Cell patterning method and cell patterning device

Abstract

Provided is a cell patterning method and a cell patterning device which make it possible to control cell proliferation and to easily pattern the shape of a cell population. The cell patterning method comprises: a step for allowing cells to adhere to a cell cultivation substrate 1, on one surface thereof having a first region 11 which blocks ultraviolet light and a second region 12 which allows ultraviolet light to pass through; and a step for irradiating the cell cultivation substrate 1 with ultraviolet light, from the surface opposite to the side that has the cells adhered thereto. Among the cells adhered to the cell cultivation substrate, cells on the second region 12 are removed by the step for irradiating ultraviolet light.

Description

Cell patterning method and cell patterning device

[0001] The present invention relates to a cell patterning method and a cell patterning device.

[0002] For quantitative cell-based experiments such as drug evaluation, it is necessary to form cellular tissues consisting of cell populations containing a certain number of cells and to precisely control the size of the cellular tissues. One known method for controlling cellular tissues is cell patterning, which controls the cell adhesion area by dividing the surface of a substrate into adhesive and non-adhesive areas.

[0003] For example, a technique is known in which a microstructured 2-methacryloyloxyethyl phosphorylcholine (MPC) polymer film is molded into a polydimethylsiloxane (PDMS) micromold formed on a silicon wafer by pouring an ethanol solution of MPC polymer into the mold, thereby forming a desired pattern (see, for example, Non-Patent Document 1). The areas covered with MPC polymer are areas to which cells do not adhere, and the areas not covered with MPC polymer are areas to which cells do adhere, thereby producing a microdevice for cell patterning that is separated into areas to which cells adhere and areas to which cells do not adhere.

[0004] N. Tanaka, et al., Vacuum microcasting of 2-methacryloyloxyethylphosphorylcholine polymer for stable cell patterning, BioTechniques, Vol. 69, No. 3, 2020

[0005] When culturing adherent cells, the surface of a cell culture substrate may be coated with cell adhesion / growth factors such as extracellular matrix (ECM) (e.g., collagen or fibronectin). However, in the aforementioned Non-Patent Document 1, an MPC polymer is used to form a pattern that separates areas where cells adhere and areas where they do not, making it difficult to use a coating agent for cell adhesion / growth, such as ECM, in combination.

[0006] The present invention has been made in consideration of the above-described circumstances, and its purpose is to provide a cell patterning method and a cell patterning device that can perform good cell patterning according to various cells.

[0007] According to one embodiment, the present invention relates to a cell patterning method, comprising the steps of: attaching cells to one surface of a cell culture substrate having a first region that blocks ultraviolet light and a second region that transmits ultraviolet light; and irradiating ultraviolet light from the surface of the cell culture substrate opposite to the surface to which the cells are attached, wherein the ultraviolet light irradiation step removes cells on the second region from among the cells attached to the cell culture substrate.

[0008] According to another embodiment, the present invention relates to a cell patterning device comprising: a cell culture substrate having a first region that blocks ultraviolet light and a second region that transmits ultraviolet light; and an ultraviolet irradiation device that can irradiate the cell culture substrate with ultraviolet light, wherein the cell patterning device removes cells on the second region from among cells attached to the cell culture substrate by irradiating the ultraviolet light using the ultraviolet irradiation device.

[0009] According to yet another embodiment, the present invention relates to a method for producing a patterned cell population, comprising: attaching cells to one surface of a cell culture substrate having a first region that blocks ultraviolet light and a second region that transmits ultraviolet light; and culturing the attached cells, wherein the culturing step comprises irradiating the cells with ultraviolet light from the surface of the cell culture substrate opposite to the surface to which the cells are attached.

[0010] According to the present invention, it is possible to provide a cell patterning method and cell patterning device, which can control cell proliferation and easily pattern the shape and size of cellular tissue consisting of the cell population obtained by proliferation, as well as a method for producing a patterned cell population.

[0011] 1(a) and 1(b) are a top view and a cross-sectional view schematically illustrating the configuration of a cell culture substrate used in a cell patterning method according to one embodiment of the present invention. FIG. 2 is a diagram illustrating the schematic configuration of a cell patterning device according to one embodiment. FIGS. 3(a) to 3(d) are diagrams illustrating the flow of a cell patterning method according to one embodiment. FIG. 4 is a graph showing the relationship between the wavelength of irradiated light and the transmittance for various materials. FIG. 5 shows (a) a state immediately after cells are seeded on a cell culture substrate without a second layer, and (b) a state immediately after cells are seeded on a cell culture substrate with no second layer, and (c) a state immediately after cells are seeded on a cell culture substrate with no second layer, and (d) a state immediately after cells are seeded on a cell culture substrate with no second layer, and (e) a state immediately after cells are seeded on a cell culture substrate with no second layer, and (f ... ２ (b) is a fluorescence image of the cell culture substrate on the fourth day after culturing, with the cumulative exposure dose calculated using the energy amount of light at a wavelength of 254 nm. Figure 6 shows the results of (a) immediately after seeding cells on the cell culture substrate provided with the second layer containing Poly-D-Lysine, and (b) immediately after seeding cells on the second layer provided with Poly-D-Lysine, with the cumulative exposure dose of ultraviolet light at 0 mJ / cm. ２ 7 shows a fluorescence image of the cell culture substrate (a) immediately after seeding cells on a cell culture substrate without a second layer, and (b) on the fourth day after culturing with an integrated dose of ultraviolet light of 1038 mJ / cm. ２ 8 shows a fluorescence image of the cell culture substrate on the fourth day after culturing (b) with the second layer containing collagen, taken immediately after seeding the cells (a), and after culturing the cells with the second layer containing collagen at an integrated dose of ultraviolet light of 1038 mJ / cm. ２ FIG. 9 shows a fluorescence image of the cell culture substrate on the fourth day after culturing (b) when the diameter X of the first region, which is the cell proliferation area, was changed and the cell patterning method was performed. FIG. 10 shows the results of evaluating the cell proliferation rate in the first region and the number of cells binding to the second region when no ultraviolet light was irradiated. FIG. 11 shows the results of evaluating the cell proliferation rate in the first region and the number of cells binding to the second region when the integrated ultraviolet light exposure was 519 mJ / cm. ２The results of evaluating the cell proliferation rate in the first region and the number of cells bound to the second region when the cumulative exposure dose of ultraviolet light was 1038 mJ / cm ２ 13 is a diagram illustrating the design of a cell culture substrate having a honeycomb-shaped cell growth area in which the first region is a regular hexagon and multiple regular hexagons are connected with the second region as a boundary. FIG. 14 is an image of a cell culture substrate actually manufactured based on the design drawing of FIG. 13 , showing the honeycomb-shaped cell growth area (a) and an enlarged image (b) of region Z in (a). FIG. 15 is a diagram illustrating the cell culture substrate (a) immediately after seeding cells on the cell culture substrate having the honeycomb-shaped cell growth area shown in FIG. 14 and the cell culture substrate (b) immediately after seeding cells on the honeycomb-shaped cell growth area shown in FIG. 14 and the cell culture substrate (b) after seeding with cells with an integrated dose of ultraviolet light of 0 mJ / cm. ２ 16 shows a fluorescence image of the substrate immediately after seeding cells on the same cell culture substrate as in FIG. 15 (a), and on the second day after culturing with an integrated dose of ultraviolet light of 2076 mJ / cm (b). ２ 17 shows a fluorescence image of the substrate on the second day after culturing with the substrate set at 0 mJ / cm after the cumulative exposure of ultraviolet light (a) and immediately after seeding cells on a cell culture substrate having a single hexagonal cell growth area, in which only one regular hexagonal first region is provided and a third region is provided around the second region (b). ２ 18 shows a fluorescence image of the substrate immediately after seeding cells on the same cell culture substrate as in FIG. 17 (a), and on the second day after culturing with an integrated dose of ultraviolet light of 2076 mJ / cm (b). ２ 19 shows the results of evaluating the cell proliferation rate in the first region and the number of cells bound to the second region when the design value A of the circumscribed circle diameter of the first region is set to 750 μm in a cell culture substrate similar to that shown in FIG. 14 , and the cumulative exposure dose of ultraviolet light is 0 mJ / cm. ２ (a), 1038 mJ / cm ２ (b), and 2076 mJ / cm ２Fig. 20 shows the results of evaluating the cell proliferation rate in the first region and the number of cells bound to the second region when the design value A of the circumscribed circle diameter of the first region is set to 750 µm in a cell culture substrate similar to that in Fig. 17, and the cumulative exposure dose of ultraviolet light is 0 mJ / cm. ２ (a), 1038 mJ / cm ２ (b), and 2076 mJ / cm ２ 21 shows the results of evaluating the cell proliferation rate in the first region and the number of cells bound to the second region when the design value A of the circumscribed circle diameter of the first region is set to 450 μm in a cell culture substrate similar to that in FIG. 14 , and the cumulative exposure dose of ultraviolet light is 0 mJ / cm ２ (a), 1038 mJ / cm ２ (b), and 2076 mJ / cm ２ 22 shows the results of evaluating the cell proliferation rate in the first region and the number of cells bound to the second region when the design value A of the circumscribed circle diameter of the first region is set to 450 μm in a cell culture substrate similar to that in FIG. 17 , and the cumulative exposure dose of ultraviolet light is 0 mJ / cm ２ (a), 1038 mJ / cm ２ (b), and 2076 mJ / cm ２ (c) Fluorescence image of the substrate.

[0012] The cell patterning method and cell patterning device according to one embodiment of the present invention perform cell patterning by controlling the cell adhesion area using a cell culture substrate whose surface is divided into areas where cells adhere and areas where cells do not adhere. The control of the cell adhesion area is achieved by forming an ultraviolet-shielding thin film on the substrate and irradiating it with localized ultraviolet light.

[0013] Specifically, cells are attached to a cell culture substrate having an ultraviolet-shielding region that blocks ultraviolet light and an ultraviolet-transmitting region that transmits ultraviolet light, and ultraviolet light is irradiated onto the cell culture substrate. The ultraviolet light irradiation removes cells on the ultraviolet-transmitting region from among the cells attached to the cell culture substrate, thereby performing cell patterning.

[0014] A cell patterning method and a cell patterning device according to one embodiment will be described in detail below with reference to the drawings. Fig. 1(a) is a top view schematically showing the configuration of a cell culture substrate according to one embodiment, and Fig. 1(b) is a cross-sectional view taken along line A-A in Fig. 1(a).

[0015] 1(a) and 1(b), the cell culture substrate 1 has a first region 11, which is an ultraviolet-shielding region that blocks ultraviolet light, and a second region 12, which is an ultraviolet-transmitting region that transmits ultraviolet light, and is configured so that cells attached to the cell culture substrate 1 can be locally irradiated with ultraviolet light without precise alignment. The cell culture substrate 1 may further have a third region 13, which is an ultraviolet-shielding region. In this specification, the shape formed by the combination of one first region 11 (and optionally the third region 13) and the second region 12 is also referred to as a pattern.

[0016] The first region 11 is a cell growth area for forming a cell colony and is formed, for example, in a circular shape with a diameter X. However, the shape of the first region 11 is not limited thereto, and it can also be a polygon, including a regular polygon such as a regular hexagon. The shape and size of the first region 11 can be appropriately determined by those skilled in the art to correspond to the desired shape and size of the formed cell colony. When the first region 11 is circular, the lower limit of the diameter X is not particularly limited and can be appropriately determined depending on the number of cells to be grown, etc. For example, a single cell may be placed in the first region 11, in which case the diameter X may be approximately 10 μm. When multiple cells are placed in the first region 11, the diameter X may be, for example, approximately 50 μm or more, or may be 100 μm or more, and preferably approximately 150 μm or more. The upper limit of the diameter X is not particularly limited, but can be, for example, approximately 1000 μm. Even when the shape of the first region 11 is other than a circle, the size of the first region 11 can be determined so that the first region 11 has an area similar to that of the circle. For example, when the shape of the first region is a regular polygon, the diameter of the circumscribing circle of the regular polygon can be set to the above value.

[0017] The second region 12 is an area that contacts the outer periphery of the first region 11 and defines the boundary of the first region 11. The second region 12 is formed, for example, in a ring shape with a width Y to match the shape of the first region 11. The third region 13 is optionally provided in an area outside the second region 12 and may be a region that can replace the first region 11. The width Y is not limited to a specific width as long as it is wide enough to prevent contact between cells cultured in the first region 11 and cells cultured in an area outside the second region 12. In certain embodiments, the width Y is preferably a width that allows ultraviolet light to pass through and can be wider than the wavelength of the ultraviolet light to be irradiated. For example, when 254 nm ultraviolet light is used for irradiation, the lower limit of the width Y may be approximately 300 nm. Theoretically, the upper limit of the width Y is not particularly limited.

[0018] Although FIG. 1( a) shows only one first region 11, i.e., one cell growth area, the number of cell growth areas provided on the cell culture substrate 1 is not limited to one. The cell culture substrate 1 may be configured to have multiple first regions 11 and multiple second regions 12 that define their boundaries, thereby providing multiple cell growth areas. When multiple cell growth areas are provided, the first regions 11 and second regions 12 may be identical in shape and size or may be different. An example of a pattern having multiple first and second regions of the same shape and size is a pattern in which the first region is a regular hexagon and the second region is a honeycomb shape in which multiple regular hexagons are connected together, with the second region as a boundary. Furthermore, multiple different patterns may be provided on a single cell culture substrate 1. A third region 13 may occupy the space between one second region 12 and another second region 12.

[0019] As shown in FIG. 1(b), the cell culture substrate 1 includes a substrate 10 that transmits visible light and ultraviolet light, a first layer 20 formed on the substrate 10 and having a portion with ultraviolet light blocking properties, and a second layer 30 formed on the first layer 20 for supporting cell growth.

[0020] The substrate 10 is not particularly limited as long as it is a material that transmits visible light and ultraviolet light, but is selected from materials suitable for patterning the first layer 20. For example, quartz glass is preferable. The substrate 10 is preferably a flat member with a substantially uniform thickness, and its thickness is not particularly limited. Considering that the thicker the substrate 10, the lower its ultraviolet transmittance, the thickness of the substrate 10 can be, for example, approximately 1 mm. The length and width dimensions of the substrate 10 are not particularly limited, but can be, for example, approximately the same as a regular glass slide (length approximately 76 mm, width approximately 26 mm) in consideration of cell observation using a microscope.

[0021] The first layer 20 has a first portion 21 in which a material having ultraviolet ray blocking properties is disposed, corresponding to the first region 11, and a second portion 22 in which a material having ultraviolet ray blocking properties is not disposed, corresponding to the second region 12. The first portion 21 is a portion having ultraviolet ray blocking properties and can also be referred to as an ultraviolet ray blocking layer. The second portion 22 is provided around the first portion 21 so as to define the boundary of the first region 11. Furthermore, the first layer 20 may have a third portion 23 in which a material having ultraviolet ray blocking properties is disposed, corresponding to the third region 13. The third portion 23 is also a portion having ultraviolet ray blocking properties and can also be referred to as an ultraviolet ray blocking layer.

[0022] Ultraviolet light generally has a wavelength of approximately 10 nm to 400 nm. Light with a wavelength of approximately 315 nm or less is classified as UVB (wavelength of approximately 280 to approximately 315 nm) or UVC (wavelength of approximately 200 to approximately 280 nm), and generally has a bactericidal effect. In one embodiment, the material with UV-blocking properties is preferably selected from materials that have an absorption peak for the wavelength of UV light emitted from the UV irradiation device described below (e.g., wavelengths of approximately 315 nm or less corresponding to UVB or UVC). A material suitable for patterning the first layer 20 is also preferably selected. Furthermore, the material with UV-blocking properties is preferably transparent so as not to interfere with cell observation. Here, transparency means that the transmittance of light necessary for cell observation, such as visible light with a wavelength of approximately 360 nm to 830 nm and excitation light / fluorescence with a wavelength of approximately 340 to 740 nm, is preferably 70% or more, more preferably 80% or more, and more preferably 90% or more. An example of a material having such ultraviolet blocking properties is indium tin oxide (ITO). The thickness of the ITO can also be selected from the viewpoints of ultraviolet blocking properties and transmittance of observation light. In particular, it is preferable that the thickness of the ITO is about 4000 Å (400 nm) or more and about 30000 Å (3000 nm) or less, but there is no particular thickness limitation as long as it can absorb and block ultraviolet rays. As an example, another material having ultraviolet blocking properties of about 254 nm is silicon dioxide (SiO ２ In the following, an example in which ITO is used as a material having ultraviolet blocking properties will be described.

[0023] The formation of the UV-shielding layer constituting the first portion 21 or the third portion 23 on the substrate 10 is performed using a method appropriate for the material having UV-shielding properties. When forming an ITO film as the first portion 21 or, optionally, the third portion 23, methods such as sputtering or photolithography, which are used in semiconductor manufacturing techniques, can be used. As a specific example of a manufacturing method, an ITO film is formed by sputtering in the area of ​​the substrate 10 where the pattern is to be formed, a resist is applied to the ITO film, and then exposure and development are performed to remove the resist in the area corresponding to the second region 12 of the pattern. The ITO from the area where the resist was removed is removed with hydrochloric acid, and finally, the resist remaining in the first region 11 and, optionally, the third region 13 is removed with a remover. This results in the formation of a UV-shielding layer of the desired shape on the substrate 10, including the first portion 21 and, optionally, the third portion 23. Note that ITO, which is cited as an example of a material having UV-shielding properties in the present invention, does not function as an electrode. That is, the first portion 21 and the third portion 23 where the ITO is arranged are not connected to a power source by wiring or the like, and are not configured to be able to apply a voltage.

[0024] The second layer 30 is an optional layer provided to support cell adhesion and proliferation in the first region 11 and can also be referred to as a layer made of a coating material. The second layer 30 can be formed, for example, by placing the substrate 10 with the above-described ultraviolet-shielding layer formed thereon in a container such as a petri dish with the ultraviolet-shielding layer facing downward, and then introducing a coating agent, for example, dissolved in ultrapure water, between the surface of the petri dish and the substrate 10, thereby adhering the coating agent to the surface of the substrate. As shown in FIG. 1( b), the second layer 30 is disposed on and in contact with the first portion 21 and the third portion 23 of the first layer 20. The second layer 30 is also disposed on the second portion 22, and in the second region 12, the coating material is also filled in the second portion 22 and contacts the substrate 10. The coating material constituting the second layer 30 can be selected appropriately depending on the cell type to be cultured. Examples include extracellular matrices (ECMs) such as collagen and fibronectin, and Matrigel, a reconstituted basement membrane isolated from EHS mouse Engelbreth-Holm-Swarm sarcoma cells. Alternatively, the second layer 30 need not be formed; instead, the first layer 20 can be surface-modified by plasma treatment or other methods without forming the second layer 30. Surface modification of the first layer 20 by plasma treatment can be combined with the formation of a second layer 30 made of collagen or other materials. The thickness of the second layer 30 on the first portion 21 and the third portion 23 is not particularly limited as long as it supports cell adhesion and proliferation. For example, it can be approximately 0.1 to 3 μm, and more preferably approximately 0.1 to 1 μm. The second layer 30 constitutes the surface of the cell culture substrate 1 that comes into contact with cells and functions as the surface on which the cells are seeded. When the second layer 30 is provided, the surface on which the cells are seeded is preferably a flat surface, but is not particularly limited thereto.

[0025] Depending on the cell type to be cultured and the culture environment, the second layer 30 may not be provided. In a cell culture substrate (not shown) without a second layer, the first region may be a region in which a substrate and a first portion (ultraviolet-shielding layer) are stacked in this order perpendicular to the main surface of the cell culture substrate, and the second region may consist of only the substrate. The third region, which may be optionally provided, may be a region in which a substrate and a third portion (ultraviolet-shielding layer) are stacked in this order. In this case, the surface on which the cells are seeded may have irregularities due to the first and third portions. Although not shown, the second layer may also be formed on the surface of the substrate opposite to the surface on which the first layer is formed. In this case, the second layer can be formed directly on the substrate, and cells can be seeded so that they are in contact with the second layer. The formation method for forming the second layer on the surface of the substrate opposite to the surface on which the first layer is formed can be the same as the formation method for forming the second layer on the first layer, except that the substrate on which the ultraviolet-shielding layer is formed is placed in a container such as a petri dish with the ultraviolet-shielding layer facing upward.

[0026] Next, the configuration of a cell patterning device for performing cell patterning using a cell culture substrate 1 will be described with reference to Fig. 2. As shown in Fig. 2, the cell patterning device 100 is composed of the cell culture substrate 1 and an ultraviolet irradiation device 110 capable of irradiating the cell culture substrate 1 with ultraviolet light UV. As described above in detail, the ultraviolet light UV emitted from the ultraviolet irradiation device 110 is not particularly limited as long as it has a wavelength that can kill cells when irradiated onto them, but for example, the device is configured to irradiate ultraviolet light with a wavelength of approximately 254 nm.

[0027] 2 , the cell culture substrate 1 is placed vertically below the ultraviolet irradiation device 110. Therefore, the cell culture substrate 1 is placed in a container 120 such as a cell culture dish, with the substrate 10 on the upper side and the second layer 30 to which the cells 50 are attached on the lower side. The container 120 is configured to be able to hold a culture medium 121 and is provided with a support member 122 made of, for example, silicone rubber. The cell culture substrate 1 placed in the container 120 is supported by the support member 122, and at this time, the second layer 30 to which the cells 50 are attached comes into contact with the culture medium 121.

[0028] The ultraviolet rays UV emitted from the ultraviolet irradiation device 110 are incident on the substrate 10 side of the cell culture substrate 1 placed in the container 120. That is, the ultraviolet rays UV are incident on the surface opposite to the surface of the cell culture substrate 1 on which the cells 50 are attached. The ultraviolet rays UV incident on the cell culture substrate 1 are transmitted through the second region 12, which is an ultraviolet-transmitting region, and are blocked by the first region 11, which is an ultraviolet-blocking region, so that the cells are not irradiated with the ultraviolet rays.

[0029] 2, the cell culture substrate 1 is disposed vertically below the ultraviolet irradiation device 110, but this is not limiting and the cell culture substrate 1 may be disposed vertically above the ultraviolet irradiation device 110. In this case, the cell culture substrate 1 is disposed with the substrate 10 on the bottom side and the second layer 30 to which the cells 50 are attached on the top side so that ultraviolet light UV is incident from the substrate 10 side.

[0030] Next, the flow of a cell patterning method using the cell patterning device 100 will be described with reference to FIGS.

[0031] First, as shown in Figure 3(a), a cell culture substrate 1 is prepared, which has a first region 11 that blocks ultraviolet light UV and a second region 12 that transmits ultraviolet light UV. The cell culture substrate 1 may also have a third region 13 that blocks ultraviolet light UV. As described above, the cell culture substrate 1 includes a first layer 20 and a second layer 30 formed on a substrate 10.

[0032] Next, as shown in FIG. 3( b), cells 50 are seeded on the cell culture substrate 1 and allowed to adhere to the second layer 30. At this time, it is preferable that the cells 50 are seeded approximately uniformly in the first region 11, the second region 12, and optionally the third region 13. The number and concentration of cells seeded may vary depending on the purpose and are not particularly limited. As an example, cells may be seeded so that only one cell is placed in the first region 11. As another example, when a cell population is seeded using a pipette or the like, for example, 10 or more, or 100 or more cells can be seeded in the first region 11. Assuming that 1 mL of cell suspension is used, the concentration can be converted to approximately 1.0 × 10 ３ For example, the concentration can be about 1.0 × 10 cells / mL or more. １０ cells / mL or less, preferably about 1.0 x 10 ６ cells / mL or less, or about 1.0 x 10 ５ The concentration can be about 1000 cells / mL or less. A culture medium may be included as one type of solution used for seeding.

[0033] When multiple first regions 11 are provided on one cell culture substrate 1, the same cells can be seeded simultaneously in the multiple first regions depending on the purpose. Alternatively, different cells can be seeded in each of the multiple first regions 11. In this case, the number of cells to be placed in the first region and the cell concentration to be seeded may be the same or different among the multiple first regions 11 depending on the purpose.

[0034] During the subsequent culture period, as shown in FIG. 3( c), the cell culture substrate 1 is irradiated with ultraviolet light UV for a predetermined time using the ultraviolet irradiation device 110 described above. The ultraviolet light UV is incident on the substrate 10 side of the cell culture substrate 1. The incident ultraviolet light UV is blocked by the first portion 21 and the third portion 23 of the first layer 20, where ITO is disposed, and is transmitted through the second portion 22, where ITO is not disposed. Cells 50 attached to the second region 12 corresponding to the second portion 22 of the first layer 20 are killed by the incident ultraviolet light UV and removed from the cell culture substrate 1. On the other hand, cells 50 attached to the first region 11 corresponding to the first portion 21 of the first layer 20 and the third region 13 corresponding to the optional third portion 23 are not irradiated with ultraviolet light UV, and therefore the cells 50 remain attached and are cultured and proliferate on the cell culture substrate 1.

[0035] The cell culture substrate 1 is periodically irradiated with ultraviolet light UV during the culture period. The periodic irradiation conditions for ultraviolet light UV, such as irradiation time and frequency, can be determined appropriately depending on the cell type, chip design, etc. As a result, as shown in Figure 3(d), the second region 12 is used as the boundary, so cells 50 on the region 12 are removed, and a cell colony can be formed in the first region 11, which is the cell proliferation area.

[0036] According to the cell patterning method and cell patterning device 100 according to the embodiment described above, the following effects can be achieved.

[0037] The cell patterning device 100 includes a cell culture substrate 1 having a first region 11 that blocks ultraviolet rays UV and a second region 12 that transmits ultraviolet rays UV, and an ultraviolet irradiation device 110 that can irradiate ultraviolet rays UV onto the cell culture substrate 1, and is configured to remove cells 50 on the second region 12 of the cells 50 attached to the cell culture substrate 1 by irradiating ultraviolet rays UV using the ultraviolet irradiation device 110.

[0038] By irradiating the cell culture substrate 1 with cells 50 attached thereto with ultraviolet light UV, only the cells 50 on the second region 12 that transmits ultraviolet light UV can be removed, while the cells 50 on the first region 11 that blocks ultraviolet light UV can be left unremoved. In this way, by irradiating the cell culture substrate 1 locally with ultraviolet light UV and controlling the irradiation range of ultraviolet light UV, the cell proliferation range can be limited, and good cell patterning can be achieved.

[0039] The cell culture substrate 1 includes a substrate 10 that transmits visible light and ultraviolet (UV) light, and a first layer 20 formed on the substrate 10 and including a portion made of a material with ultraviolet blocking properties. The material with ultraviolet blocking properties may be ITO, and the first layer 20 of the cell culture substrate 1 includes a first portion 21 in which ITO is disposed corresponding to the first region 11, and a second portion 22 in which indium tin oxide is not disposed corresponding to the second region 12. The second portion 22 is disposed around the first portion 21 so as to define the boundary of the first region 11.

[0040] A transparent ITO thin film is formed on the substrate 10 as a material with ultraviolet blocking properties, so that the first region 11, which blocks ultraviolet UV, does not affect cell observation. A second region 22, in which no ITO is disposed, is formed around the first region 21, so that the second region 22 defines the boundary of the first region 11, allowing the first region 11 to be formed as a single cell colony.

[0041] The cell culture substrate 1 further includes a second layer 30 formed on the first layer 20 to support cell growth. In conventional cell culture substrates in which an adhesive substance is coated on the substrate for cell patterning, it can be difficult to use cell adhesion / growth factors to support cell growth. This is because the adhesive substance for cell patterning and the cell adhesion / growth factors for supporting cell growth cancel each other out. The adhesive substance may include, for example, a biocompatible synthetic polymer such as the MPC polymer described in Non-Patent Document 1. In contrast, in one embodiment of the present invention, the cell culture substrate 1 does not include an adhesive substance for cell patterning. Therefore, even if a second layer 30 made of cell adhesion / growth factors is formed, it is possible to create a culture environment suitable for the cell type without affecting cell patterning.

[0042] From another perspective, the cell patterning method having the above-described effects can also be considered as a method for producing a patterned cell population. The method for producing a patterned cell population includes the following steps: attaching cells to one surface of a cell culture substrate having a first region that blocks ultraviolet light and a second region that transmits ultraviolet light, and culturing the attached cells, the culturing step including irradiating the cells with ultraviolet light from the surface of the cell culture substrate opposite to the surface to which the cells are attached.

[0043] The step of attaching cells to one surface of the cell culture substrate can be carried out in the same manner as the cell patterning method described above. The number of cells to be attached may be one or more, and can be determined appropriately depending on the purpose. The cell culturing step involves culturing and growing the attached cells under conditions suitable for the cells until a predetermined cell population is generated. Then, in the culturing step, ultraviolet light is irradiated from the surface opposite to the surface to which the cells are attached for a predetermined period of time and a predetermined number of times.

[0044] The method for producing a patterned cell population can provide a cell population that is attached to a cell culture substrate and prepared to a desired shape and size, and optionally, multiple cell populations, i.e., multiple cell colonies, that are derived from the same or different cells or cell populations and grown and prepared to a desired shape and size can be obtained. The resulting multiple cell colonies can be used for various applications, such as screening, while still attached to the cell culture substrate. Alternatively, they can be separated from the cell culture substrate 1 and used for analysis or other applications.

[0045] As a more specific application example, the use of patterned multiple cell populations when the seeded cells are derived from malignant tumors will be described. Malignant tumor-derived cells may be collected from the peripheral blood of a human suffering from a malignant tumor. For example, circulating cancer cells (CTCs) can be collected from human peripheral blood. Alternatively, malignant tumor-derived cells can be collected from tissue sections of malignant glioma tumor tissue. Using these cells, a cell seeding step and a cell culturing step are performed. In the cell culturing step, an anticancer drug, for example, is applied to the first region. After a predetermined period of culture, the state of the cell population is observed and evaluated, allowing, for example, the identification of anticancer drug-resistant strains. Similarly, a step of confirming the sensitivity of cells to multiple anticancer drugs can also be performed. This enables the culture and evaluation of cell populations derived from specific patients, which can greatly contribute to cancer prognosis and the determination of treatment strategies.

[0046] Example I. Cell Culture Substrate Having a Circular Pattern in the First Region The cell culture substrate shown in FIG. 1 and a cell culture substrate without a second layer were produced, and cells were patterned on them.

[0047] (1) Selection of Materials with UV Blocking Properties Materials capable of absorbing UV and transmitting visible light were selected for the first and third portions. Candidate materials were 854.7 Å (85.47 nm) thick ITO, 4204 Å (420.4 nm) thick ITO, and 8334 Å (833.4 nm) thick ITO films deposited on 1 mm thick synthetic quartz glass. Additionally, 1000 μm thick polystyrene (PS), 1000 μm thick borosilicate glass, and 1 mm thick synthetic quartz glass were prepared. The relationship between the wavelength of irradiated light and the transmittance of these materials was investigated using a spectrophotometer (UV2700, Shimadzu Corporation).

[0048] Figure 4 is a graph showing the relationship between the wavelength of irradiated light and the transmittance of these materials. Figure 4 confirms that ITO, PS, and borosilicate glass (silica glass) can be used as materials that block ultraviolet light with a wavelength of 254 nm and transmit visible light. In subsequent experiments, ITO with a thickness of 8334 Å was selected as the material for the first and third portions.

[0049] (2) Cell Patterning Using Cell Culture Substrates Cell culture substrates were fabricated with the first, second, and third regions shown in Figure 1. The substrate was made of 1 mm-thick synthetic quartz glass, with the first and third regions composed of 8334 Å-thick ITO. Three patterns were fabricated: one without a second layer, one with a layer containing Type I collagen (Merck, product number C9791-10MG), and one with a layer containing Poly-D-Lysine (Sigma-Aldrich, product number P7280). In all cases, the diameter X of the first region was designed to be 1000 μm, and the width Y of the second region was designed to be 100 μm. The ITO was deposited by sputtering. Specifically, the synthetic quartz substrate was washed with sulfuric acid and hydrogen peroxide for 10 minutes. Using a sputtering system (iMiller, Shibaura Mechatronics) and an ITO sputtering target, an ITO film was formed on the area of ​​the substrate where the pattern was to be formed. Next, a positive photoresist (S1818, Rohm and Haas Electronic Materials LLC) was formed on the ITO film by spin coating at 4000 rpm and heated at 115°C for 3 minutes. The photoresist was exposed to light and developed to remove the ITO from the area corresponding to the second area of ​​the pattern. The substrate was then immersed in an ITO etchant (hydrochloric acid, 083-03435, Fujifilm Wako Pure Chemical Industries, Ltd.), and the ITO from the area where the photoresist was removed was etched. Finally, the photoresist remaining in the first and third regions was removed using a photoresist remover (Microposit Remover 1165, Rohm and Haas Electronic Materials LLC), yielding a cell culture substrate composed of a synthetic quartz substrate and a patterned first layer of ITO thereon. The second layer was prepared by mixing Type I collagen or Poly-D-lysine with ultrapure water, adjusting the concentration of Type I collagen to 100 μg / ml and Poly-D-lysine to 50 μg / ml.The substrate obtained above was placed in a petri dish with the first layer facing the dish, and a Type I collagen solution or a Poly-D-lysine solution was introduced into the gap between the surface of the petri dish and the first layer. The substrate was then placed in a refrigerator at 4°C and left to stand overnight.

[0050] These three types of cell culture substrates were cultured at 5.0 × 10 ５ A cell suspension containing human glioblastoma cell line U-87 modified to express fluorescent protein (GFP) was seeded at 1000 cells / mL and cultured for 4 days. From the start of culture, the cells were irradiated with ultraviolet light with a peak wavelength of 254 nm for a predetermined period of time once daily. The cells on the substrate were observed immediately after seeding and 4 days later. The observation results are shown in Figures 5 to 8.

[0051] FIG. 5 shows the results of (a) immediately after seeding cells on a cell culture substrate without a second layer, and (b) when the cumulative exposure dose of ultraviolet light was 0 mJ / cm. ２ (b) is a fluorescence image of the cell culture substrate on day 4 of culture. In this example, the cumulative exposure dose was calculated using the energy amount of light with a wavelength of 254 nm. No ultraviolet light was irradiated during culture, and it was confirmed from Figure 5 that cells proliferated in all three regions (first, second, and third).

[0052] FIG. 6 shows the results of (a) immediately after seeding cells on a cell culture substrate provided with a second layer made of Poly-D-Lysine, and (b) immediately after seeding cells on a cell culture substrate provided with a second layer made of Poly-D-Lysine. ２ 6 shows a fluorescence image of the cell culture substrate on day 4 of culture (a) and (b). Similarly to FIG. 5, no ultraviolet light was irradiated during culture, and it was confirmed that cells had proliferated in all three regions (first, second, and third).

[0053] FIG. 7 shows the state immediately after seeding cells on a cell culture substrate without a second layer (a) and the state immediately after seeding cells on a cell culture substrate with a cumulative exposure dose of ultraviolet light of 1038 mJ / cm ２ 7 shows a fluorescence image of the cell culture substrate on day 4 (b) after culturing with an exposure time of 600 seconds, once a day. It was confirmed from Fig. 7 that ultraviolet irradiation killed the cells in the second region and generated a cell colony isolated from the surrounding area in the first region.

[0054] FIG. 8 shows the results of (a) immediately after seeding cells on a cell culture substrate provided with a second layer made of collagen, and (b) immediately after seeding cells on a cell culture substrate provided with a second layer made of collagen and (c) immediately after seeding cells on a cell culture substrate provided with a second layer made of collagen and (d) immediately after seeding cells on a cell culture substrate provided with a second layer made of collagen and (e) immediately after seeding cells on a cell culture substrate provided with a second layer made of collagen and (f) immediately after seeding cells ２ 8 shows a fluorescence image of the cell culture substrate on the fourth day after culturing with an exposure time of 600 seconds, once a day (b). It was confirmed from Figure 8 that ultraviolet irradiation killed the cells in the second region, as in Figure 7. It was also confirmed that a cell colony isolated from the surrounding area was formed in the first region, demonstrating a greater proliferation effect than in Figure 7.

[0055] (3) Study on the dimensions of the first region The diameter X of the first region, which is the cell proliferation area, was changed, and cells were seeded and cultured under the same conditions as in (2) above, without providing a second layer. The cumulative exposure dose of ultraviolet light was 1038 mJ / cm ２ The exposure time was 600 seconds per day. Figure 9 shows a fluorescent image of the substrate on the fourth day after culture. In Figure 9, the four patterns in the top row of the image had a first region diameter X of 965 μm. The three patterns from the right in the middle row of the image had a first region diameter X of 713 μm. The patterns from the left in the middle row of the image and the two patterns from the right in the bottom row of the image had a first region diameter X of 455 μm. The two patterns from the left in the bottom row of the image had a first region diameter X of 198 μm. The square patterns in the image are patterns used to measure the actual measured values ​​of the different patterns in Figure 9. Figure 9 shows that cell patterning was possible in all cases where the first region was circular, the diameter X was approximately 200 to 1000 μm, and the width Y was 100 μm. More specifically, cells were grown in the first and third regions, cells were killed and removed in the second region, and colonies of cells were successfully generated in the first region.

[0056] (4) Study of Cell Proliferation Rate in the First Region and Number of Cells Attached to the Second Region The type of coating material and the diameter X of the first region were varied to investigate the cell proliferation rate in the first region and the number of cells attached to the second region. The type of coating material and application method were the same as in (2). The diameter X of the first region was targeted at 500 μm, 750 μm, and 1000 μm. The cell proliferation rate in the first region was measured by the ratio of the cell area to the area of ​​the first region. The cell area was measured at the time of cell seeding, on the second day of culture, and on the fourth day of culture. The cell area was measured by measuring the fluorescent area using image analysis. The number of cells attached to the second region was evaluated by counting the number of points on the image where the inner circle and the outer circle were connected by cells. The boundary between the first and second regions was defined as the inner circle, and the boundary between the second and third regions as the outer circle. The presence of a point where the inner and outer circles are connected by cells means that the cell colony in the first region is not isolated from other cells in the third region.

[0057] The evaluation results are shown in Figures 10, 11, and 12. The legend in each figure indicates the diameter (μm) of the culture region. In each figure, the upper row (a) shows the evaluation results for a pattern provided with a layer made of collagen (Collagen), the middle row (b) shows the evaluation results for a pattern provided with a layer made of Poly-D-Lysine (Poly-D-Lysine), and the lower row (c) shows the evaluation results for a pattern without a second layer (NON). The cumulative exposure dose of ultraviolet light was 0 mJ / cm in Figure 10. ２ , and Fig. 11 is 519 mJ / cm ２ (Exposure time: 300 sec) ２ (Exposure time: 600 sec) with exposure once a day. In each graph, the white plot indicates the cell proliferation rate within the first region, expressed as the ratio of the cell area occupied by the cells to the area of ​​the first region on the left axis. The black plot indicates the number of cells binding to the second region on the right axis. Cells binding to the second region with a number of more than 10 cells are represented as 10.

[0058] 10 to 12, it was confirmed that increasing the amount of UV exposure significantly reduced the number of cells bound to the second region, enabling cell proliferation in the first region. In particular, it was confirmed that using a cell culture substrate provided with a coating material killed and removed cells in the second region, enabling the generation of cell colonies, without reducing the cell proliferation rate in the first region.

[0059] Example II. Cell Culture Substrate with a First Region Having a Regular Hexagonal Pattern (1) Design and Manufacturing of a Honeycomb-Type Cell Culture Substrate A cell culture substrate was designed in which the first region was a regular hexagon, and a second region was provided around the regular hexagon as a boundary. Sixteen regular hexagons were arranged vertically and horizontally, with a third region provided around the periphery of these regular hexagons. In this example, a cell culture substrate having a cell growth area of ​​this shape is referred to as a honeycomb-type cell culture substrate. Figure 13 is a diagram illustrating the design of a honeycomb-type cell culture substrate. In Figure 13, A is defined as the design value of the circumscribed circle diameter of the first region consisting of a regular hexagon, B is the design value of the gap length in the diameter direction of the circumscribed circle, C is the design value of the width of the second region, and D is the design value of the distance between two parallel sides of the regular hexagon.

[0060] Six types of samples were designed, with variations A to D, and the ITO first layer was patterned based on the design in the same manner as in Example I (2). The second layer contained Type I collagen (Merck, product number C9791-10MG). Figure 14 shows an image of a cell culture substrate actually manufactured based on the design diagram of Figure 13, including (a) the honeycomb-shaped cell growth area and (b) an enlarged photograph of region Z in (a). In Figure 14, the measured value of the diameter of the circumscribed circle of the regular hexagonal first region was defined as E, the measured value of the gap length in the diametric direction of the circumscribed circle as F, the measured value of the width of the second region as G, the measured value of the distance between the two parallel sides of the regular hexagon as H, and the measured value of the width of the ITO layer partially etched by undercut as I.

[0061] The design values ​​and actual measurement values ​​of six types of samples are shown in Table 1. In the table, the design values ​​are abbreviated as Des and the actual measurement values ​​are abbreviated as Mea. The actual measurement values ​​Mea in the table represent the average values ​​of the results measured at three different locations.

[0062] As can be seen from Table 1, although undercuts were unavoidable in the manufacturing process, the product was manufactured almost exactly as designed.

[0063] (2) Design and Fabrication of a Single Hexagonal Cell Culture Substrate A cell culture substrate with the same pattern as that shown in FIG. 1 or 3 was designed, except that the circular first region 11 in FIG. 1 or 3 was changed to a regular hexagon and a second region of uniform width was provided around the periphery of the regular hexagon. Only one first region was provided, and a third region was provided around the periphery of the second region. In this example, a cell culture substrate with a cell growth area of ​​this shape is referred to as a single hexagonal cell culture substrate. Based on the design, the first layer, ITO, was patterned in the same manner as in Example I (2). The second layer was a layer containing Type I collagen (Merck, product number C9791-10MG).

[0064] (3) Cell Patterning Using Cell Culture Substrates Sample No. 1 honeycomb-type cell culture substrate and a single hexagonal-type cell culture substrate with a circumscribed circle diameter design value A of the first region set to 450 μm and a gap length design value B of 50 μm were used. ５ A cell suspension containing human glioblastoma cell line U-87 modified to express fluorescent protein (GFP) was seeded at 1000 cells / mL and cultured for two days. From the start of culture, the cells were exposed to ultraviolet light with a peak wavelength of 254 nm for a predetermined period of time once a day. The cells on the substrate were observed immediately after seeding and two days later. The observation results are shown in Figures 15 to 18.

[0065] FIG. 15 shows the results of (a) immediately after seeding cells on a honeycomb-type cell culture substrate and (b) when the integrated exposure dose of ultraviolet light was 0 mJ / cm. ２15 shows a fluorescence image taken two days after culturing (b). No ultraviolet light was irradiated during culturing, and it was confirmed from FIG. 15 that cells had proliferated in both the first and second regions. Here, in the second region, the ITO layer does not absorb the fluorescence of the human glioblastoma cell line U-87, so the U-87 cells in the second region appear relatively brighter than the U-87 cells in region 1. FIG. 16 shows a fluorescence image taken immediately after seeding the cells on the honeycomb-type cell culture substrate (a), and after the cumulative ultraviolet light exposure was 2076 mJ / cm. ２ 16 shows a fluorescence image taken on the second day after culturing with an exposure time of 1,200 seconds, once a day (b). It was confirmed from Fig. 16 that ultraviolet irradiation killed the cells in the second region and generated cell colonies in the multiple hexagonal first regions that were isolated from the other first regions.

[0066] FIG. 17 shows the results immediately after seeding cells on a single hexagonal cell culture substrate (a) and after the cumulative exposure dose of ultraviolet light was 0 mJ / cm ２ 17 shows a fluorescence image taken two days after culturing with the single hexagonal cell culture substrate (a) and (b) on the second day. No ultraviolet light was irradiated during culturing, and it was confirmed from Figure 17 that cells had proliferated in both the first and second regions. Figure 18 shows a fluorescence image taken immediately after seeding the cells on the single hexagonal cell culture substrate (a) and after the cumulative ultraviolet light exposure was 2076 mJ / cm. ２ 18 shows a fluorescence image taken on the second day after culturing with an exposure time of 1,200 seconds, once a day (b). It was confirmed that ultraviolet irradiation killed the cells in the second region and generated a cell colony isolated from the surrounding area in a single first region.

[0067] 15 to 18, it was confirmed that cell patterning was possible using a regular hexagonal ITO layer as well as a circular pattern. It was also confirmed that similar results could be obtained when regular hexagonal ITO layers were densely arranged in a honeycomb pattern.

[0068] (4) Study on cell proliferation rate in the first region and number of cells attached to the second region For the honeycomb type and single hexagon type cell culture substrates, the design value A of the circumscribed circle diameter of the first region and the design value B of the gap length were changed, and after a predetermined amount of UV exposure, the cell proliferation rate in the first region and the number of cells attached to the second region were investigated. The type and application of the coating material were the same as in (1) and (2) of Example II, and the cell culture conditions were the same as in (3) of Example II. Exposure to UV light was performed once a day. UV exposure dose: 0 mJ / cm ２ , 1038mJ / cm ２ (Exposure time 600 sec), or 2076 mJ / cm ２ The results were evaluated for a case in which the exposure time was 1,200 seconds. The cell proliferation rate in the first region was measured using the ratio of the cell area to the area of ​​the first region. The cell area was measured at the time of cell seeding and on the second day of culture. The method for measuring the cell area was the same as in (4) of Example I. For honeycomb-type cell culture substrates, the cell area was measured using three randomly selected regular hexagons other than the outermost regular hexagon, and the average value was calculated. The number of cells bound to the second region was evaluated based on the number of connections between the periphery of a first region and the periphery of another adjacent first region or the third region. For honeycomb-type cell culture substrates, the number of bound cells was measured using three randomly selected regular hexagons other than the outermost regular hexagon, and the average value was calculated.

[0069] The evaluation results are shown in Figures 19 to 22. The legend in the upper right corner indicates the gap length design value B (μm). In addition, the ultraviolet exposure dose in each figure is 0 mJ / cm for the upper row (a). ２ , middle row (b) is 1038 mJ / cm ２ , and the lower row (c) is 2076 mJ / cm ２ The results are shown in Table 1. In each graph, the white plot indicates the cell proliferation rate in the first region, expressed as the ratio of the cell area occupied by the cells to the area of ​​the first region on the left axis. The black plot indicates the number of cells binding to the second region on the right axis. If the number of cells binding to the second region is more than 10, it is represented as 10.

[0070] Fig. 19 shows the evaluation results using a honeycomb-type cell culture substrate with a circumscribing circle diameter design value A of 750 μm, Fig. 20 shows the evaluation results using a single hexagonal-type cell culture substrate with a circumscribing circle diameter design value A of 750 μm, Fig. 21 shows the evaluation results using a honeycomb-type cell culture substrate with a circumscribing circle diameter design value A of 450 μm, and Fig. 22 shows the evaluation results using a single hexagonal-type cell culture substrate with a circumscribing circle diameter design value A of 450 μm.

[0071] 19 to 22, it was confirmed that, as with the circular first region, increasing the UV exposure significantly reduced the number of cells binding to the second region, enabling cell proliferation in the first region, even when the first region was a regular hexagon. However, the cumulative exposure dose in this experiment may not be sufficient to inhibit cell binding when the gap length was 25 μm. Therefore, when the gap length was 25 μm or less, increasing the cumulative exposure dose is thought to more reliably inhibit cell binding.

[0072] REFERENCE SIGNS LIST 1 cell culture substrate 10 substrate 11 first region 12 second region 20 first layer 21 first portion 22 second portion 30 second layer 100 cell patterning device 110 ultraviolet irradiation device

Claims

1. A step of attaching cells to one surface of a cell culture substrate having a first region that blocks ultraviolet light and a second region that transmits ultraviolet light; irradiating the cell culture substrate with ultraviolet light from a surface opposite to the surface to which the cells are attached; A cell patterning method, comprising removing cells on the second region among the cells attached to the cell culture substrate by the step of irradiating with ultraviolet light, The cell culture substrate comprises: a substrate that is transparent to visible light and ultraviolet light; a first layer formed on the substrate and including a portion having an ultraviolet ray shielding property; Equipped with A cell patterning method, wherein the portion having ultraviolet ray shielding ability is indium tin oxide.

2. The first layer of the cell culture substrate has a first portion in which the indium tin oxide is arranged corresponding to the first region, and a second portion in which the indium tin oxide is not arranged corresponding to the second region; The cell patterning method of claim 1 , wherein the second portion is provided around the first portion so as to define a boundary of the first region.

3. The cell patterning method of claim 1, wherein the cell culture substrate further comprises a second layer formed on the first layer or on a surface of the substrate opposite the first layer for supporting cell growth.

4. A cell culture substrate having a first region that blocks ultraviolet light and a second region that transmits ultraviolet light; an ultraviolet irradiation device capable of irradiating ultraviolet light onto the cell culture substrate; Equipped with The cell culture substrate comprises: a substrate that is transparent to visible light and ultraviolet light; a first layer formed on the substrate and including a portion having an ultraviolet ray shielding property; Equipped with The portion having ultraviolet ray shielding ability is indium tin oxide, A cell patterning apparatus that removes cells on the second region from among the cells attached to the cell culture substrate by irradiating the cell with ultraviolet light using the ultraviolet irradiation device.

5. A method for producing a patterned cell population, comprising: A step of attaching cells to one surface of a cell culture substrate having a first region that blocks ultraviolet light and a second region that transmits ultraviolet light; Culturing the attached cells; Including, The cell culture substrate comprises: a substrate that is transparent to visible light and ultraviolet light; a first layer formed on the substrate and including a portion having an ultraviolet ray shielding property; Equipped with The portion having ultraviolet ray shielding ability is indium tin oxide, The method, wherein the culturing step comprises a step of irradiating the cells with ultraviolet light from a surface of the cell culture substrate opposite to the surface to which the cells are attached.