Cell culture substrate with tumor cell clusters as well as method for evaluating malignancy of tumor cells and method for evaluating test substances using the same

Abstract

To provide a cell culture substrate with tumor cell clusters having three-dimensional tumor cell clusters, in which tumor cell clusters that more closely reflect the pathological tumor tissue structure are formed; and furthermore, to provide a method for evaluating malignancy of tumor cells and a method for evaluating test substances sing the cell culture substrate with tumor cell clusters.SOLUTION: Provided is a cell culture substrate with a tumor cell cluster having a cell culture substrate and a three-dimensional tumor cell cluster. Therein: three-dimensional tumor cell cluster is attached to a surface of at least one side of the cell culture substrate; the cell culture substrate has an uneven structure with multiple convex portions or multiple concave portions on the surface (cell culture surface) of the cell culture substrate to which the three-dimensional tumor cell cluster is attached; and an average pitch between the multiple convex portions or the multiple concave portions is 200 nm or more and 650 nm or less.SELECTED DRAWING: None

Description

Technical Field

[0001] The present invention relates to a cell culture substrate with tumor cell masses, and a method for evaluating the malignancy of tumor cells and a method for evaluating a test substance using the same.

Background Art

[0002] In recent years, cell culture techniques have been used in various industrial fields (for example, Patent Documents 1 and 2). Patent Document 1 proposes a cell culture container that can be suitably used in biochemical experiments, clinical trials, drug development research, etc. Further, Patent Document 2 proposes a cell culture substrate for forming cell masses having morphological polarity and tissue movement characteristics as observed in cancer tissues in vivo.

Prior Art Documents

Patent Documents

[0003]

Patent Document 1

Patent Document 2

Summary of the Invention

Problems to be Solved by the Invention

[0004] On the bottom surface of the well of the cell culture container of Patent Document 1, a hydrophilic film with dot-shaped holes formed in a pattern is provided. This hydrophilic film suppresses non-specific adhesion of cells, and the cells adhere to the dot-shaped hole portions. However, since the dot-shaped holes are dispersed and arranged in the well as a result of pattern formation, it is not possible to culture cells so that cell masses are formed. In addition, in the cell culture substrate of Patent Document 2, cells are cultured on the rough surface portion not covered with the biocompatible polymer layer to form cell masses. However, since the step of selectively applying the biocompatible polymer uses a photolithography technique and is not suitable for mass production, there is room for improvement in industrial mass productivity during manufacturing.

[0005] An object of the present invention is to provide a cell culture substrate with tumor cell masses having three-dimensional tumor cell masses, in which tumor cell masses reflecting the tumor tissue structure of the pathological state are formed. Furthermore, an object of the present invention is to provide a method for evaluating the malignancy of tumor cells and a method for evaluating a test substance using the above-mentioned cell culture substrate with tumor cell masses.

Means for Solving the Problems

[0006] The above problems of the present invention can be solved by the following configurations <1> to <8>. <1> A cell culture substrate and a cell culture substrate with tumor cell masses having three-dimensional tumor cell masses, wherein the three-dimensional tumor cell masses adhere to the surface of at least one surface of the cell culture substrate, and the cell culture substrate has an uneven structure having a plurality of convex portions or a plurality of concave portions on the surface (cell culture surface surface) of the cell culture substrate to which the three-dimensional tumor cell masses adhere, and the average pitch between the plurality of convex portions or between the plurality of concave portions is 200 nm or more and 650 nm or less. A cell culture substrate with tumor cell masses. <2> The cell culture substrate with tumor cell masses according to <1>, wherein the cell culture substrate has an uneven structure continuous over the entire surface of the cell culture surface surface. <3> The cell culture substrate with tumor cell masses according to <1> or <2>, wherein the three-dimensional tumor cell mass has a thickness of 10 μm or more in the height direction. <4> The cell culture substrate with tumor cell masses according to any one of <1> to <3>, wherein the tumor cells constituting the three-dimensional tumor cell mass are epithelial tumor cells. <5> The cell culture substrate with tumor cell masses according to <4>, wherein the three-dimensional tumor cell mass has a lumen structure. <6> The cell culture substrate with tumor cell mass according to <4> or <5>, wherein vimentin-positive cells and CD68-positive cells are observed in the tumor cells in the three-dimensional tumor cell mass. <7> A method for evaluating the malignancy of tumor cells, comprising the steps of three-dimensionally culturing tumor cells on a cell culture substrate having an uneven structure with a plurality of convex portions or a plurality of concave portions on the surface of the cell culture substrate to obtain a cell culture substrate with tumor cell mass, and evaluating the malignancy of the tumor cells constituting the three-dimensional tumor cell mass on the obtained cell culture substrate with tumor cell mass, wherein the average pitch between the plurality of convex portions or between the plurality of concave portions is 200 nm or more and 650 nm or less. <8> A method for evaluating a test substance, comprising the steps of three-dimensionally culturing tumor cells on a cell culture substrate having an uneven structure with a plurality of convex portions or a plurality of concave portions on the surface of the cell culture substrate to obtain a cell culture substrate with tumor cell mass, allowing a test substance to be evaluated to act on the three-dimensional tumor cell mass on the obtained cell culture substrate with tumor cell mass, and evaluating the test substance based on the response of the three-dimensional tumor cell mass or the tumor cells constituting the three-dimensional tumor cell mass, wherein the average pitch between the plurality of convex portions or between the plurality of concave portions is 200 nm or more and 650 nm or less.

Advantages of the Invention

[0007] According to the present invention, there is provided a cell culture substrate with tumor cell mass having a three-dimensional tumor cell mass, in which a tumor cell mass reflecting the tumor tissue structure of a more pathological condition is formed. Further, according to the present invention, there are provided a method for evaluating the malignancy of tumor cells using the above cell culture substrate with tumor cell mass, and a method for evaluating a test substance.

Brief Description of the Drawings

[0008]

Figure 1

Figure 2

Figure 3-1

Figure 3-2

Figure 4

Figure 5

Mode for Carrying Out the Invention

[0009] Hereinafter, preferred embodiments of the present invention will be described. In this specification, "X to Y" indicating a range means "X or more and Y or less". When numerical ranges are described stepwise, the upper and lower limits of each numerical range can be arbitrarily combined.

[0010] [Cell Culture Substrate with Tumor Cell Aggregates] The cell culture substrate with tumor cell aggregates of the present invention is a cell culture substrate with a cell culture substrate and three-dimensional tumor cell aggregates, wherein the three-dimensional tumor cell aggregates adhere to the surface of at least one surface of the cell culture substrate, and the cell culture substrate has a concavo-convex structure having a plurality of convex portions or a plurality of concave portions on the surface (cell culture surface surface) of the cell culture substrate to which the three-dimensional tumor cell aggregates adhere, and the average pitch between the plurality of convex portions or between the plurality of concave portions is 200 nm or more and 650 nm or less. When a conventional planar cell culture substrate is used, tumor cell aggregates are cultured in a planar manner and do not have a three-dimensional structure. In contrast, in the cell culture substrate with tumor cell aggregates of the present embodiment, it is considered that tumor cell aggregates are three-dimensionally cultured and a tumor tissue closer to the tumor tissue in vivo is constructed. As a result, it can be used as a three-dimensional culture model, and the following effects are expected. · It becomes possible to construct a tissue structure of a millimeter-level microtumor similar to the patient's pathological condition · Since it does not have a micro pattern that serves as a scaffold for cell adhesion, it is possible to construct a spontaneous and free tumor tissue of tumor cells without being restricted in behavior by the pattern design. ·Enabling live cell imaging and three-dimensional analysis of tumor cell masses ·Enabling the reproduction of tissue architecture images similar to the thin cross-sections observed in patient pathological tissue specimens ·It is possible to construct cell masses with a three-dimensional structure on the order of micrometers, enabling the observation of sheet-like microtissues with a three-dimensional structure In addition, the present inventors have found that when adenocarcinoma cells are used as tumor cells in the cell culture substrate with tumor cell masses of the present embodiment, a luminal glandular structure, which is a characteristic of adenocarcinoma, is induced. As described above, this means that tumor cells are cultured in a form closer to the pathological condition in patients. Therefore, more specifically, it is considered that the effects of gene abnormalities and test substances such as anticancer agents on the construction of tumor tissues of tumor cells can be evaluated and analyzed

[0011] The detailed reasons for obtaining the above effects are unclear, but some are considered as follows In the present embodiment, the cell culture substrate has an uneven structure having a plurality of convex portions or a plurality of concave portions on the surface of the cell culture substrate (the surface of the cell culture surface) to which three-dimensional tumor cell masses are attached, and the average pitch of the plurality of convex portions or the plurality of concave portions is 200 nm or more and 650 nm or less Hereinafter, the present embodiment will be described in more detail

[0012] <Cell culture substrate> In the present embodiment, the cell culture substrate has an uneven structure having a plurality of convex portions or a plurality of concave portions on the surface of the cell culture substrate (the surface of the cell culture surface) to which three-dimensional tumor cell masses are attached, and the average pitch of the plurality of convex portions or the plurality of concave portions is 200 nm or more and 650 nm or less Incidentally, the above concavo-convex structure only needs to be formed on at least a part of the cell culture substrate. However, it is preferably not formed in a pattern, and more preferably has a continuous concavo-convex structure, that is, it is more preferably to have a continuous concavo-convex structure on the entire surface of the cell culture surface. Incidentally, "continuous on the entire surface of the cell culture surface" means that the concavo-convex structure is not formed in a pattern. For example, when placing a film having a concavo-convex structure on the bottom surface of a petri dish used for cell culture, it does not mean that the film covers the entire bottom surface of the petri dish.

[0013] From the viewpoint of stable adhesion of tumor cells, the average pitch of the plurality of convex portions or the plurality of concave portions is 200 nm or more and 650 nm or less, preferably 225 nm or more, more preferably 250 nm or more, still more preferably 275 nm or more, and preferably 625 nm or less, more preferably 600 nm or less, still more preferably 575 nm or less, even more preferably 550 nm or less, and particularly preferably 525 nm or less.

[0014] The average pitch of the plurality of convex portions is determined as follows. The cell culture substrate is observed in plan view using a scanning electron microscope (SEM). In a plurality of images in a magnification range in which 50 to 100 convex portions are imaged, 20 convex portions are randomly selected, and the arithmetic mean value of the shortest distances between the centers of the circumscribed ellipses of the minimum areas in plan view of two adjacent convex portions is taken as the average pitch of the plurality of convex portions. The average pitch of the plurality of concave portions is determined in the same manner as the average pitch of the convex portions, except that the shortest distance between the centers of the circumscribed ellipses of the minimum areas in plan view of two adjacent concave portions is measured. The details and preferred embodiments of the average pitch of the plurality of concave portions are the same as those described for the average pitch of the plurality of convex portions.

[0015] An uneven structure having a plurality of convex portions or a plurality of concave portions, for example, a plurality of concave portions are formed between the plurality of convex portions. Or, continuous convex portions are formed between the plurality of concave portions. Such an uneven structure having a plurality of convex portions and a plurality of concave portions can function as a scaffold for cells like the extracellular matrix. Therefore, it is considered that by having an uneven structure, high cell adhesiveness is exhibited and tumor cells can form a three-dimensional structure. The shapes of the convex and concave portions of the uneven structure are not particularly limited. The shapes of the convex and concave portions may be conical, cylindrical, frustoconical, pyramidal, or bell-shaped. Also, it is not limited to these exemplified shapes. Among these, from the viewpoint of obtaining stable cell adhesiveness, it is preferable that the cell culture substrate is a cell culture substrate having a plurality of convex portions.

[0016] The average height of the plurality of convex portions or the average depth of the plurality of concave portions is preferably 250 nm or more, more preferably 300 nm or more, still more preferably 400 nm or more, from the viewpoints of stable adhesion of tumor cells and ease of manufacture, and is preferably 2 μm or less, more preferably 1.2 μm or less, still more preferably 800 nm or less. Incidentally, the average height of the plurality of convex portions and the average depth of the plurality of concave portions are obtained as follows. The culture substrate is cut perpendicularly to the surface at an arbitrary position by a microtome or CP processing (ion milling), etc., and the cross section is observed with a scanning electron microscope (SEM). The convex or concave portions are randomly selected, the height of the convex portion or the depth of the concave portion is measured, and the arithmetic mean thereof is taken as the average height of the plurality of convex portions or the average depth of the plurality of concave portions. Here, the height of each convex portion is measured as the shortest distance in the thickness direction of the substrate between the apex of the convex portion and the base surface, and the height of each concave portion is measured as the shortest distance in the thickness direction of the substrate between the apex of the concave portion and the substrate surface. In addition, the "base surface of the substrate" means the flat surface of the substrate itself when the surface of the substrate is a flat surface. When a fine uneven structure is formed on the surface of the substrate, the "base surface of the substrate" means the surface obtained by removing all the convex portions from the surface, or the surface formed by filling all the concave portions of the surface. Instead of the average height of the plurality of convex portions, the average depth of the plurality of concave portions may be measured. The average depth of the plurality of concave portions is obtained in the same manner as the average height of the convex portions, except that the depth of each concave portion is measured as the shortest distance between the base surface of the concave portion at the same height position as the apex of the adjacent convex portion and the base surface of the uneven structure. The details and preferred embodiments of the average depth of the plurality of concave portions are the same as those described for the average height of the plurality of convex portions.

[0017] The material of the cell culture substrate is not particularly limited, and examples thereof include a glass substrate made of inorganic glass and a resin substrate made of an organic polymer resin. Among these, as the cell culture substrate, a hydrophobic substrate is preferable, and from the viewpoint of ease of manufacture (moldability, manufacturing cost), a resin substrate made of an organic polymer resin is preferable. Examples of the substance constituting the organic polymer resin substrate include a thermoplastic resin, a cured product of a thermosetting resin, and a cured product of a photocurable resin. Among these, a substrate containing at least one resin selected from the group consisting of polyethylene terephthalate (PET), triacetyl cellulose (TAC), polycarbonate (PC), cycloolefin polymer (COP), cycloolefin copolymer (COC), acrylic resin (such as polymethyl methacrylate (PMMA)), polystyrene (PS), and polydimethylsiloxane (PDMS) is preferable. However, the resin constituting the organic polymer resin substrate is not limited to these examples. Among these, from the viewpoint of moldability, PC, COP, COC, and PS are more preferable, and COP, COC, and PS are even more preferable.

[0018] Examples of the substance constituting the inorganic glass substrate include, but are not limited to, quartz glass, various alkali glasses, various alkali-free glasses, etc. When forming the fine uneven structure by the glass imprint process, lead borosilicate glass which is a low melting point glass and lead-free low melting point glass are preferably used. In addition, for example, a method of forming a fine structure by UV imprinting in which a UV curable resin is applied on an alkali glass and exposed in a state where a plate of the fine structure is pressed against the resin coating layer is also possible. A combination of an inorganic glass substrate and an organic polymer material as in this example is also effective.

[0019] The overall shape of the substrate is not particularly limited. For example, film shape, sheet shape, plate shape, block shape, etc. may be mentioned. The overall shape of the substrate can be appropriately selected according to the use of cultured cells, cell aggregates, etc. For example, when manufacturing a cell sheet in which cell aggregates are formed on the surface, the overall shape of the substrate is preferably sheet-shaped.

[0020] A hydrophilic coating layer may be formed on the surface of the cell culture surface of the substrate for the purpose of suppressing non-specific adhesion of cells. The hydrophilic coating layer is a layer of a hydrophilic material and imparts hydrophilicity to the surface of the cell culture substrate. The hydrophilic coating layer is preferably a biocompatible polymer, and "biocompatible polymer" means a polymer compound that does not have a harmful effect on cells. The hydrophilic material constituting the hydrophilic coating layer is not particularly limited as long as it has hydrophilic functional groups such as a hydroxyl group, a carbonyl group, a carboxy group, etc. Examples of the biocompatible polymer include polydimethylsiloxane (PDMS), polyethylene glycol (PEG), oligoethylene glycol (OED), 2-methacryloyloxyethyl phosphorylcholine (MPC), poly(MPC-co-butyl methacrylate) (PBM), poly(MPC-co-dodecyl methacrylate) (PMD), etc.

[0021] The thickness of the hydrophilic coating layer is preferably 0.005 μm or more and 0.500 μm or less, more preferably 0.010 μm or more, and more preferably 0.300 μm or less, and still more preferably 0.100 μm or less, from the viewpoints of suppressing non-specific adhesion and productivity. The thickness of the hydrophilic coating layer is measured as the shortest distance between the surface of the substrate and the upper surface of the hydrophilic coating layer. In addition, in the present embodiment, it was confirmed that there is no significant difference in the morphology and adhesiveness of the three-dimensional tumor cell mass depending on the presence or absence of the hydrophilic coating layer. Therefore, the hydrophilic coating layer may or may not be provided.

[0022] When the substrate is a resin substrate (organic polymer resin substrate), a hydrophilic functional group may be introduced onto the surface of the resin substrate. For example, by irradiation treatment such as plasma irradiation, ion irradiation, radical irradiation, ultraviolet irradiation, etc., the chemical bonds of the molecules on the surface of the substrate are broken, and depending on the type of resin, a hydrophilic functional group is introduced onto the surface of the resin substrate. The portion where the hydrophilic functional group is introduced on the surface of the resin substrate exhibits hydrophilicity. Among these, plasma irradiation and ultraviolet irradiation are preferable from the viewpoints of less physical damage to the substrate surface and ease of manufacture.

[0023] An adhesion factor layer may be provided on the cell culture surface of the cell culture substrate to enhance cell adhesiveness. Examples of the adhesion factor include extracellular matrices such as laminin, collagen, gelatin, fibronectin, polylysine (PDL, PLL), hyaluronic acid, polymers, gels, and the like.

[0024] Furthermore, in order to facilitate detachment and recovery of the cultured cells, a layer made of a stimulus-responsive material may be formed on the cell culture surface. As the stimulus-responsive material, a temperature-responsive polymer whose hydrophilicity changes with temperature change is preferable, and poly-N-isopropylacrylamide (PIPAAm) is preferably exemplified. The layer made of the stimulus-responsive material may be formed by applying the stimulus-responsive material to the surface of the substrate, may form the concavo-convex structure after applying the stimulus-responsive material to the substrate, or may apply the stimulus-responsive material to the substrate after forming the concavo-convex structure. In addition, when applying the stimulus-responsive material to the substrate after forming the concavo-convex structure, it is preferable to apply the stimulus-responsive material to the substrate before the hydrophilic treatment.

[0025] (Method for producing the substrate) The method for forming the concavo-convex structure on the surface of the resin substrate is not particularly limited. For example, a method of producing an original plate (X1) having a concavo-convex structure on its surface and producing a mold (Y1) with the surface shape of the original plate (X1) reversed from the original plate (X1) is preferable. This is because industrial mass productivity is further excellent in that the fine concavo-convex structure can be transferred to the substrate by repeatedly using the mold (Y1) as a stamper. The method for producing the original plate (X1) is not particularly limited. A fine concavo-convex structure may be formed on the surface of a substrate (for example, a Si substrate) by methods such as vapor phase etching, colloidal lithography, anodic oxidation, and interference exposure. For the production of the original plate (X1), for example, a single-particle film etching mask made of colloidal silica described in JP-A-2009-034630 may be used. A fine concavo-convex structure can be uniformly and highly accurately formed on the substrate by vapor phase etching using the single-particle film etching mask.

[0026] The method for producing the mold (Y1) is not particularly limited. For example, a metal layer is formed on the surface of the original plate (X1) having a fine concavo-convex structure or the like, and a fine structure or the like is transferred to the metal layer. Examples of the method for forming the metal layer include a method of providing the metal layer on the surface of the original plate (X1) by electroless plating or metal evaporation and increasing the thickness of the metal layer by electroplating. However, the method for forming the metal layer is not limited to this method at all. Also, the material of the metal layer is not particularly limited. For example, nickel, copper, gold, silver, platinum, titanium, cobalt, tin, zinc, chromium, gold-cobalt alloy, gold-nickel alloy, solder, copper / nickel / chromium alloy, tin-nickel alloy, nickel-palladium alloy, nickel / cobalt / phosphorus alloy, etc. can be mentioned. After forming the metal layer, when separating the metal layer from the original plate (X1), a mold (Y1) with the surface shape and surface structure of the original plate (X1) inverted can be obtained. This mold (Y1) may be used for nanoimprinting, injection molding, etc. to form a fine uneven structure region including a plurality of convex portions or a plurality of concave portions on the surface of the base material.

[0027] (Tumor cells) The cell culture substrate with tumor cell mass of the present embodiment can be obtained by culturing tumor cells on the above-described cell culture substrate. Note that tumor cells mean a group of cells that grow autonomously and aimlessly, and include all of epithelial, non-epithelial, benign, and malignant types. Although not particularly limited as the tumor cells to be used, from the viewpoint of culturing on the cell culture substrate, it is preferably adherent tumor cells, more preferably epithelial tumor cells, and even more preferably malignant epithelial tumor cells. For example, cervical cancer cells such as HeLa cells, pancreatic cancer cells, lung cancer cells, colorectal cancer cells, head and neck cancer cells, etc. can be mentioned. Among these, pancreatic cancer cells, lung cancer cells, colorectal cancer cells, and head and neck cancer cells are preferred, and pancreatic duct adenocarcinoma cells are more preferred.

[0028] Tumor cells are cultured in a state where they can come into contact with the cell culture substrate. Typically, they are cultured in a cell culture container provided with a cell culture substrate having an uneven structure on the surface of the cell culture surface. Culture conditions such as the medium other than the cell culture substrate, culture temperature, culture time, CO2 concentration, etc. may be those commonly used in the culture of the tumor cells to be used. Note that when using, for example, pancreatic cancer cells, lung cancer cells, colorectal cancer cells, or head and neck cancer cells that do not require coating with extracellular matrix (ECM), by culturing in DMED supplemented with fetal bovine serum at 37°C for about 1 night to 48 hours, a three-dimensional tumor cell mass composed of these cells can be obtained. Since the time until the formation of three-dimensional tumor cell aggregates varies depending on the type of tumor cells, the seeding concentration, etc., the culture time is appropriately set according to the type of tumor cells used, seeding conditions, culture conditions, etc. In addition, the morphology of the three-dimensional tumor cell aggregates may also vary depending on the tumor cells used.

[0029] The three-dimensional tumor cell aggregates preferably have a thickness of 10 μm or more in the height direction. This reveals that the tumor cell aggregates are three-dimensionally constructed. When tumor cells are cultured on a cell culture substrate with a flat cell culture surface as in the prior art, the tumor cells adhere to the surface of the culture substrate and proliferate two-dimensionally, so the formation of tumor cell aggregates in the height direction was not observed. In the present embodiment, it is preferable that three-dimensional tumor cell aggregates having a thickness of 10 μm or more in the height direction are formed. The thickness of the three-dimensional tumor cell aggregates in the height direction is preferably 10 μm or more, more preferably 30 μm or more, and even more preferably 50 μm or more. The thickness of the three-dimensional tumor cell aggregates is measured by the method described in the examples. Also, the thickness of the three-dimensional tumor cell aggregates may be measured by three-dimensional construction of Z-axis continuous fragment images using a confocal laser microscope.

[0030] As described above, the cell culture substrate with tumor cell aggregates of the present embodiment can be used as a cell adhesion type three-dimensional culture model, and it is considered that a tissue structure of a microtumor similar to the pathological condition presented by the tumor cells is induced. Also, by having it on one side (continuously) without forming the concavo-convex structure in a pattern, the construction of the tumor tissue becomes possible without being restricted in behavior by the pattern design. Furthermore, with the cell culture substrate with tumor cell aggregates, observation of living cells is possible, and live imaging and three-dimensional analysis of living cells are also possible. On the other hand, on the base surface (XY axis) of the substrate, culturing can be carried out at the millimeter level and centimeter level, and in the thickness direction (Z axis), culturing is carried out at the micro level. Therefore, while having a large-scale tumor tissue structure, a thin three-dimensional structure that can be observed with a microscope or lens is formed, and a three-dimensional tumor cell mass suitable for observation is formed.

[0031] As the tumor cells, adenocarcinoma cells are particularly suitable. When adenocarcinoma cells are cultured on the uneven substrate of the present embodiment, a lumen structure peculiar to adenocarcinoma cells is formed in a few days. That is, by culturing adenocarcinoma cells on an uneven substrate, spontaneous and free tumor tissue construction is induced, and it is considered that the entire glandular tissue structure can be reproduced. The lumen structure in the cancer tissue of a living body refers to a lumen structure by Hollowing-type lumen formation in which constituent cells expand to form a lumen, a lumen structure by cavitation-type lumen formation in which the lumen of a solid part forms a lumen by causing cell death, and other comedo patterns, cribriform, papillary patterns, etc. in the tumor structure patterns observed in the tumor lesions of pathological tissue sections of cancer patients are cited as examples. In the present invention, the lumen structure means a circular or hole-shaped structure with a diameter of 30 μm or more surrounded by cells, where the cell mass shows the shape of an island structure on the cell culture substrate. That is, when there is a circular structure or hole-shaped structure with a diameter of 30 μm or more in a shape with a size of 500 μm or more having a continuous shape of cells, it is considered to have a lumen structure. The lumen structure formed by the three-dimensional tumor cell mass is considered to reflect the differentiation type of the tumor cells. In highly differentiated tumor cells, a distinct lumen structure is formed, while in moderately differentiated tumor cells, disruption of the lumen structure is observed. Therefore, the cell culture substrate with tumor cell masses cultured on a substrate having an uneven structure is effective for evaluating the degree of atypia, differentiation, and malignancy of tumor cells.

[0032] When using adenocarcinoma cells, particularly pancreatic cancer cells, as tumor cells, it is preferable that vimentin-positive cells and CD68-positive cells are observed in the tumor cells in the three-dimensional tumor cell mass. Generally, in pathological tissue examination, cells stained with the CD68(+) vimentin(+) phenotype accumulated in the tumor site and its luminal structure are diagnosed as infiltration and accumulation of tumor-associated macrophages (TAM). The presence of vimentin-positive and CD68-positive cells indicates that a large number of pancreatic cancer cells with a phenotype similar to that of tumor-associated macrophages appear inside the lumen, and it is considered that a three-dimensional cell mass closer to the patient's pathological tissue is formed. In addition, the detection of vimentin-positive and CD68-positive cells can be performed by the method described in the examples.

[0033] <Use of cultured cell sheet> The cell culture substrate with tumor cell mass of this embodiment can be used for various applications. For example, it can be used for evaluating the malignancy of tumor cells. That is, the method for evaluating the malignancy of tumor cells in this embodiment includes a step of three-dimensionally culturing tumor cells on a cell culture substrate having an uneven structure with a plurality of convex portions or a plurality of concave portions on the surface of the cell culture substrate to obtain a cell culture substrate with tumor cell mass, and a step of evaluating the malignancy of the tumor cells constituting the three-dimensional tumor cell mass on the obtained cell culture substrate with tumor cell mass, and the average pitch between the plurality of convex portions or between the plurality of concave portions is 200 nm or more and 650 nm or less. In this embodiment, by observing the three-dimensional tumor cell mass, for example, in the case of adenocarcinoma cells, it is possible to evaluate the degree of differentiation of tumor cells by the ability to form lumens, etc., and thereby evaluate the malignancy. The evaluation of malignancy is not limited to this, and it is also possible to evaluate the malignancy based on the difference in the degree of atypia of the cell population structure.

[0034] In addition, the cell culture substrate with tumor cell mass of this embodiment may be used for evaluating a test substance. That is, the method for evaluating a test substance according to the present embodiment includes a step of three-dimensionally culturing tumor cells on a cell culture substrate having an uneven structure with a plurality of convex portions or a plurality of concave portions on the surface of the cell culture substrate to obtain a cell culture substrate with tumor cell clusters, a step of allowing a test substance to be evaluated to act on the three-dimensional tumor cell clusters on the obtained cell culture substrate with tumor cell clusters, and a step of evaluating the test substance based on the response of the three-dimensional tumor cell clusters or the tumor cells constituting the three-dimensional tumor cell clusters, and the average pitch between the plurality of convex portions or between the plurality of concave portions is 200 nm or more and 650 nm or less. By treating the test substance on the three-dimensional tumor cell clusters and evaluating the effects on the tumor cell clusters from various viewpoints, the test substance can be evaluated. In the present embodiment, it is considered that the tumor cells form three-dimensional tumor cell clusters and form a tumor tissue closer to that in vivo. Not only the morphological changes and life and death of each individual tumor cell are evaluated, but also the shape changes of the three-dimensional tumor mass, the changes in the cell population motility in the three-dimensional tumor mass, the changes in the lumen shape, and the changes in the expression levels of various proteins expressed in the three-dimensional tumor mass are evaluated. By doing so, it is possible to evaluate the effects of the test substance on tumor cells in more detail.

Example

[0035] Examples are given below to specifically explain the present invention, but the present invention is not limited to these examples.

[0036] <Fabrication of convex cell culture substrate> A single-particle film etching mask made of colloidal silica was fabricated on a Si wafer by the method described in JP-A-2009-034630. By using this single-particle film etching mask, a fine uneven structure was formed on the Si substrate by a vapor etching method, and a convex Si-made surface fine structure master (1) with a plurality of convex structures arranged was fabricated. In the convex Si-made surface fine structure master (1), a plurality of convex portions having a substantially conical shape were included, the average pitch of the plurality of convex portions was 400 nm, and the average height of the plurality of convex portions was 500 nm. Subsequently, a concave Ni electroformed stamper (2) was fabricated from a convex Si surface microstructure master (1). Thereafter, using the concave Ni electroformed stamper (2), a convex structure serving as a placement portion was transferred onto the surface of a cycloolefin polymer film with a thickness of 188 μm by a hot nanoimprint method to fabricate a convex resin transfer member (3). Specifically, the structural surface of the concave Ni electroformed stamper (2) was opposed to the surface of a cycloolefin polymer film with a thickness of 188 μm and set in the pressurizing section of a nanoimprint apparatus. In that state, heating was started, and pressurization was performed at 4 MPa for 3 minutes at 168°C. After 3 minutes had elapsed, after cooling the pressurizing section to room temperature, the concave Ni electroformed stamper (2) was released from the surface of the cycloolefin polymer film. Also, as the Si surface micro-roughness structure master (1), a master with an average pitch of the plurality of convex portions of 150 nm and an average height of the plurality of convex portions of 180 nm, a master with an average pitch of the plurality of convex portions of 300 nm and an average height of the plurality of convex portions of 340 nm, and a master with an average pitch of the plurality of convex portions of 700 nm and an average height of the plurality of convex portions of 760 nm were used. Similarly, convex resin transfer members were fabricated and used as cell culture substrates. (Without Lipidure coating) The obtained convex resin transfer member (3) was subjected to a hydrophilic treatment by a UV ozone method and then used for cell culture. (With Lipidure coating) After the obtained convex resin transfer member (3) was subjected to a hydrophilic treatment by a UV ozone method, Lipidure (model number CM5206, registered trademark, manufactured by NOF Corporation) was coated by a spin coating method and used for cell culture. The Lipidure coating was performed by the following method. A film with a size of 20 mm × 20 mm was set in a spin coater, and 0.2 ml of a 0.5% (w / v) Lipidure solution (solvent: ethanol) was dropped. It was rotated at 1000 rpm for 10 seconds to remove the excess Lipidure solution and dry the surface.

[0037] <Fabrication of concave cell culture substrate> In the above <Production of convex cell culture substrate>, from the original silicon surface microstructure master (1) with a convex shape produced by the hot nanoimprint method, a concave structure serving as a placement part was transferred onto the surface of a cycloolefin polymer film with a thickness of 188 μm, and a concave resin master (1R) was produced. From the concave resin master (1R), a convex nickel electroformed stamper (2R) was produced. Then, except for using the convex nickel electroformed stamper (2R), a concave resin transfer member (3R) was produced by the same production method as in <Production of convex cell culture substrate> and used for cell culture. (Without Lipidure coating) The obtained convex resin transfer member (3R) was subjected to a hydrophilic treatment by the UV ozone method and then used for cell culture. (With Lipidure coating) After the obtained convex resin transfer member (3R) was subjected to a hydrophilic treatment by the UV ozone method, Lipidure (model number CM5206, registered trademark, manufactured by NOF Corporation) was coated by the spin coating method and used for cell culture. In the following description, a cell culture substrate having a concavo-convex structure with a plurality of convex portions or a plurality of concave portions on the surface, as described above, is also referred to as a "concavo-convex substrate", and a cell culture substrate with a flat surface is also referred to as a "flat substrate".

[0038] Example 1 The resin transfer member (concavo-convex substrate) (film with a size of 20 mm × 20 mm) produced as described above was placed in a 35 mm dish, and 3 ml of DMEM medium (containing 10% FBS) containing 2.5 × 10 6 cells of the PCI-55 pancreatic cancer cell line (human pancreatic duct adenocarcinoma cell line) were seeded. It was cultured for 7 days in an incubator with a CO2 concentration of (5%), a humidity of (90 - 95%), and a temperature of (37°C), and cell masses were observed on the 3rd day and the 7th day. The results are shown in Table 1 below. Also, the actual state of the cells on the 3rd day of culture without coating is shown in Figure 1.

[0039]

Table 1

[0040] The adhesiveness of the cells was evaluated as follows. On the 3rd to 7th days after cell seeding, the state where the cells (cell aggregates) did not adhere to the substrate and were floating was regarded as "none". Also, on the substrate to which the cells adhered, when pipetting with PBS (Phosphate-buffered saline) or medium was performed 3 times and the cells (cell aggregates) were detached from the substrate and released by the water flow, it was regarded as "weak". When the cells (cell aggregates) adhered to the substrate after pipetting was performed 3 times, it was regarded as "present". Regarding the lumen structure, it was described whether it had a shape similar to the comedo pattern, cribriform, or papillary pattern in the tumor structure pattern described above.

[0041] According to the results in Table 1 and FIG. 1, when using a cell culture substrate having an uneven structure with a plurality of convex portions or a plurality of concave portions and an average pitch between the plurality of convex portions or between the plurality of concave portions being 200 nm or more and 650 nm or less, it was confirmed that the adhesiveness of the cells to the substrate was good, and a lumen structure was formed and a three-dimensional tumor cell mass was formed.

[0042] Example 1 The convex resin transfer member (3) (uneven substrate) (film with a size of 20 mm × 20 mm) with a 400 nm pitch without Lipidure coating prepared as described above was placed in a 35 mm dish, and 3 ml of DMEM medium (containing 10% FBS) containing 2.5 × 10 6 PCI-55 pancreatic cancer cell lines were seeded. They were cultured for 5 days in an incubator with a CO2 concentration of 5%, humidity of 90 - 95%, and temperature of 37°C to form cell masses on the film. The cell mass cultured for 5 days was fixed with a 4% paraformaldehyde solution for 20 minutes after removing the culture medium. To stain the inside of the cell mass, the cells were treated with PBS containing 0.5% Triton-X-100 for 20 minutes and then fixed with ice-cold 70% methanol for 5 minutes. PBT (0.05% Tween-20 in PBS) containing 0.1% goat serum was used for blocking non-specific binding. For immunofluorescence double staining, CD68 (macrophage marker) and Vimentin (mesenchymal marker) were detected. After incubation with the primary antibody for each target protein, an Alexa Fluor-labeled goat polyclonal antibody was used as the secondary antibody. The nuclei were stained with Hoechst 33258. After staining, the cell mass was enclosed with a cover glass using an embedding agent, and 3D continuous cross-sections were photographed with a confocal laser microscope (Nikon Confocal Laser Microscope System A1; manufactured by Nikon Instech Co., Ltd.), and then 3D reconstruction was performed using image analysis software (NIS-Elements AR Ver 5.30.00; manufactured by Nikon Solutions Co., Ltd.). As a control, a 35-mm dish for cell culture with a flat culture surface was used, and pancreatic cancer cell lines were seeded and cultured in the same manner, followed by immunofluorescence double staining. The results are shown in Figure 2 below.

[0043] The distance in the height (Z-axis) direction of the acquired 3D images was compared between the cell mass cultured on the flat substrate and the cell mass cultured on the uneven substrate. The distance in the height (Z-axis) direction was an average of 5 μm for the cell mass cultured on the flat substrate, but an average of 50 μm for the tumor cell mass cultured on the uneven substrate. Since the stained cell mass is enclosed with a cover glass, the cell mass is in a compressed (crushed) state in the height direction due to the enclosure with the cover glass, and it is considered that the actual height of the cell mass is about 100 μm. In addition, lumen formation was observed in the culture on the uneven substrate.

[0044] Example 2 The resin transfer member (3) (concave-convex base material) with a pitch of 400 nm without the convex-type Lipidure coating prepared as described above (a film with a size of 20 mm × 20 mm) was placed in a 35-mm dish, and 3 ml of DMEM medium (containing 10% FBS) containing 2.5×10 6 PCI-55 pancreatic cancer cell lines were seeded. They were cultured in an incubator with a CO2 concentration of 5%, a humidity of 90 - 95%, and a temperature of 37°C for 5 days, and cell masses were formed on the film. The cell masses cultured for 5 days were fixed with a 4% paraformaldehyde solution for 20 minutes after removing the culture medium. To stain the inside of the cell masses, the cells were treated with PBS containing 0.5% Triton-X-100 for 20 minutes and then fixed with ice-cold 70% methanol for 5 minutes. PBT (0.05% Tween-20 in PBS) containing 0.1% goat serum was used for blocking non-specific binding. Immunofluorescence double staining detected CD68 (green: macrophage marker) and Vimentin (red: mesenchymal marker). After incubation with the primary antibody for each target protein, Alexa Fluor-labeled goat polyclonal antibody was used as the secondary antibody. The nuclei were stained with Hoechst 33258. The stained film (and cell masses) was sealed with a cover glass using a mounting agent, and 3D continuous cross-sections were photographed with a confocal laser microscope (Nikon Confocal Laser Microscope System A1; manufactured by Nikon Instech Co., Ltd.), and then 3D reconstruction was performed using image analysis software (NIS-Elements AR Ver 5.30.00; manufactured by Nikon Solutions Co., Ltd.).

[0045] As a control, a 35-mm dish for cell culture with a flat culture surface was used, and pancreatic cancer cell lines were seeded, cultured, and immunofluorescence double staining was performed in the same manner. In the obtained 3D images, the localization of the proteins detected by the markers was compared between the cell masses cultured on the flat base material and the cell masses cultured on the concave-convex base material. The results are shown in Figures 3-1 and 3-2.

[0046] When cultured on a flat substrate, cells in which the macrophage marker CD68 and the mesenchymal marker Vimentin were simultaneously detected (CD68+, Vimentin+) were rarely observed. When cultured on the textured substrate, cells expressing both the macrophage marker CD68 and the mesenchymal marker Vimentin (CD68+, Vimentin+) were frequently observed within the luminal structures. Although the cells observed within the luminal structures were pancreatic cancer cells, they showed a CD68(+) vimentin(+) phenotype. Cells that are generally stained with CD68(+) vimentin(+) are activated macrophages. Cells with a similar phenotype are frequently observed in histopathological images of cancer lesions removed from pancreatic cancer patients. In general, in pathological tissue examinations, cells stained with the CD68(+) vimentin(+) phenotype that have accumulated in tumor sites or within their luminal structures are diagnosed as infiltration and accumulation of tumor-associated macrophages (TAMs). In pathological tissue images of cancer lesions removed from patients, TAMs refer to macrophages that have infiltrated into cancer tissues, and are thought to promote carcinogenesis and malignancy through various functions such as angiogenesis, growth factor production, immunosuppression, and metastasis promotion. These results demonstrated that when only pancreatic cancer cell lines were cultured on the textured substrate, cancer cells exhibiting the CD68(+) vimentin(+) phenotype accumulated inside the luminal structures, and that large numbers of pancreatic cancer cells with a phenotype similar to tumor-associated macrophages appeared inside the luminal structures. CD68 (macrophage marker) and Vimentin (mesenchymal marker) detected in the three-dimensional cell masses cultured on the textured substrate showed a similar localization to tissue sections from cancer patients, suggesting that the cell masses cultured on the textured substrate have a morphology and physiological activity similar to cancer tissue in the body.

[0047] Example 3 The resin transfer member (3) (concavo-convex base material) with a pitch of 400 nm without the coating of the convex Lipidure produced as described above (a film with a size of 20 mm × 20 mm) was placed in a 35 mm dish, and 3 ml of DMEM medium (containing 10% FBS) containing 2.5×10 6 PCI-55 pancreatic cancer cell lines were seeded. They were cultured for 4 days in an incubator with a CO2 concentration of (5%), a humidity of (90 - 95%), and a temperature of (37°C), and cell masses were formed on the film. As a test substance, PCI-55 dead cell residues induced apoptosis by UV irradiation were added and reacted at 37°C for 24 hours. The preparation of PCI-55 dead cell residues was carried out by the following method. Pancreatic cancer cells PCI-55 were cultured overnight in a 60 mm cell culture dish. After washing twice with PBS, ultraviolet light was irradiated at an intensity of 250 mJ / cm 2 using a Bio-Rad GS Gene Linker (Bio-Rad, San Jose, CA). After washing once with PBS, they were incubated in serum-free DMEM medium for 2 hours. The dead cell residues of PCI-55 were collected with a cell scraper and used. Generally, tests that give dead cells to cancer cells are used as an evaluation method to clarify endocytosis (Reference: Scientific Reports 8, Article number: 14054). Endocytosis is a function of taking in nutrients and hormones into cells and is an essential process for many signal transmissions mediated by cell growth factors and their receptors, ion channels, adhesion proteins, synaptic vesicle proteins, etc. Inhibiting endocytosis blocks many types of cell signal transmissions and also prevents the invasion of pathogens. Such tests to clarify endocytosis are also useful for the development of anticancer drugs and anti-infective drugs.

[0048] Twenty-four hours after adding PCI-55 dead cell residues, the cell mass was fixed with 4% paraformaldehyde, and then double immunofluorescence staining was performed. For the double immunofluorescence staining, AE1AE3 (green: pan-epithelial marker) and Vimentin (red: mesenchymal marker) were used, and the nuclei were stained with Hoechst 33258. Images were taken as 3D continuous cross-sections with a confocal laser microscope and then 3D reconstructed. The results are shown in Figure 4.

[0049] As shown in Figure 4, in the cell mass to which PCI-55 dead cell residues were added, compared with the untreated cell mass, the localization of the mesenchymal marker Vimentin increased in the cells on the surface of the cell mass, and furthermore, the morphology of the lumen-like structure was disrupted. This indicates that when dead cell residues are administered as a test substance, it is possible to evaluate the degree of enhancement of the motility of epithelial cancer cells (acquisition of mesenchymal traits) and the changes in structural abnormalities. In normal two-dimensional culture (culturing of tumor cells using a flat substrate), the uptake of dead cancer cells is known as cannibalism. However, in the culture of tumor cells on the uneven substrate on which the three-dimensional tumor cell mass of this embodiment is formed, a novel phenomenon of phagocytosis of dead cells with active motility by the tumor mass is observed, which is considered to be advantageous for the evaluation of endocytosis at the tissue level that has never been seen before. In this example, the cells were treated with dead cell residues. However, by treating with other test substances such as anticancer agents, compared with the evaluation of test substances using conventional flat cell culture substrates, it becomes possible to evaluate the acquisition of stromal traits of tumor cells (enhancement of motility (enhancement of invasive ability)) and the evaluation of structural abnormalities of tumor tissues due to exposure to test substances, and it is considered that the influence of test substances on tumor tissues can be evaluated in more detail.

[0050] Example 4 The resin transfer member (3) (uneven substrate) (film with a size of 20 mm × 20 mm) with a 400-nm pitch without a convex Lipidure coating prepared as described above was placed in a 35-mm dish, and the PCI-55 pancreatic cancer cell line was seeded at 2.5×10 63 ml of DMEM medium (containing 10% FBS) containing [[number]] cells was seeded. It was cultured for 4 days in an incubator with a CO2 concentration of (5%), humidity of (90 - 95%), and temperature of (37°C), and cell masses were formed on the film. Similarly, 2.5×10 6 PCI-6 pancreatic cancer cell lines containing [[number]] cells were seeded in 3 ml of DMEM medium (containing 10% FBS). The observation results of the cells on the 5th day of culture are shown in Fig. 5.

[0051] As shown in Fig. 5, the highly differentiated PCI-55 cell line formed a lumen structure (glandular structure) with a clear contour. On the other hand, it was confirmed that the moderately differentiated PCI-6 cell line formed a lumen structure (glandular structure), but the shape was unclear. By detecting the difference in tumor structure construction, it is considered possible to evaluate the degree of differentiation of tumor cell lines.

[0052] From the above results, the cell culture substrate with tumor cell mass of the present embodiment reflects the tumor tissue of actual patients compared to the conventional ones. As a result, by using the cell culture substrate with tumor cell mass, it is considered possible to perform an evaluation closer to the in vivo situation as a method for evaluating the malignancy of the tumor cells themselves and the test substance to be evaluated.

Claims

1. A cell culture substrate and a cell culture substrate with tumor cell masses having three-dimensional tumor cell masses, wherein the three-dimensional tumor cell masses adhere to the surface of at least one surface of the cell culture substrate, the cell culture substrate has a concavo-convex structure having a plurality of convex portions or a plurality of concave portions on the surface (cell culture surface surface) of the cell culture substrate to which the three-dimensional tumor cell masses adhere, the average pitch between the plurality of convex portions or between the plurality of concave portions is 200 nm or more and 650 nm or less, A cell culture substrate with tumor cell masses.

2. The cell culture substrate with tumor cell masses according to claim 1, wherein the cell culture substrate has a concavo-convex structure continuous over the entire surface of the cell culture surface surface.

3. The cell culture substrate with tumor cell masses according to claim 1 or 2, wherein the three-dimensional tumor cell masses have a thickness of 10 μm or more in the height direction.

4. The cell culture substrate with tumor cell masses according to claim 1 or 2, wherein the tumor cells constituting the three-dimensional tumor cell masses are epithelial tumor cells.

5. The cell culture substrate with tumor cell masses according to claim 4, wherein the three-dimensional tumor cell masses have a lumen structure.

6. The cell culture substrate with tumor cell masses according to claim 4, wherein vimentin-positive cells and CD68-positive cells are recognized in the tumor cells in the three-dimensional tumor cell masses.

7. A step of three-dimensionally culturing tumor cells on a cell culture substrate having a concavo-convex structure having a plurality of convex portions or a plurality of concave portions on the surface of the cell culture substrate to obtain a cell culture substrate with tumor cell masses, and a step of evaluating the malignancy of the tumor cells constituting the three-dimensional tumor cell masses on the obtained cell culture substrate with tumor cell masses, wherein the average pitch between the plurality of convex portions or between the plurality of concave portions is 200 nm or more and 650 nm or less, A method for evaluating the malignancy of tumor cells.

8. A step of three-dimensionally culturing tumor cells on a cell culture substrate having a concavo-convex structure having a plurality of convex portions or a plurality of concave portions on the surface of the cell culture substrate to obtain a cell culture substrate with tumor cell masses, a step of allowing a test substance to be evaluated to act on the three-dimensional tumor cell masses on the obtained cell culture substrate with tumor cell masses, and a step of evaluating the test substance based on the response of the three-dimensional tumor cell masses or the tumor cells constituting the three-dimensional tumor cell masses, wherein the average pitch between the plurality of convex portions or between the plurality of concave portions is 200 nm or more and 650 nm or less, A method for evaluating a test substance.

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