JP7807774B2 - Cell culture substrate and cell culture method - Google Patents

Description

The present invention relates to a cell culture substrate and a cell culture method.

In the cultivation of general anchorage-dependent cells for the purpose of expansion, cell culture substrates made of plastics such as polystyrene (PS), which are easily surface-modified, or cell culture substrates coated with an extracellular matrix (ECM) composed of biomolecules such as polylysine and collagen as an adhesive substrate are used (e.g., Non-Patent Document 1). On the other hand, cell culture substrates used for culturing cells for imaging purposes are often made of glass, which has excellent optical properties, so that the cultured cells can be placed on the substrate and observed using an optical microscope. However, many established cell lines, primary cultured cells, embryonic stem (ES) cells, induced pluripotent stem (iPS) cells, etc. generally have difficulty adhering to glass surfaces. Furthermore, even if seeded cells do adhere to the glass surface, they detach from the adhesive surface during division, and are unable to re-adhere to the glass surface, resulting in death and difficulty in proliferation. Therefore, cell culture substrates made of plastics with excellent optical properties, such as cycloolefin resin (COP), have been developed (e.g., Patent Document 1 and Non-Patent Document 2).

Patent No. 4391523

Masamasa Taniguchi and Kiyotoshi Sekiguchi, "Development of culture substrates for stem cells: current status and challenges," Bioengineering, Vol. 92, No. 9, pp. 491-494, 2014 Aurora Microplates Catalog, Wakembtech Co., Ltd., [Retrieved December 1, 2020], Internet <URL: https://www.wakenbtech.co.jp/wp/wp-content/uploads/2016/03/1604-Aurora_Catalog-J-1.pdf>

For the cultivation of some established cell lines and primary cultured cells such as neurons and hepatocytes, glass substrates can be used by coating them with an optimized extracellular matrix. However, for human ES/iPS cells and their differentiated cells, which are used in research in the field of regenerative medicine, glass substrates provide insufficient cell adhesion, making proliferation difficult. Furthermore, cycloolefin resins are inferior to glass in terms of optical properties such as autofluorescence, making them insufficient for optical observation using confocal laser microscopes. Therefore, in order to stably cultivate and continuously observe poorly adherent human ES/iPS cells and their differentiated cells, a cell culture substrate is needed that has optical properties equivalent to glass substrates but also possesses the cell adhesion properties of plastic substrates.

The present invention was made in consideration of the above-mentioned problems, and aims to provide a cell culture substrate and cell culture method that can be widely used for culturing anchorage-dependent cells, including ES/iPS cells derived from mammals such as humans and mice, and that are suitable for optical observation.

To solve the above problems, the cell culture substrate of the present invention comprises a substrate made of glass and a thin resin film covering the upper surface of the substrate, the surface on which the thin resin film is provided is used as a cell culture surface, and the thickness of the thin resin film is smaller than half the wavelength of light for optical observation of placed cells . With this configuration, while using glass, which has excellent optical properties, as the substrate, the resin coating on the surface can impart cell adhesiveness to the glass substrate without impairing the optical properties of the glass.

To solve the above-mentioned problems, the cell culture method of the present invention involves a seeding step in which cells are seeded on the resin thin film side of a cell culture substrate, which is a glass substrate coated with a resin thin film; a culture step in which the cells are grown; and an observation step in which the cells are optically observed on the cell culture substrate, wherein the thickness of the resin thin film on the cell culture substrate is less than half the wavelength of light used in the observation step. Through these steps, the cell culture method enables the cultivation of anchorage-dependent cells on a cell culture substrate suitable for optical observation.

The present invention makes it possible to culture many anchorage-dependent cells and facilitates optical observation.

1 is a schematic diagram illustrating the configuration of a cell culture substrate according to an embodiment of the present invention. FIG. 1 is a flowchart illustrating a method for producing a cell culture substrate according to an embodiment of the present invention. 1 is a schematic diagram illustrating the configuration of a thin resin film for cell culture according to an embodiment of the present invention. FIG. 1 is a flowchart illustrating a cell culture method according to an embodiment of the present invention. Photographs of human ES cells cultured using test materials from examples and comparative examples of cell culture substrates according to the present invention, where (a) is an example (SPL1), (b) is a reference example (SPL2) of a polystyrene substrate, and (c) is a comparative example (SPL3) of a glass substrate without a thin resin film. Photographs of human iPS cells cultured using test materials from examples and comparative examples of cell culture substrates according to the present invention, where (a) is an example (SPL1), (b) is a reference example (SPL2) of a polystyrene substrate, and (c) is a comparative example (SPL3) of a glass substrate without a thin resin film. 1 is a graph showing the number of human ES cells cultured using an example (SPL1), a reference example (SPL2), and a comparative example (SPL3) of the cell culture substrate according to the present invention. 1 is a graph showing the number of human iPS cells cultured using an example (SPL1), a reference example (SPL2), and a comparative example (SPL3) of the cell culture substrate according to the present invention. 1 shows confocal micrographs of RNA-FISH analysis of human ES cells on test materials of examples and comparative examples of cell culture substrates according to the present invention, where (a) is an example (SPL1), (b) is a comparative example (SPL4) of a cycloolefin resin substrate, and (c) is a reference example (SPL3) of a glass substrate without a thin resin film. FIG. 1 is a distribution diagram of DAPI fluorescence intensity in RNA-FISH analysis of human ES cells on each test material of the cell culture substrate according to the present invention, in Examples and Comparative Examples.

Modes for implementing the cell culture substrate and cell culture method of the present invention will be described with reference to the drawings. The cell culture substrate and its elements shown in the drawings may be exaggerated in size, positional relationship, etc., and may be simplified in shape for clarity of explanation.

[Cell culture substrate]
As shown in Figure 1, a cell culture substrate 1 according to an embodiment of the present invention comprises a substrate 2 made of glass and a thin resin film 3 covering one surface (top surface) of the substrate 2. The cell culture substrate 1 is used for monolayer culture, and cells isolated from tissue or the like are seeded on the surface on which the thin resin film 3 is provided, which serves as a cell culture surface. Each element will be described in detail below.

(Base material)
The substrate 2 is the main component of the cell culture substrate 1. The substrate 2 is made of glass suitable for optical observation and is processed into a shape suitable for optical observation using a high-magnification optical microscope, a fluorescence microscope, or the like. The glass material and shape of the substrate 2 are not particularly limited as long as it can be used for optical observation. Furthermore, commercially available glass slides or cover slips for optical observation may also be used for the substrate 2. The substrate 2 has optical properties (high transmittance, low birefringence, low autofluorescence, etc.) appropriate for the type of optical observation. For example, borosilicate glass or quartz glass is used for the substrate 2. It is preferably flat and can have any desired planar shape, such as a rectangle or circle. The substrate 2 has the strength to support the resin thin film 3 and/or the extracellular matrix, while having a thickness suitable for optical observation. Specifically, the thickness of the substrate 2 is preferably 0.08 mm or more, more preferably 0.12 mm or more, and even more preferably 0.15 mm or more. Furthermore, a thickness of 1 mm or less is preferred for low-magnification observation, and 0.20 mm or less is preferred for high-magnification observation. In particular, it is preferable that the cell culture substrate 1, in which the substrate 2 is less than 1 mm thick, is sized so that it can be accommodated in a lidded culture vessel such as a dish (petri dish) or a well plate, or that it be attached to a plastic frame or the like as a bottom plate, as described below.

(thin resin film)
The resin thin film 3 is provided on the culture surface of the cell culture substrate 1. By covering one surface of the substrate 2, the resin thin film 3 facilitates adhesion and spreading of cells to be cultured. The resin thin film 3 is formed of a non-toxic, durable, and transparent resin, such as that used in common plastic dishes and well plates used for monolayer culture. Specifically, any of the following resins can be used: polystyrene (PS), polymethyl methacrylate (PMMA), polyethylene terephthalate (PET), polydimethylsiloxane (PDMS), polycarbonate (PC), polytetrafluoroethylene (PTFE), or cycloolefin resin (COP), with polystyrene being particularly preferred. Alternatively, any of the above resins can be copolymerized with a hydrophilic group-containing monomer to form the resin thin film 3 with a hydrophilic surface. To impart cell adhesiveness, the resin thin film 3 has a thickness of 5 nm or more, preferably 10 nm or more, more preferably 20 nm or more, and even more preferably 50 nm or more. On the other hand, although it depends on the method of coating the substrate 2, if the coating is too thick, adhesion to the substrate 2 may decrease and peeling may occur, so the thickness is preferably 300 nm or less, more preferably 200 nm or less.

Furthermore, when cells on the cell culture substrate 1 are optically observed using an optical microscope or visually, the resin thin film 3 has inferior optical properties compared to the glass substrate 2, so to avoid any adverse effects, it is preferable that the resin thin film 3 be thinner, with a thickness less than half the wavelength of light. For example, when observing using a fluorescence microscope with an excitation wavelength of 370 nm, this refers to less than 185 nm. When observing using a confocal laser microscope or other device that uses multiple single-wavelength light sources (e.g., 405 nm, 488 nm, 546 nm, 633 nm), this refers to less than 202.5 nm, which is less than half the 405 nm wavelength of the shortest single-wavelength light source. When observing using light with a wavelength range similar to visible light (e.g., 380-780 nm), this refers to less than 202.5 nm, which is less than half the central wavelength of 405 nm in the short-wavelength violet wavelength band (380-430 nm), and preferably less than 190 nm, which is less than half the lower limit of the wavelength band, 380 nm. Furthermore, when observing with a scanning multiphoton laser microscope using a near-infrared laser light source with a wavelength of 700 to 1100 nm, the thickness refers to less than 500 nm, which is smaller than half of 1000 nm, and preferably less than 350 nm, which is smaller than half of the lower limit of 700 nm, so that observation is possible with light in the 1000 to 1100 nm wavelength range. In other words, observation with light in a wavelength range exceeding twice the thickness of the resin thin film 3 is possible without being optically affected by the resin thin film 3. To ensure versatility of the cell culture substrate 1 and to ensure adhesion to the substrate 2, it is particularly preferable that the resin thin film 3 have a thickness of 200 nm or less. Note that the resin thin film 3 does not have to cover the entire top surface of the cell culture substrate 1; it is sufficient that it is provided in at least the region where cells are desired to spread. For example, it can be configured so that it does not cover the periphery in plan view.

To ensure adhesion to the substrate 2, the resin thin film 3 is preferably annealed, as described in the cell culture substrate manufacturing method described below. The resin thin film 3 may also be surface-modified as needed. Resins such as polystyrene and polyethylene terephthalate are hydrophobic and may have low affinity for cells and extracellular matrices, potentially resulting in insufficient adhesion to certain cells. Therefore, when culturing such cells on the cell culture substrate 1, the resin thin film 3 is preferably surface-modified to impart appropriate hydrophilicity by introducing hydrophilic functional groups to the surface. Treatments for imparting hydrophilicity include plasma irradiation, exposure to active oxygen via ultraviolet irradiation, and graft polymerization. Alternatively, the resin thin film 3 may be modified by silane coupling, with the amino groups further modified with glutaraldehyde. This treatment allows for strong crosslinking of various extracellular matrices. Known coupling agents can be used for the silane coupling treatment. The entire surface of the resin thin film 3 does not need to be surface modified; at least the area where cells are desired to spread needs to be surface modified.

(extracellular matrix)
Some cells to be cultured require an adhesive substrate (scaffold) on the culture surface, and therefore the cell culture substrate 1 for culturing such cells may have an extracellular matrix coated on the thin resin film 3 (not shown). The extracellular matrix is made of biomolecules that correspond to the cells to be cultured, and examples of such materials that can be used include fibronectin, laminin, vitronectin, collagen, poly-D-lysine, and Corning Matrigel (registered trademark) prepared from these.

(Method for producing cell culture substrate)
As shown in FIG. 2 , the cell culture substrate 1 is manufactured by performing a resin thin film formation step S1 in which a resin thin film 3 is formed on a substrate 2, followed by a surface modification treatment step S2 in which the surface of the resin thin film 3 is modified, and an adhesive substrate coating step S3a in which an extracellular matrix is coated on the resin thin film 3, as needed. The resin thin film formation step S1 can be performed using a known method appropriate for the material and film thickness of the resin thin film 3. For example, a coating step S11 is performed in which an organic solvent solution of the resin is applied to a uniform thickness on the substrate 2, and then dried to volatilize the solvent, thereby forming a resin thin film 3 of the desired thickness. Examples of solution application methods include spin coating, dip coating, and spraying. The resin thin film formation step S1 preferably includes an annealing step S12 in which the resin thin film 3 is annealed under conditions appropriate for the material after the coating step S11.

The surface modification process S2 is performed after the resin thin film formation process S1, or before the adhesive substrate coating process S3a if it is performed. Surface modification is performed according to the material of the resin thin film 3, the cells to be cultured, and the extracellular matrix used for that. The surface modification process S2 can be performed using surface modification methods similar to those used on general plastic dishes. To impart hydrophilicity, plasma irradiation, exposure to active oxygen via ultraviolet irradiation, or graft polymerization can be applied. Known coupling agents can be used for the silane coupling process. The adhesive substrate coating process S3a involves coating the resin thin film 3 with an extracellular matrix, similar to general plastic dishes coated with an extracellular matrix.

The cell culture substrate 1 can also be used in combination with culture vessels such as plastic dishes made of polystyrene or other materials, or well plates. Specifically, in dishes such as glass-bottom dishes, which are composed of a plastic body with holes formed in the bottom surface and a glass plate that is attached to the bottom surface of the body to cover the holes, the glass plate can be replaced with the cell culture substrate 1. With this configuration, cells can be cultured integrally with the culture vessel using the cell culture substrate 1, and even if the cell culture substrate 1 (substrate 2) is thin, it can support cells and medium. Furthermore, the cultured cells can be observed without removing them from the culture vessel together with the cell culture substrate 1.

[Resin thin film for cell culture]
Next, the cell culture resin thin film 3A will be described. As shown in FIG. 3 , the cell culture resin thin film 3A according to an embodiment of the present invention is a sheet-like member that covers the culture surface of a substrate 2A to form a cell culture substrate. The resulting cell culture substrate is used for monolayer culture. In particular, by covering a glass substrate 2A having the same configuration as the substrate 2 of the cell culture substrate 1, optical observation is possible, as with the cell culture substrate 1 according to the above embodiment. That is, the cell culture resin thin film 3A has the same configuration as the resin thin film 3 of the cell culture substrate 1 and is configured to be formed as a single unit. Therefore, the cell culture resin thin film 3A is formed from a non-toxic, durable, and transparent resin that is commonly used in plastic dishes, well plates, and the like, used for monolayer culture. Specifically, any of the following resins can be used: polystyrene (PS), polymethyl methacrylate (PMMA), polyethylene terephthalate (PET), polydimethylsiloxane (PDMS), polycarbonate (PC), polytetrafluoroethylene (PTFE), or cycloolefin resin (COP), with polystyrene being particularly preferred. Alternatively, any of the above resins can be copolymerized with a hydrophilic group-containing monomer to form a resin thin film for cell culture with a hydrophilic surface. Furthermore, the resin thin film for cell culture 3A preferably has a thickness of 50 nm or more to ensure sufficient strength to prevent breakage. Furthermore, the resin thin film for cell culture preferably has a thickness of 200 nm or less to be attached to a substrate without the use of an adhesive, as described below.

(Method for producing a resin thin film for cell culture)
The cell culture resin thin film 3A is produced by forming it on a substrate made of silicon or the like and then peeling it off from the substrate. The method for forming the cell culture resin thin film on the substrate is the same as the coating step S11 in the cell culture substrate manufacturing method. Before applying an organic solvent solution of the resin that forms the cell culture resin thin film 3A, it is preferable to use a release agent, a material that is insoluble in the organic solvent of the organic solvent solution but soluble in a solvent (aqueous solvent) that is insoluble in the resin, and apply the solution to the substrate in the same manner as in the coating step S11 to form a film. Examples of materials that can be used as release agents include acrylic acid-based water-soluble polymers such as polyacrylic acid (PAA), polymethacrylic acid, and polyacrylamide, polystyrene sulfonate (PSS), polyethylene glycol (PEG), polyvinyl alcohol (PVAL), and nonionic water-soluble polymers of polysaccharides such as starch and cellulose acetate. The release agent preferably has a thickness of 0.01 to 10 μm. After forming the cell culture resin thin film 3A on the substrate with a release agent, the substrate is immersed in an aqueous solvent (e.g., water) to dissolve the release agent and peel the cell culture resin thin film 3A from the substrate. The cell culture resin thin film 3A is then lifted out of the aqueous solvent and dried. The cell culture resin thin film 3A may be stored and distributed while attached to the substrate. In this case, the substrate preferably has a small thickness, preferably a few mm or less, more preferably 1 mm or less, and more preferably 10 μm or more.

The cell culture resin thin film 3A is attached to the substrate 2A by contacting the culture surface side. The cell culture resin thin film 3A exhibits high adhesion when it is approximately 200 nm or less in thickness, and is attached to the substrate 2A solely by physical adsorption, such as van der Waals forces and electrostatic interactions. Furthermore, the cell culture resin thin film 3A that has coated the substrate 2A is preferably subjected to an annealing treatment to ensure adhesion to the substrate 2A. The annealing method is the same as the annealing step S12 in the manufacturing method of the cell culture substrate. The substrate 2A coated with the cell culture resin thin film 3A can be used in the same way as the cell culture substrate 1.

[Cell culture method]
A cell culture method using a cell culture substrate according to an embodiment of the present invention will be described. As shown in FIG. 4 , the cell culture method according to an embodiment of the present invention includes a seeding step S4 in which cells are seeded on the resin thin film side of the cell culture substrate according to the embodiment, a culture step S5 in which the cells are grown, and an observation step S6 in which the cells are optically observed on the cell culture substrate, and is characterized in that the thickness of the resin thin film of the cell culture substrate is smaller than half the wavelength of the light used in the observation step S6. The cell culture method further includes, as necessary, an adhesive substrate preparation step S3b in which an extracellular matrix is coated on the resin thin film side of the cell culture substrate before the seeding step S4. Steps S3b, S4, and S5 can be performed in the same manner as known monolayer culture.

The cells to be cultured using the cell culture method according to this embodiment are not particularly limited as long as they can be cultured in a monolayer on a plastic dish or plate such as polystyrene, and are suitable for optical observation using a confocal laser microscope or super-resolution microscope while placed (adhered) on a cell culture substrate. Cells that are difficult to culture on glass due to their anchorage dependency are particularly suitable. Specific examples include primary culture cells, ES/iPS cells and differentiated cells from animals such as mice and humans, and various established cell lines.

(Adhesive substrate preparation process)
In the adhesive substrate preparation step S3b, an extracellular matrix is applied to the resin thin film 3 of the cell culture substrate 1. The adhesive substrate preparation step S3b is performed when the cell culture substrate 1 is not pre-coated with an extracellular matrix and cells requiring an adhesive substrate are to be cultured.

(Seeding process)
In the seeding step S4, cells to be cultured are seeded on the culture surface of the cell culture substrate 1, and a medium is supplied at the same time. At this time, the number of cells seeded is determined based on the area of the cell culture substrate 1 so as to achieve a predetermined seeding density. The medium may be supplied to the cell culture substrate 1 before the cells are seeded, or the cells may be suspended in the medium and then seeded.

(Culture process)
In the culture step S5, the cell culture substrate 1 on which the cells have been seeded is placed in an incubator or the like and exposed to a predetermined environment (temperature, humidity, _CO2 concentration, etc.). If necessary, the culture medium is added or replaced after a predetermined time has elapsed. The culture step S5 is performed at its longest for the cells to spread over the entire culture surface of the cell culture substrate 1 (the area covered with the resin thin film 3 and extracellular matrix).

(Observation process)
In the observation step S6, the cultured cells are observed under an optical microscope while still placed on the cell culture substrate 1. The optical microscope may be a confocal microscope, a total internal reflection microscope, a stimulated emission depletion (STED) microscope, or the like. The cells are also stained as necessary. If the cell culture substrate 1 has been cultured in a culture vessel, the cell culture substrate 1 together with the cells is removed from the culture vessel and placed on the stage of the optical microscope.

The above describes embodiments for carrying out the cell culture substrate and cell culture method according to the present invention. Below, we will explain examples that confirmed the effectiveness of the present invention. Note that the present invention is not limited to these examples and the above embodiments, and various modifications are possible within the scope of the claims.

[Cell culture experiment]
To evaluate cell adhesiveness, anchorage-dependent human ES cells and human iPS cells were cultured in monolayers, and the cell spreading state and cell number were observed.

(sample material)
A 0.17 mm thick, 18 mm x 18 mm (culture area 3.24 cm ² ) Zeiss cover glass, #0109030091 (manufactured by ZEISS), was used as the substrate. One side of the glass was spin-coated with a toluene solution of polystyrene (30 mg/mL, manufactured by Acros Organics) with a molecular weight of 260,000 at 4,000 rpm for 60 seconds, and then annealed overnight (approximately 8 hours) at 100 °C in a dryer to form a 160 nm thick polystyrene resin thin film. This produced a sample material (SPL1) according to an embodiment of the present invention. As a reference example, a polystyrene well plate (6 wells, no surface treatment) (#353046, manufactured by FALCON) with a diameter of 35 mm (culture area 9.62 cm ² ) was used as sample material SPL2. As a comparative example, the cover glass of sample material SPL1 was used as sample material SPL3. Furthermore, test materials SPL1 and SPL3 were placed in the same well plate as test material SPL2. Three wells of each test material were prepared for culturing human ES cells and human iPS cells, respectively.

(seeding, culturing)
Corning Matrigel® #354277 was applied as an extracellular matrix to the entire surface of one side of the test material (SPL1: the thin resin film side, SPL2: the entire bottom surface). StemFlex #A3349401 (Thermo Fisher Scientific) medium, supplemented with Rock inhibitor +, was then applied on top of the material, and human ES cells or human iPS cells were seeded. Human ES cells (SEES1) were seeded onto ^each test material at a seeding density of 5.2 x ¹⁰ cells/cm. Human iPS cells (ADSC-iPS) were seeded onto each test material at ^a seeding density of 1.0 x ¹⁰ cells/cm. The cells seeded onto the test material were cultured in an incubator adjusted to 37°C, ₅ % CO, 95% air, and 100% humidity. 24 hours after the start of the culture, the medium was replaced with a medium containing a rock inhibitor, and the cells were cultured for another 9 days.

(evaluation)
After a total of 10 days of culture, the cells on the test material were stained with Crystal Violet and visually observed. Furthermore, as an index of cell number per area, the signal intensity of the stained area was quantified using ImageJ (https://imagej.nih.gov/ij/) to calculate the value per culture area. Photographs of stained human ES cells are shown in Figures 5(a), (b), and (c), and photographs of human iPS cells are shown in Figures 6(a), (b), and (c). The dark areas on the test material are stained cells. Furthermore, a graph of the cell number of human ES cells is shown in Figure 7, and a graph of the cell number of human iPS cells is shown in Figure 8.

By using the sample material SPL1, a cell culture substrate according to an embodiment of the present invention, cells spread and proliferated on the culture surface, similar to the polystyrene sample material SPL2, as shown in Figures 5(a), (b) and 6(a), (b). Furthermore, as shown in Figures 7 and 8, it was confirmed that the example (sample material SPL1) was capable of culturing cells at a level comparable to that of sample material SPL2. In contrast, as shown in Figures 5(c) and 6(c), the seeded cells hardly spread or proliferated on sample material SPL3 (comparison example), which was made solely of glass. This confirmed that sufficient adhesiveness could be imparted to human ES/iPS cells using only a thin polystyrene film on the surface, without any surface modification treatment.

[Observation by optical microscope]
To evaluate the optical properties, cells seeded on the test material were observed before they began to divide, so that the state of the cells would not be affected by adhesion.

(sample material)
Sample material SPL1 used in Example 1 was prepared as a cell culture substrate according to an embodiment of the present invention, and sample material SPL3 (cover glass) was prepared as a reference example. Furthermore, a commercially available special polymer bottom petri dish BC-SFTD27 (manufactured by Biomedical Science Co., Ltd.) with a 0.1 mm-thick cycloolefin resin bottom plate for optical measurements was prepared as a sample material (SPL4) according to a comparative example.

(seeding)
As with the human ES cell culture in Example 1, Corning Matrigel (registered trademark) #354277 was applied as an extracellular matrix to the entire surface of one side of the test material, and StemFlex #A3349401 (manufactured by Thermo Fisher Scientific) medium supplemented with Rock inhibitor + was applied thereon, and human ES cells (SEES1) were seeded at a seeding density of 5.2 x 10 ⁴ cells/cm ^2. The cells seeded on each test material were exposed for a short period (2 hours) in an incubator prepared in the same manner as in Example 1, and then subjected to optical observation.

(evaluation)
Cells on the test material were stained blue with DAPI (4',6-diamidino-2-phenylindole) and subjected to RNA-FISH (Fluorescence in situ Hybridization) analysis. Optical observations were performed using a ZEISS LSM 880 confocal laser microscope with Airyscan (#LSM880) (manufactured by ZEISS) with a 100x objective lens. The laser light sources of the confocal laser microscope were 405 nm (blue-violet), 488 nm (blue), 546 nm (green), and 633 nm (red). Confocal micrographs of human iPS cells are shown in Figures 9(a), (b), and (c). Figure 10 shows the distribution of DAPI fluorescence intensity along the line crossing a single cell, represented by the white dashed line in Figures 9(a), (b), and (c).

As shown in Figures 9(a) and (c), using sample material SPL1, a cell culture substrate according to an embodiment of the present invention, cell tissue could be observed in the same way as with sample material SPL3, which is made solely of glass. Furthermore, in the microscopic image, bright blue nuclei, green XACT RNA, and red ubiquitin ligase HUWE1 were observed as dots within distorted oval cells stained purplish blue. In contrast, as shown in Figure 9(b), with sample material SPL4 (comparison example), made of a resin with improved optical properties, a clear image could not be obtained due to light absorption, and cells were barely visible with low contrast, making it difficult to see nuclei and other elements within the cells. Furthermore, as shown in Figure 10, the fluorescence brightness of the example (sample material SPL1) was equivalent to that of sample material SPL3, while that of sample material SPL4 was significantly lower.

The cell culture substrate of the present invention can be used in research and industry in the fields of molecular biology, stem cell biology, cell and tissue engineering, regenerative medicine, bioimage analysis, drug discovery, and more.

1 Cell culture substrate 2 Substrate 2A Substrate 3 Resin thin film 3A Resin thin film for cell culture S1 Resin thin film formation process S2 Surface modification treatment process S3a Adhesive substrate coating process S3b Adhesive substrate preparation process S4 Seeding process S5 Cultivation process S6 Observation process

Claims (11)

A cell culture substrate comprising a substrate made of glass and a thin resin film made of polystyrene covering the upper surface of the substrate,
The surface of the cell culture substrate on which the resin thin film is provided is used as a cell culture surface,
A cell culture substrate, characterized in that the thickness of the resin thin film is smaller than 1/2 of the wavelength of light used in optical observation of the cells placed thereon. the light is visible light,
The cell culture substrate according to claim 1 , wherein the thickness of the resin thin film is less than half the lower limit of the wavelength range of visible light. The cell culture substrate according to claim 1 or 2, wherein the thickness of the resin thin film is less than half the wavelength of the light emitted by the light source of the optical microscope used for the optical observation. The cell culture substrate according to any one of claims 1 to 3, wherein the resin thin film has a thickness of 200 nm or less. The cell culture substrate according to claim 1 , wherein the resin thin film has been subjected to an annealing treatment. The cell culture substrate according to claim 1 , wherein the resin thin film has been subjected to a surface modification treatment. The cell culture substrate according to claim 6 , wherein the surface modification treatment of the resin thin film is plasma irradiation, ultraviolet irradiation, or silane coupling treatment. The cell culture substrate according to claim 1 , further comprising an extracellular matrix on the resin thin film. The cell culture substrate according to claim 1 , wherein the substrate is in the form of a flat plate. a seeding step of seeding cells onto the resin thin film side of a cell culture substrate, the upper surface of which is made of glass and coated with a resin thin film made of polystyrene ;
a culturing step for growing the cells;
an observation step of optically observing the cells on the cell culture substrate,
The cell culture method is characterized in that the thickness of the resin thin film of the cell culture substrate is smaller than 1/2 of the wavelength of light used in the observation step. The cell culture method according to claim 10 , wherein an adhesive substrate preparation step is carried out before the seeding step, in which an extracellular matrix is coated on the surface of the cell culture substrate on the side of the resin thin film.