in the above-described embodiments can be employed in any other embodiment. The specific configurations of the components are not limited to the above-described embodiments, and various modifications are possible within the scope of the present invention.

[0080] The above examples are for the cell culture substrate 1 according to the fourth embodiment, but it is presumed that the penetration of ECM9a into the recesses 3 in the cell culture substrates 1 according to the first to third embodiments also produces effects similar to those produced when ECM9a penetrates into the valleys 5 between multiple protrusions 4. [Explanation of symbols]

[0081] 1: Substrate for cell culture 2: Surface 3: Recess 3a:Aperture 4: Protrusion 4a: Vertex 5: Valley 9: Cell 9a: ECM 9b: Integrin 31: 1st groove 32: 2nd groove 33: Rikube

Claims

1. A surface structure of a cell culture substrate on which cells are cultured, The surface structure of the cell culture substrate has recesses in at least the region where cells come into contact, into which biomolecules can enter to allow cells to adhere to the surface.

2. The surface structure of the cell culture substrate according to claim 1 , wherein the recesses have openings with widths of 30 nm or more.

3. The surface structure of the cell culture substrate according to claim 1 , wherein the recesses have a depth of 1.5 nm or more.

4. The surface structure of the cell culture substrate according to claim 1 , wherein the recessed portion comprises a first groove extending along a first direction.

5. The surface structure of the cell culture substrate according to claim 4, wherein the recess comprises a plurality of the first grooves and a plurality of second grooves extending along a second direction different from the first direction and intersecting the first grooves.

6. A surface structure of a cell culture substrate on which cells are cultured, the surface comprises a plurality of protrusions at least in the region where the cells contact; The surface structure of the cell culture substrate, wherein the plurality of protrusions form valleys between the protrusions into which biomolecules can penetrate to allow cells to adhere to the surface.

7. The surface structure of the cell culture substrate according to claim 6 , wherein the adjacent protrusions are arranged so that the distance between their apexes is 30 nm or more.

8. The surface structure of the cell culture substrate according to claim 6 or 7, wherein the protrusions have a height of 1.5 nm or more.

9. The surface structure of the cell culture substrate according to claim 6 or 7, wherein the skewness (Ssk) of the surface is 2 nm or more.

10. The surface Spd (Density of peaks) is 100 / μm ２ The surface structure of the cell culture substrate according to claim 6 or 7, wherein the surface structure is as described above.

11. A cell culture chip comprising the surface structure of the cell culture substrate according to claim 1 or 2.

12. A cell culture chip comprising the surface structure of the cell culture substrate according to claim 6 or 7.

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