Cell patterning method and cell patterning apparatus

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

[Technical Field]

[0001] This invention relates to a cell patterning method and a cell patterning apparatus. [Background technology]

[0002] Quantitative experiments such as drug evaluation using cells require the formation of cell tissues consisting of a certain number of cells and precise control over the size of these cell tissues. One known method for controlling cell tissue is cell patterning, which involves dividing the surface of the substrate to which cells adhere into areas where cells adhere and areas where they do not, thereby controlling the cell adhesion area.

[0003] For example, a technique is known in which a microstructured MPC polymer film is formed by pouring an ethanol solution of 2-methacryloyloxyethyl phosphorylcholine (MPC) polymer into a polydimethylsiloxane (PDMS) micromold formed on a silicon wafer, thereby creating a desired pattern (see, for example, Non-Patent Literature 1). Since the areas covered with the MPC polymer become areas where cells do not adhere, and the areas not covered with the MPC polymer become areas where cells do adhere, a microdevice for cell patterning is fabricated in which the areas where cells adhere and the areas where they do not are separated. [Prior art documents] [Non-patent literature]

[0004] [Non-Patent Document 1] N. Tanaka, et al., Vacuum microcasting of 2-methacryloyloxyethylphosphorylcholine polymer for stable cell patterning, BioTechniques, Vol. 69, No. 3, 2020 [Overview of the project] [Problems that the invention aims to solve]

[0005] When culturing adhesive cells, cell adsorption and growth factors such as Extracellular Matrix (ECM) (e.g., collagen or fibronectin) are sometimes coated onto the surface of the cell culture substrate. However, in Non-Patent Document 1 mentioned above, a pattern is formed using MPC polymer to separate areas where cells adhere from areas where they do not, making it difficult to use in combination with coating agents for cell adsorption and growth such as ECM.

[0006] The present invention has been made in view of the above-described circumstances, and its purpose is to provide a cell patterning method and a cell patterning apparatus that can perform good cell patterning according to various types of cells. [Means for solving the problem]

[0007] According to one embodiment, the present invention is a method for patterning cells, A step of attaching cells to one side of a cell culture substrate having a first region that blocks ultraviolet light and a second region that transmits ultraviolet light, The process includes irradiating the cell culture substrate with ultraviolet light from the side opposite to the side to which the cells are attached, The present invention relates to a cell patterning method comprising the step of irradiating with ultraviolet light to remove cells from the cell culture substrate that are attached to the cell culture substrate, specifically cells located in the second region.

[0008] According to another embodiment, the present invention is a cell patterning apparatus, A cell culture substrate having a first region that blocks ultraviolet light and a second region that transmits ultraviolet light, A UV irradiation device capable of irradiating the cell culture substrate with ultraviolet light, Equipped with, The present invention relates to a cell patterning apparatus that removes cells from the cell culture substrate that are attached to the cell culture substrate from the second region by irradiating it with ultraviolet light using the ultraviolet light irradiation device.

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

Advantages of the Invention

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

Brief Description of the Drawings

[0011] [Figure 1] FIG. 1(a) and (b) are top views and cross-sectional views schematically showing the configuration of a cell culture substrate used in a cell patterning method according to an embodiment of the present invention. [Figure 2] FIG. 2 is a diagram showing a schematic configuration of a cell patterning device according to an embodiment. [Figure 3] FIGS. 3(a) to (d) are diagrams for explaining the process of a cell patterning method according to an embodiment. [Figure 4] FIG. 4 is a graph showing the relationship between the wavelength of irradiation light and the transmittance for various materials. [Figure 5] FIG. 5 is a fluorescence image of a cell culture substrate immediately after seeding cells on a cell culture substrate without a second layer (a), and on the fourth day after culturing with an integrated exposure amount of ultraviolet light of 0 mJ / cm2 (b). The integrated exposure amount was calculated based on the energy amount of light with a wavelength of 254 nm. [Figure 6]Figure 6 shows fluorescence images of the cell culture substrate immediately after seeding cells on a substrate containing a second layer of poly-D-lysine (a), and on day 4 after culturing with an integrated ultraviolet exposure of 0 mJ / cm2 (b). [Figure 7] Figure 7 shows fluorescence images of the cell culture substrate immediately after seeding cells in a cell culture substrate without a second layer (a), and on day 4 after culturing with an integrated ultraviolet exposure of 1038 mJ / cm2 (b). [Figure 8] Figure 8 shows fluorescence images of the cell culture substrate immediately after seeding cells on a cell culture substrate containing a second layer of collagen (a), and on day 4 after culturing with an integrated ultraviolet exposure dose of 1038 mJ / cm2 (b). [Figure 9] Figure 9 shows the fluorescence image of the substrate on day 4 after culturing, when the cell patterning method was performed by changing the diameter X of the first region, which is the cell proliferation area. [Figure 10] Figure 10 shows the results of evaluating the cell proliferation rate in the first region and the number of cells bound to the second region when ultraviolet light was not irradiated. [Figure 11] Figure 11 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 cumulative ultraviolet exposure dose was 519 mJ / cm2. [Figure 12] Figure 12 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 cumulative ultraviolet exposure dose was 1038 mJ / cm2. [Figure 13] Figure 13 illustrates the design of a cell culture substrate having a honeycomb-shaped cell proliferation area, where the first region is a regular hexagon and the second region is the boundary between multiple connected regular hexagons. [Figure 14] Figure 14 is an image of a cell culture substrate actually manufactured based on the design drawing in Figure 13, showing a honeycomb-shaped cell proliferation area (a) and a magnified image of region Z of (a) (b). [Figure 15]Figure 15 shows fluorescence images of the cell culture substrate having a honeycomb-shaped cell proliferation area as shown in Figure 14, immediately after seeding cells (a), and on day 2 after culturing with an integrated ultraviolet exposure of 0 mJ / cm2 (b). [Figure 16] Figure 16 shows fluorescence images of the cell culture substrate immediately after seeding cells on the same substrate as in Figure 15 (a), and on day 2 after culturing with an integrated ultraviolet exposure dose of 2076 mJ / cm2 (b). [Figure 17] Figure 17 shows fluorescence images of a cell culture substrate having a single hexagonal cell proliferation area, where a first regular hexagonal region is provided, and a third region is provided around the periphery of a second region, immediately after seeding cells (a) and on day 2 after culturing with an integrated ultraviolet exposure of 0 mJ / cm2 (b). [Figure 18] Figure 18 shows fluorescence images of the cell culture substrate immediately after seeding cells on the same substrate as in Figure 17 (a), and on day 2 after culturing with an integrated ultraviolet exposure dose of 2076 mJ / cm2 (b). [Figure 19] Figure 19 shows the results of evaluating the cell proliferation rate in the first region and the number of cells bound to the second region in a cell culture substrate similar to that in Figure 14, with a design value A for the circumscribed diameter of the first region set to 750 μm. The images are fluorescence images of the substrate with integrated ultraviolet exposure doses of 0 mJ / cm2 (a), 1038 mJ / cm2 (b), and 2076 mJ / cm2 (c). [Figure 20] Figure 20 shows the results of evaluating the cell proliferation rate in the first region and the number of cells bound to the second region in a cell culture substrate similar to that in Figure 17, with a design value A for the circumscribed diameter of the first region set to 750 μm. The images are fluorescence images of the substrate with integrated ultraviolet exposure doses of 0 mJ / cm2 (a), 1038 mJ / cm2 (b), and 2076 mJ / cm2 (c). [Figure 21] Figure 21 shows the results of evaluating the cell proliferation rate in the first region and the number of cells bound to the second region in a cell culture substrate similar to that in Figure 14, with a design value A for the circumscribed diameter of the first region set to 450 μm. The images are fluorescence images of the substrate with integrated ultraviolet exposure doses of 0 mJ / cm2 (a), 1038 mJ / cm2 (b), and 2076 mJ / cm2 (c). [Figure 22] Figure 22 shows the results of evaluating the cell proliferation rate in the first region and the number of cells bound to the second region in a cell culture substrate similar to that in Figure 17, with a design value A for the circumscribed diameter of the first region set to 450 μm. The images are fluorescence images of the substrate with integrated ultraviolet exposure doses of 0 mJ / cm2 (a), 1038 mJ / cm2 (b), and 2076 mJ / cm2 (c). [Modes for carrying out the invention]

[0012] A cell patterning method and a cell patterning apparatus according to one embodiment of the present invention perform cell patterning to control 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. Control of the cell adhesion area is achieved by forming a UV-shielding thin film on the substrate and irradiating it with localized ultraviolet light.

[0013] Specifically, the system is configured to perform cell patterning by attaching cells to a cell culture substrate having an ultraviolet-blocking region that blocks ultraviolet light and an ultraviolet-transmitting region that transmits ultraviolet light, irradiating the cell culture substrate with ultraviolet light, and removing the cells attached to the cell culture substrate that are located on the ultraviolet-transmitting region by ultraviolet light irradiation.

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

[0015] As shown in Figures 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, so that localized irradiation of cells attached to the cell culture substrate 1 with ultraviolet light is possible 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 a combination of one first region 11 (and optionally a third region 13) and the second region 12 is also called a pattern.

[0016] The first region 11 is a cell proliferation area for forming a cell colony, and is formed, for example, as a circle with a diameter of X. However, it is not limited to this, and the shape of the first region 11 can also be a polygon including regular polygons such as a regular hexagon. The shape and size of the first region 11 can be appropriately determined by a person skilled in the art to correspond to the desired shape and size of the cell colony after formation. When the first region 11 is circular, the lower limit of the diameter X is not particularly limited and can be appropriately determined by the number of cells to be proliferated, etc. For example, one cell may be placed in the first region 11, in which case the diameter X may be about 10 μm. When multiple cells are placed in the first region 11, the diameter X may be, for example, about 50 μm or more, or 100 μm or more, and preferably about 150 μm or more. The upper limit of the diameter X is not particularly limited, but for example it can be about 1000 μm. Even when the shape of the first region 11 is not circular, the size of the first region 11 can be determined so that it has an area of ​​roughly the same size as the first region 11. For example, if the shape of the first region is a regular polygon, the diameter of the circumscribed circle of the regular polygon can be set to the above value.

[0017] The second region 12 is adjacent to the outer periphery of the first region 11 and defines the boundary of the first region 11. The second region 12 is formed in an annular shape, for example, with a width Y, to match the shape of the first region 11. The third region 13 is optionally provided in the area outside the second region 12 and may be a region that can substitute for the first region 11. The width Y is not limited to a specific width, and should be such that cells cultured in the first region 11 and cells cultured in the area outside the second region 12 do not come into contact. In some embodiments, the width Y is preferably such that ultraviolet light can be transmitted, and can be wider than the wavelength of ultraviolet light used for irradiation. For example, when 254 nm ultraviolet light is used for irradiation, the lower limit of the width Y may be about 300 nm. The upper limit of the width Y is not particularly limited in theory.

[0018] Figure 1(a) shows only one first region 11, i.e., one cell proliferation area, but the number of cell proliferation areas on the cell culture substrate 1 is not limited to one. The cell culture substrate 1 may be configured to have multiple cell proliferation areas by forming multiple first regions 11 and multiple second regions 12 that define their boundaries. When multiple cell proliferation areas are provided, the shape and size of the multiple first regions 11 and second regions 12 may be the same or different. An example of a pattern with multiple first and second regions of the same shape and size is a honeycomb shape in which the first region is a regular hexagon and the second region is a boundary between multiple regular hexagons. Alternatively, multiple different patterns may be provided on a single cell culture substrate 1. The space between one second region 12 and another second region 12 may be occupied by a third region 13.

[0019] As shown in Figure 1(b), the cell culture substrate 1 comprises a substrate 10 that transmits visible light and ultraviolet light, a first layer 20 formed on the substrate 10 and having a portion that provides ultraviolet light shielding, and a second layer 30 formed on the first layer 20 to assist in cell proliferation.

[0020] The substrate 10 is not particularly limited as long as it is a material that transmits visible light and ultraviolet light, but it is selected from materials suitable for patterning the first layer 20. For example, quartz glass is preferred. The substrate 10 is preferably a flat plate-shaped member with a substantially uniform thickness, and its thickness is not particularly limited. Considering that the transmittance of ultraviolet light decreases as the thickness of the substrate 10 increases, the thickness of the substrate 10 can be, for example, about 1 mm. The length and width dimensions of the substrate 10 are not particularly limited, but considering cell observation with a microscope, they can be, for example, similar to the dimensions of a standard microscope slide (length about 76 mm, width about 26 mm).

[0021] The first layer 20 has a first portion 21 in which a material having ultraviolet shielding properties is arranged corresponding to the first region 11, and a second portion 22 in which a material having ultraviolet shielding properties is not arranged corresponding to the second region 12. The first portion 21 is the portion having ultraviolet shielding properties and can also be referred to as an ultraviolet shielding 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 shielding properties is arranged corresponding to the third region 13. The third portion 23 is also a portion having ultraviolet shielding properties and can also be referred to as an ultraviolet shielding layer.

[0022] Ultraviolet light is generally light with wavelengths of approximately 10 nm to 400 nm. Of these, light with wavelengths of approximately 315 nm or less is classified as UVB (wavelengths of approximately 280 to 315 nm) and UVC (wavelengths of approximately 200 to 280 nm), and generally has a bactericidal and sterilizing effect. In one embodiment, the material having ultraviolet shielding performance is preferably selected from among materials that have an absorption peak for the wavelength of ultraviolet light irradiated from the ultraviolet irradiation device described later (for example, wavelengths of approximately 315 nm or less corresponding to UVB or UVC), and is suitable for patterning the first layer 20. Furthermore, the material having ultraviolet shielding performance is preferably transparent so as not to affect cell observation. Here, transparency means that the transmittance to light necessary for cell observation, such as visible light with wavelengths of approximately 360 nm to 830 nm and excitation light / fluorescence with wavelengths of approximately 340 to 740 nm, is preferably 70% or more, more preferably 80% or more, and preferably 90% or more. Examples of materials with such UV shielding properties include indium tin oxide (ITO). The thickness of the ITO can be selected from the viewpoint of UV shielding performance and the transmittance of observation light. In particular, it is preferable that the thickness of the ITO be about 4000 Å (400 nm) or more and about 30000 Å (3000 nm) or less, but it is not limited to a specific thickness as long as it can absorb and shield ultraviolet rays. As an example, silicon dioxide (SiO2) and polystyrene are also included as materials with UV shielding performance of about 254 nm. Below, an example in which ITO is used as a material with UV shielding properties will be described.

[0023] Forming the ultraviolet shielding layer constituting the first portion 21 or the third portion 23 on the substrate 10 is done using a method appropriate to the material having ultraviolet shielding properties. When forming an ITO film as the first portion 21 or optionally the third portion 23, for example, sputtering or photolithography used in semiconductor manufacturing technology can be used. As an example of a specific manufacturing method, an ITO film is deposited on the pattern-forming region of the substrate 10 by sputtering, a resist is applied to the ITO film, and then exposure and development are performed to remove the resist in the portion corresponding to the second region 12 of the pattern. The ITO in the area where the resist has been 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. As a result, an ultraviolet shielding layer of the desired shape, including the first portion 21 and optionally the third portion 23, is formed on the substrate 10. Note that ITO, which is given as an example of a material having ultraviolet shielding properties in this invention, is not used as an electrode. In other words, the first portion 21 and the third portion 23 on which the ITO is located are not connected to a power supply by wiring or the like, and are not configured to allow voltage to be applied.

[0024] The second layer 30 is an optional layer provided to assist in the adhesion of cells to the first region 11 and the proliferation of attached cells, and can also be described as a layer made of a coating material. The second layer 30 can be formed, for example, by placing the substrate 10 on which the above-mentioned ultraviolet shielding layer is formed into a container such as a petri dish with the ultraviolet shielding layer facing downwards, introducing a coating agent dissolved in ultrapure water, for example, between the surface of the petri dish and the substrate 10, and adhering the coating agent to the surface of the substrate. As shown in Figure 1(b), the second layer 30 is placed on the first portion 21 and the third portion 23 of the first layer 20 in contact with them. It is also placed on the second portion 22, and in the second region 12, the coating material is also filled into the second portion 22 and in contact with the substrate 10. The coating material constituting the second layer 30 can be selected to suit the cell type being cultured. Examples include extracellular matrix (ECM) such as collagen and fibronectin, and Matrigel, a reconstituted basement membrane isolated from EHS mouse sarcoma (mouse Engelbreth-Holm-Swarm sarcoma) cells. Alternatively, the second layer 30 does not need to be formed; 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 also be combined with the formation of the second layer 30, such as collagen. 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 can assist in cell adhesion and the proliferation of attached cells. For example, it can be about 0.1 to 3 μm, and more preferably about 0.1 to 1 μm. The second layer 30 constitutes the surface in the cell culture substrate 1 that comes into contact with cells and functions as the surface on which cells are seeded. When a second layer 30 is provided, the surface on which cells are seeded is preferably a flat surface, but is not particularly limited.

[0025] Depending on the cell type to be cultured and the culture environment, the second layer 30 may be omitted. In a cell culture substrate without a second layer (not shown), the first region may be a region in which the substrate and the first part (ultraviolet shielding layer) are sequentially stacked in a direction perpendicular to the main surface of the cell culture substrate, and the second region may consist only of the substrate. A third region, which may be optionally provided, may be a region in which the substrate and the third part (ultraviolet shielding layer) are sequentially stacked. In this case, the surface on which cells are seeded may have irregularities caused by the first and third parts. Although not shown, the second layer can 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 as to be in contact with the second layer. When forming the second layer on the side of the substrate opposite to the side on which the first layer is formed, the method of formation is the same as when 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 so that the ultraviolet shielding layer faces upward.

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

[0027] In the example shown in Figure 2, the cell culture substrate 1 is positioned 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 facing upwards and the second layer 30 to which the cells 50 are attached facing downwards. The container 120 is configured to hold a culture medium 121 and is provided with a support member 122, for example, made of 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 is in contact with the culture medium 121.

[0028] The ultraviolet (UV) light emitted from the UV irradiation device 110 is incident on the cell culture substrate 1 placed inside the container 120 from the substrate 10 side. In other words, the UV light is incident on the cell culture substrate 1 from the side opposite to the side to which the cells 50 are attached. The UV light incident on the cell culture substrate 1 is transmitted through the second region 12, which is an ultraviolet transmission region, and blocked in the first region 11, which is an ultraviolet shielding region, so that the cells are not irradiated.

[0029] In the example shown in Figure 2, the cell culture substrate 1 is positioned vertically below the ultraviolet irradiation device 110. However, the configuration is not limited to this, and the cell culture substrate 1 may be positioned vertically above the ultraviolet irradiation device 110. In this case, the cell culture substrate 1 is positioned with the substrate 10 facing downwards and the second layer 30 to which the cells 50 are attached facing upwards, so that ultraviolet UV light is incident from the substrate 10 side.

[0030] Next, with reference to Figures 3(a) to 3(d), the flow of the cell patterning method using the cell patterning device 100 will be explained.

[0031] First, as shown in Figure 3(a), a cell culture substrate 1 is prepared having a first region 11 that blocks ultraviolet UV rays and a second region 12 that transmits ultraviolet UV rays. The cell culture substrate 1 may also have a third region 13 that blocks ultraviolet UV rays. 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 Figure 3(b), cells 50 are seeded onto the cell culture substrate 1 and attached to the second layer 30. At this time, it is preferable to seed the cells 50 substantially 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 seeding a cell population using a pipette or the like, for example, 10 or more cells, or 100 or more cells, can be seeded in the first region 11. Assuming the use of 1 mL of cell suspension, the concentration can be converted to approximately 1.0 × 10⁻⁶. 3 It can be 1.0 × 10⁻⁶ or more. 10 cells / mL or less, preferably about 1.0 × 10⁶ 6 cells / mL or less, or approximately 1.0 × 10⁻⁶ 5 The concentration can be reduced to approximately cells / mL or less. A culture medium may be included as one of the solutions used for sowing.

[0033] When multiple first regions 11 are provided on a single cell culture substrate 1, the same cells can be seeded simultaneously in 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 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 Figure 3(c), the cell culture substrate 1 is irradiated with ultraviolet (UV) light by the UV irradiation device 110 described above for a predetermined time. The UV light enters the cell culture substrate 1 from the substrate 10 side. The incident UV light is shielded in the first portion 21 and the third portion 23 of the first layer 20 where ITO is placed, and transmitted through the second portion 22 where ITO is not placed. Cells 50 attached to the second region 12 corresponding to the second portion 22 of the first layer 20 are killed by the incident UV light 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 optionally provided third portion 23 are not irradiated with UV light, so the cells 50 remain attached and cultured and proliferate on the cell culture substrate 1 without being killed.

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

[0036] The cell patterning method and cell patterning apparatus 100 according to the above-described embodiment can produce the following effects.

[0037] The cell patterning apparatus 100 comprises a cell culture substrate 1 having a first region 11 that blocks ultraviolet UV light and a second region 12 that transmits ultraviolet UV light, and an ultraviolet irradiation device 110 capable of irradiating the cell culture substrate 1 with ultraviolet UV light. The apparatus is configured to remove cells 50 attached to the cell culture substrate 1 that are located on the second region 12 by irradiating them with ultraviolet UV light using the ultraviolet irradiation device 110.

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

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

[0040] Since a transparent ITO thin film is formed on the substrate 10 as a material with ultraviolet shielding properties, the observation of cells is not affected by the first region 11 that shields from ultraviolet UV. A second region 22 in which ITO is not present is formed around the first region 21, so that the boundary of the first region 11 is defined by the second region 22, and the first region 11 can be formed as a single cell colony.

[0041] The cell culture substrate 1 is formed on a first layer 20 and further comprises a second layer 30 for assisting cell proliferation. In the case of conventional cell culture substrates coated with an adhesive substance on the substrate for cell patterning, it was sometimes difficult to use cell adsorption and growth factors to assist cell proliferation. This is because the adhesive substance for cell patterning and the cell adsorption and growth factors for assisting cell proliferation cancel each other out. The adhesive substance may include, for example, a biocompatible synthetic polymer such as the MPC polymer described in Non-Patent Literature 1. In contrast, in one embodiment of the present invention, the cell culture substrate 1 does not have an adhesive substance for cell patterning. Therefore, even if a second layer 30 consisting of, for example, a cell adsorption and growth factor is formed, a culture environment suitable for the cell type can be constructed without affecting cell patterning.

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

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

[0044] The method for producing patterned cell populations allows for obtaining cell populations prepared to a desired shape and size, attached to a cell culture substrate. Furthermore, it is possible to selectively obtain multiple cell populations, i.e., multiple cell colonies, prepared to a desired shape and size, derived from the same or different cells or cell populations. These 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 and used for analysis or other applications.

[0045] As a more specific application example, we will describe the use of patterned cell populations when the cells to be seeded are cells derived from malignant tumors. Cells derived from malignant tumors may be collected from the peripheral blood of a person affected by a malignant tumor. For example, circulating cancer cells (CTCs) can be collected from human peripheral blood. Alternatively, cells derived from malignant tumors can be collected from tissue sections of malignant glioma tumor tissue. Using these cells, the steps of seeding and culturing are carried out. In the cell culturing step, for example, an anticancer drug is applied to the first region. After culturing for a predetermined period, the state of the cell population is observed and evaluated to identify, for example, anticancer drug-resistant strains. Similarly, a step to confirm the sensitivity of cells to multiple types of anticancer drugs can be carried out. This makes it possible to culture and evaluate cell populations derived from specific patients, which can greatly contribute to prognosis diagnosis and treatment strategy determination for cancer. [Examples]

[0046] Example I. Cell culture substrate having a circular pattern in the first region. Cell culture substrates as shown in Figure 1 and cell culture substrates without the second layer were prepared, and cell patterning was performed.

[0047] (1) Selection of materials with ultraviolet shielding properties We selected materials for the first and third parts of the film that could absorb ultraviolet light and transmit visible light. As candidate materials, we prepared ITO with a thickness of 854.7 Å (85.47 nm), ITO with a thickness of 4204 Å (420.4 nm), and ITO with a thickness of 8334 Å (833.4 nm), all deposited on 1 mm thick synthetic quartz glass, as well as polystyrene (PS) with a thickness of 1000 μm, borosilicate glass with a thickness of 1000 μm, and synthetic quartz glass with a thickness of 1 mm. For these materials, we investigated the relationship between the wavelength of the irradiated light and the transmittance using a spectrophotometer (UV2700, Shimadzu Corporation).

[0048] Figure 4 is a graph showing the relationship between the wavelength of irradiated light and the transmittance for these materials. From Figure 4, it was confirmed that ITO, PS, and borosilicate glass (silica glass) can be used as materials that block ultraviolet light at 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 parts.

[0049] (2) Cell patterning using cell culture substrates A cell culture substrate was fabricated comprising the first, second, and third regions shown in Figure 1. The substrate was made of 1 mm thick synthetic quartz glass, and the first and third regions were composed of 8334 Å thick ITO. Three patterns were fabricated: one without a second layer, one with a layer containing Type I collagen (Merck, catalog number C9791-10MG), and one with a layer containing Poly-D-Lysine (Sigma-Aldrich, catalog 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 100 μm. The ITO was deposited by sputtering. Specifically, the synthetic quartz substrate was washed with sulfuric acid and hydrogen peroxide for 10 minutes. ITO was deposited onto the regions forming the patterns on the substrate using a sputtering apparatus (iMiller, Shibaura Mechatronics) and an ITO sputtering target. Next, a positive-type photoresist (S1818, Rohm and Haas Electronic Materials LLC) was deposited on the deposited ITO film by spin coating at a rotation speed of 4000 rpm and heated at 115°C for 3 minutes. The photoresist was removed from the ITO portion corresponding to the second region of the pattern by exposure and development of the photoresist, and the ITO portion from which the photoresist had been removed was etched by immersion in an ITO etching solution (hydrochloric acid, 083-03435, Fujifilm Wako Pure Chemical Industries). 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) to obtain a cell culture substrate consisting of a synthetic quartz substrate and the first layer of ITO patterned 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 previously obtained substrate was placed in a petri dish with the first layer facing the dish, and the Type I collagen solution or Poly-D-lysine solution was introduced into the gap between the surface of the petri dish and the first layer. The dish was then placed in a refrigerator at 4°C and left to stand overnight.

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

[0051] FIG. 5 is a fluorescence image of the cell culture substrate immediately after seeding cells on a cell culture substrate without a second layer (a), and on the 4th day after culture with an integrated exposure dose of ultraviolet light of 0 mJ / cm 2 . In this example, the integrated exposure dose was calculated as the energy amount of light with a wavelength of 254 nm. Since ultraviolet light was not irradiated during the culture, it was confirmed from FIG. 5 that cells grew in all of the first, second, and third regions.

[0052] FIG. 6 is a fluorescence image of the cell culture substrate immediately after seeding cells on a cell culture substrate provided with a second layer made of Poly-D-Lysine (a), and on the 4th day after culture with an integrated exposure dose of ultraviolet light of 0 mJ / cm 2 . Also in FIG. 6, since ultraviolet light was not irradiated during the culture, it was confirmed that cells grew in all of the first, second, and third regions, similar to FIG. 5.

[0053] FIG. 7 is a fluorescence image of the cell culture substrate immediately after seeding cells on a cell culture substrate without a second layer (a), and on the 4th day after culture with an integrated exposure dose of ultraviolet light of 1038 mJ / cm 2 and an exposure time of 600 sec once a day. It was confirmed from FIG. 7 that ultraviolet irradiation killed the cells in the second region and generated cell colonies isolated from the surroundings in the first region.

[0054] FIG. 8 is a fluorescence image of the cell culture substrate immediately after seeding cells on a cell culture substrate provided with a second layer made of collagen (a), and on the 4th day after culture with an integrated exposure dose of ultraviolet light of 1038 mJ / cm 2Figure 8 shows a fluorescence image of the cell culture substrate on day 4 after culturing, with an exposure time of 600 seconds once a day (b). From Figure 8, it was confirmed that UV irradiation killed the cells in the second region, similar to the case in Figure 7. In addition, it was confirmed that isolated cell colonies were formed in the first region, and a higher proliferation effect was obtained than in Figure 7.

[0055] (3) Examination of 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 creating a second layer. The cumulative ultraviolet exposure was 1038 mJ / cm². 2 (Exposure time was set to 600 sec per day). Figure 9 shows the fluorescence image of the substrate on day 4 after culture. In Figure 9, the four patterns in the upper part of the image had a first region diameter X of 965 μm. The three patterns from the right in the middle part of the image had a first region diameter X of 713 μm. The patterns on the left in the middle part of the image and the two patterns from the right in the lower part of the image had a first region diameter X of 455 μm. The two patterns from the left in the lower part 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 values ​​of multiple different patterns in Figure 9. From Figure 9, when the first region was circular, with a diameter X of approximately 200-1000 μm and a width Y of 100 μm, cell patterning was possible in all cases. More specifically, we succeeded in growing cells in the first and third regions, killing and removing cells in the second region, and generating cell colonies in the first region.

[0056] (4) Examination of cell proliferation rate in the first region and the number of cells bound to the second region. The type of coating material and the diameter X of the first region were changed, and the cell proliferation rate in the first region and the number of cells binding to the second region were investigated. The type of coating material and application method were the same as in (2). The diameter X of the first region was prepared aiming for 500 μm, 750 μm, and 1000 μm. As an indicator of the cell proliferation rate in the first region, the ratio of the area occupied by cells to the area of ​​the first region was used, and the occupied area was obtained at the time of cell seeding, on day 2 of culture, and on day 4 of culture. The method of obtaining the occupied area was by measuring the area that emits fluorescence by image analysis. The number of cells binding to the second region was evaluated by the number of places where the inner circle and the outer circle are connected by cells in the image, with the boundary between the first and second regions defined as the inner circle and the boundary between the second and third regions as the outer circle. The existence of places where the inner and outer circles are connected by cells means that the cell colonies in the first region are 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 area. In each figure, the top row (a) shows the evaluation results for a pattern with a layer made of collagen (Collagen), the middle row (b) shows the evaluation results for a pattern with a layer made of poly-D-lysine (Poly-D-Lysine), and the bottom row (c) shows the evaluation results for a pattern without a second layer (NON). The cumulative ultraviolet exposure is 0 mJ / cm² in Figure 10. 2 Figure 11 shows 519 mJ / cm². 2 (Exposure time: 300 sec), Figure 12 shows 1038 mJ / cm² 2 The exposure time was 600 seconds, and exposure was performed once a day. In each graph, white plots represent the cell proliferation rate within the first region, expressed as the percentage of cell area occupied by the cells relative to the area of ​​the first region on the left axis. Black plots represent the number of cell bindings to the second region on the right axis. If the number of cell bindings to the second region is greater than 10, it is represented as 10.

[0058] Figures 10 to 12 show that increasing the amount of ultraviolet light exposure significantly reduces 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 coated with a special material allows for the killing and removal of cells in the second region without reducing the cell proliferation rate in the first region, thereby enabling the formation of cell colonies.

[0059] Example II. Cell culture substrate having a regular hexagonal pattern in the first region. (1) Design and manufacture of honeycomb-type cell culture substrates A cell culture substrate was designed in which a first region is a regular hexagon, and 16 regular hexagons are arranged vertically and horizontally with a second region surrounding the hexagon as the boundary, and a third region is provided on the outer perimeter of these. In this embodiment, a cell culture substrate having a cell proliferation area of ​​this shape is referred to as a honeycomb-type cell culture substrate. Figure 13 is a diagram illustrating the design of the 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 regular hexagons, B is defined as the design value of the gap length in the diametrical direction of the circumscribed circle, C is defined as the design value of the width of the second region, and D is defined as the design value of the distance between two parallel sides of the regular hexagon.

[0060] Six types of samples were designed by modifying A to D, and based on the designs, the first layer, ITO, was patterned in the same manner as in (2) of Example I. The second layer was made up of Type I collagen (Merck, catalog number C9791-10MG). Figure 14 is an image of the cell culture substrate actually manufactured based on the design drawing in Figure 13, showing a honeycomb-shaped cell proliferation area (a) and a magnified photograph (b) of region Z in (a). In Figure 14, E is defined as the measured diameter of the circumscribed circle of the first region of the regular hexagon, F as the measured gap length in the diametrical direction of the circumscribed circle, G as the measured width of the second region, H as the measured distance between two parallel sides of the regular hexagon, and I as the measured width of the ITO layer that was partially etched by undercutting.

[0061] Table 1 shows the design values ​​and measured values ​​for six different samples. In the table, design values ​​are abbreviated as Des, and measured values ​​as Mea. The measured value Mea in the table represents the average value of the results measured at three different locations. [Table 1]

[0062] Table 1 shows that undercuts, which are unavoidable in the manufacturing process, occurred, but the product was manufactured almost exactly as designed.

[0063] (2) Design and manufacturing of single hexagonal cell culture substrates A cell culture substrate with a pattern similar to that in Figure 1 or Figure 3 was designed, except that the circular first region 11 in Figure 1 or Figure 3 was changed to a regular hexagon, and a second region of uniform width was provided along the periphery of the regular hexagon. Only one first region was provided, and the outer periphery of the second region became the third region. In this embodiment, a cell culture substrate having a cell proliferation area of ​​this shape is referred to as a single hexagonal type 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, catalog number C9791-10MG).

[0064] (3) Cell patterning using cell culture substrates Sample No. 1 is a honeycomb-type cell culture substrate, and a single hexagon-type cell culture substrate with a design value A of 450 μm for the circumscribed circle diameter of the first region and a design value B of 50 μm for the gap length, are used to create a 5.0 × 10⁻¹⁴ cell culture substrate. 5 A cell suspension containing human glioblastoma cell line U-87, modified to express fluorescent protein (GFP), was seeded at a concentration of cells / mL and cultured for 2 days. From the start of culture, the cells were exposed to ultraviolet light with a peak wavelength of 254 nm for a predetermined duration once daily. Cells on the substrate were observed immediately after seeding and 2 days later. The observation results are shown in Figures 15-18.

[0065] Figure 15 shows the cells immediately after seeding on a honeycomb-type cell culture substrate (a), and the cumulative ultraviolet exposure dose at 0 mJ / cm².2 Figure 15 shows the fluorescence image on day 2 after culturing (b). No ultraviolet light was irradiated during culturing, and as can be seen from Figure 15, cell proliferation was confirmed in both the first and second regions. Here, in the second region, there is no absorption of fluorescence of the human glioblastoma cell line U-87 by the ITO layer, so it is thought that the U-87 cells in the second region appear relatively brighter than the U-87 cells in region 1. Figure 16 shows the image immediately after seeding cells on a honeycomb-type cell culture substrate (a), and with an integrated ultraviolet exposure of 2076 mJ / cm². 2 Figure 16 shows a fluorescence image taken on the second day after culturing, with an exposure time of 1200 seconds once a day (b). From Figure 16, it was confirmed that UV irradiation killed the cells in the second region, and cell colonies isolated from other first regions were generated in multiple hexagonal first regions.

[0066] Figure 17 shows the cells immediately after seeding on a single hexagonal cell culture substrate (a), and the integrated ultraviolet exposure dose of 0 mJ / cm². 2 The image shows the fluorescence image on day 2 after culturing (b). No ultraviolet light was irradiated during culturing, and as can be seen from Figure 17, cell proliferation was confirmed in both the first and second regions. Figure 18 shows the cells immediately after seeding on a single hexagonal cell culture substrate (a), and with an integrated ultraviolet exposure of 2076 mJ / cm². 2 Figure 18 shows the fluorescence image on day 2 after culturing, with an exposure time of 1200 seconds once a day (b). From Figure 18, it was confirmed that UV irradiation killed the cells in the second region, and isolated cell colonies were generated in a single first region.

[0067] The results shown in Figures 15 to 18 confirm that cell patterning is possible using hexagonal ITO layers, similar to circular patterns. Furthermore, it was confirmed that similar results can be obtained by densely arranging hexagonal ITO layers in a honeycomb pattern.

[0068] (4) Examination of cell proliferation rate in the first region and the number of cells bound to the second region. For honeycomb-type and single-hexagon-type cell culture substrates, the design value A for the circumscribed circle diameter of the first region and the design value B for the gap length were changed, and after exposure to a predetermined amount of ultraviolet light, the cell proliferation rate in the first region and the number of cells bound to the second region were examined. The type and application of the coating material were the same as in Examples II (1) and (2), and the cell culture conditions were the same as in Example II (3). Exposure to ultraviolet light was performed once a day. Ultraviolet exposure dose: 0 mJ / cm 2 , 1038 mJ / cm² 2 (Exposure time: 600 sec), or 2076 mJ / cm² 2 The evaluation was performed for the case where the exposure time was 1200 sec. As an indicator of the cell proliferation rate in the first region, the ratio of the area occupied by cells to the area of ​​the first region was used, and the occupied area was obtained at the time of cell seeding and on the second day of culture. The method for obtaining the occupied area was the same as in (4) of Example I. For honeycomb-type cell culture substrates, the occupied area was evaluated using three regular hexagons randomly selected other than the outermost regular hexagon, and the average value was used. The number of cells bound to the second region was evaluated by the number of points where the outer perimeter of a certain first region and the outer perimeter of another adjacent first region or a third region are connected by cells. For honeycomb-type cell culture substrates, the number of bound cells was evaluated using three regular hexagons randomly selected other than the outermost regular hexagon, and the average value was used.

[0069] The evaluation results are shown in Figures 19-22. The legend in the upper right corner indicates the gap length design value B (μm). In each figure, the UV exposure dose is 0 mJ / cm² in the upper row (a). 2 The middle section (b) is 1038 mJ / cm². 2 The lower row (c) shows 2076 mJ / cm². 2 The results are shown below. In each graph, white plots represent the cell proliferation rate within the first region, expressed as the percentage of cell area occupied by the cells relative to the area of ​​the first region on the left axis. Black plots represent the number of cell bindings to the second region on the right axis. If the number of cell bindings to the second region is greater than 10, it is represented as 10.

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

[0071] Figures 19 to 22 show that, as with the circular case, increasing the amount of ultraviolet light exposure significantly reduces the number of cells binding to the second region, enabling cell proliferation in the first region, even when the first region is a regular hexagon. However, in this experiment, the cumulative exposure amount may not be sufficient to suppress cell binding when the gap length is 25 μm. Therefore, when the gap length is 25 μm or less, it is thought that increasing the cumulative exposure amount can more reliably suppress cell binding. [Explanation of symbols]

[0072] 1 Cell culture substrate 10 circuit boards 11. First Domain 12. Second Domain 20. Layer 1 21 Part 1 22 Part 2 30. Second layer 100 Cell Patterning Device 110 Ultraviolet irradiation device

Claims

1. A step of attaching cells to one side of a cell culture substrate having a first region that blocks ultraviolet light and a second region that transmits ultraviolet light, The process includes irradiating the cell culture substrate with ultraviolet light from the side opposite to the side to which the cells are attached, A cell patterning method comprising the step of irradiating with ultraviolet light to remove cells from the cell culture substrate that are attached to the cell culture substrate, wherein The cell culture substrate is A substrate that transmits visible light and ultraviolet light, A first layer formed on the substrate, having a portion that provides ultraviolet shielding performance. Equipped with, A cell patterning method wherein the portion having ultraviolet shielding performance is indium tin oxide.

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

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

4. A cell culture substrate having a first region that blocks ultraviolet light and a second region that transmits ultraviolet light, A UV irradiation device capable of irradiating the cell culture substrate with ultraviolet light, Equipped with, The cell culture substrate is A substrate that transmits visible light and ultraviolet light, A first layer formed on the substrate, having a portion that provides ultraviolet shielding performance. Equipped with, The portion having the aforementioned ultraviolet shielding performance is made of indium tin oxide. A cell patterning device that removes cells from the cell culture substrate that are attached to the cell culture substrate from the second region by irradiating it with ultraviolet light using the ultraviolet irradiation device.

5. A method for producing a patterned cell population, A step of attaching cells to one side of a cell culture substrate having a first region that blocks ultraviolet light and a second region that transmits ultraviolet light, A step of culturing the attached cells and Includes, The cell culture substrate is A substrate that transmits visible light and ultraviolet light, A first layer formed on the substrate, having a portion that provides ultraviolet shielding performance. Equipped with, The portion having the aforementioned ultraviolet shielding performance is made of indium tin oxide. A method comprising the step of irradiating the cell culture substrate with ultraviolet light from the side opposite to the side to which the cells are attached, in the culture step.

Images