Method for producing cell aggregates containing contrast agent and cell aggregates obtained by the same

Abstract

To provide a method for producing a cell aggregate containing a contrast agent, and to provide cell aggregates obtained by the same.SOLUTION: Provided is a method for producing a cell aggregate containing a contrast agent, comprising: (i) seeding cells in a cell culture vessel provided with a base film containing a polymer including a repeating unit represented by the following formula (Ia): [wherein, Ua1, Ua2, and Ra1 represent specific alkyl groups, and Ra2 represents specific alkylene groups]; (ii) culturing the seeded cells to form a cell monolayer on the base film; (iii) adding a contrast agent onto the cell monolayer; and (iv) continuing the culture to form a cell aggregate.SELECTED DRAWING: Figure 1

Description

[Technical field]

[0001] The present invention relates to a method for producing a cell aggregate containing a contrast agent, and a cell aggregate obtained thereby. [Background technology]

[0002] As the latest treatment for embolizing aortic aneurysms, a procedure called stent graft (SG) insertion is known, in which an artificial blood vessel with a metal spring sewn to it is inserted into the wall of the aneurysm via a catheter to block the blood flow into the aneurysm and embolize the aneurysm. However, it has been reported that in SG insertion, blood flows back in through the gap between the stent graft and the arterial wall, causing a certain percentage of endoleaks, which are continuous blood flow into the aneurysm, causing the aneurysm to expand again (see, for example, Non-Patent Document 1) (see Figure 1A).

[0003] The present inventors have reported a regenerative aneurysm treatment method in which the inside of the aneurysm is reconstructed as vascular tissue by transplanting stem cells into the aneurysm together with SG insertion to prevent the occurrence of endoleak. In this treatment method, it is considered that most of the cells are likely to diffuse outside the aneurysm if a cell suspension is simply injected into the aneurysm, and therefore, in order to keep the cells within the aneurysm, it has been considered to store the cells as a single fibrous aggregate in a catheter and place it in the aneurysm (see, for example, Non-Patent Document 2) (see Figure 1B). On the other hand, a method has not been developed that can non-invasively visually confirm whether the cell aggregate has reliably remained within the aneurysm immediately after transplantation.

[0004] A cell aggregate (also called a spheroid or cell mass, etc.) is a cell aggregate in which cells are cultured three-dimensionally and self-assembled and aggregated with each other, and its use in drug discovery research, cell therapy, and regenerative therapy has been widely considered. For the production of such a cell aggregate, for example, various methods have been reported that use a cell culture vessel whose culture surface is coated with a (co)polymer of 2-N,N-dimethylaminoethyl methacrylate (DMAEMA), which is known as a temperature-responsive polymer (see, for example, Patent Documents 1 and 2). The present inventors have reported that a shape-controlled cell aggregate, such as a fiber-like one, is formed according to the coated area by seeding cells on a cell culture vessel in which a specific polymer has been applied to any area of ​​the culture surface, whereby the cells adhere only to the coated surface of the polymer to form a monolayer, and then spontaneous aggregation is induced (see, for example, Non-Patent Document 2). [Prior art documents] [Patent documents]

[0005] [Patent Document 1] JP 2014-162865 A [Patent Document 2] International Publication No. 2020 / 040247 [Non-patent literature]

[0006] [Non-Patent Document 1] Ann Surg, 2019, 269, pp.564-573 [Non-Patent Document 2] Artificial Organs, 2022, 51(1), pp.23-24 Summary of the Invention [Problem to be solved by the invention]

[0007] An object of the present invention is to provide a method for producing a cell aggregate containing a contrast agent and a cell aggregate obtained thereby. [Means for solving the problem]

[0008] The present inventors came up with the idea of ​​combining a contrast agent with a cell aggregate to non-invasively visualize a transplanted cell aggregate, thereby producing a cell aggregate that can be detected by, for example, an imaging diagnostic system using X-rays, and conducted extensive research. As a result, they discovered that in a cell aggregate formation process using a cell culture vessel having a specific polymer on its culture surface, by adding a contrast agent during the several hours that a cell monolayer is formed, the contrast agent particles precipitate on the surface of the cell monolayer, and the cell monolayer aggregates are formed while remaining attached thereto, thereby forming a cell aggregate carrying the contrast agent, and thus completed the present invention.

[0009] The present invention is as follows. [1] (i) Formula (Ia): [ka] [In the formula, U a1 and U a2 each independently represents a hydrogen atom or a linear or branched alkyl group having 1 to 5 carbon atoms; R a1 represents a hydrogen atom or a linear or branched alkyl group having 1 to 5 carbon atoms; R a2 represents a linear or branched alkylene group having 1 to 5 carbon atoms; (ii) culturing the seeded cells to form a cell monolayer on the base membrane; (iii) adding an imaging agent onto the cell monolayer; and (iv) Continuing the culture to form cell aggregates A method for producing a cell aggregate containing a contrast agent, comprising: [2] The polymer further comprises a formula (IIa): [ka] [In the formula, R brepresents a hydrogen atom or a linear or branched alkyl group having 1 to 5 carbon atoms]. [3] The method according to [1] above, wherein the polymer further comprises a crosslinked structure. [4] The density of the contrast agent added is 500 μg / cm 2 The above is the manufacturing method described in [1] above. [5] The manufacturing method described in [1] above, wherein the contrast agent is a contrast agent that can be detected by an X-ray imaging diagnostic system. [6] The manufacturing method described in [1] above, wherein the contrast agent is titanium, tungsten or an inorganic compound thereof. [7] A cell aggregate containing a contrast agent, obtained by the manufacturing method described in [1] above. [8] The cell aggregate described in [7] above, wherein the contrast agent is a contrast agent that can be detected by an X-ray imaging diagnostic system. [9] The cell aggregate described in [7] above, wherein the contrast agent is titanium, tungsten or an inorganic compound thereof. Effect of the Invention

[0010] The method for producing a cell aggregate containing a contrast agent of the present invention focuses on the fact that cells seeded in a cell culture vessel having a base film containing a specific polymer form a cell aggregate through monolayer formation, and is characterized by adding a contrast agent during the several hours during which the cell monolayer is formed. This allows a cell aggregate carrying a desired contrast agent to be obtained simply and stably. In addition, since the obtained cell aggregate containing a contrast agent can be detected by an imaging diagnostic system using X-rays or the like, the transplanted cell aggregate can be observed non-invasively, which is extremely useful in various therapies involving cell transplantation. In addition, in the manufacturing method of the present invention, when a base film containing a specific polymer is applied to a culture surface, the application area can be appropriately selected to give a cell aggregate containing a contrast agent a desired shape. Therefore, the manufacturing method of the present invention is highly versatile because it allows appropriate selection of the types of cells and contrast agent, and the shape of the obtained cell aggregate. [Brief description of the drawings]

[0011] [Figure 1] FIG. 1A is a schematic diagram of endoleak in stent graft treatment, and FIG. 1B is a schematic diagram of a regenerative aneurysm treatment method using transplantation of a fibrous cell aggregate to prevent the occurrence of endoleak. [Diagram 2] A shows the results of comparing the appearance of the WC-supported cell aggregates obtained at each addition density in Example 1, and B shows the results of comparing the WC-supported cell aggregates obtained at each addition density in Example 1 under X-ray fluoroscopy. [Diagram 3] 1 shows the results of evaluating the cell metabolic activity of the WC-supporting cell aggregates obtained in Example 1 by MTT assay. [Figure 4] 1 shows the results of evaluating the cell proliferation activity of the WC-supporting cell aggregates obtained in Example 1. [Diagram 5] 1A shows the appearance of the fibrous WC-supported cell aggregate obtained in Example 2, and B shows an X-ray fluoroscopy image of the fibrous WC-supported cell aggregate after transplantation into a porcine aortic aneurysm model. [Figure 6] 1 shows the results of comparing the appearance of titanium-supported cell aggregates obtained at each addition density in Example 3. [Figure 7] 1 shows the appearance of a fibrous titanium-supporting cell aggregate obtained in Example 2. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0012] <Method of manufacturing cell aggregates containing a contrast agent> The method for producing a cell aggregate containing a contrast agent of the present invention is characterized by comprising the following steps (i) to (iv): (i) Formula (Ia): [ka] [In the formula, U a1 and U a2 each independently represents a hydrogen atom or a linear or branched alkyl group having 1 to 5 carbon atoms; R a1represents a hydrogen atom or a linear or branched alkyl group having 1 to 5 carbon atoms; R a2 represents a linear or branched alkylene group having 1 to 5 carbon atoms; (ii) culturing the seeded cells to form a cell monolayer on the base membrane; (iii) adding an imaging agent onto the cell monolayer; and (iv) Continuing the culture to form cell aggregates.

[0013] Process (i) (Cell culture container) The cell culture vessel according to the production method of the present invention comprises a compound represented by the following formula (Ia): [ka] [In the formula, U a1 and U a2 each independently represents a hydrogen atom or a linear or branched alkyl group having 1 to 5 carbon atoms; R a1 represents a hydrogen atom or a linear or branched alkyl group having 1 to 5 carbon atoms; R a2 represents a linear or branched alkylene group having 1 to 5 carbon atoms. The present invention is characterized in that the present invention comprises an undercoat film containing a polymer containing a repeating unit represented by the following formula:

[0014] The polymer preferably contains a repeating unit represented by the above formula (Ia) together with a repeating unit represented by the following formula (IIa): [ka] [In the formula, R b represents a hydrogen atom or a linear or branched alkyl group having 1 to 5 carbon atoms.

[0015] Such polymers typically have the following formula (I): [ka] [In the formula, U a1 , U a2 , R a1 and R a2 is obtained by polymerizing a monomer represented by the following formula (Ia):

[0016] The polymer preferably contains a cationic monomer represented by formula (I) and a cationic monomer represented by formula (II): [ka] [In the formula, R b is obtained by polymerizing an anionic monomer represented by the formula (IIa) above.

[0017] In this specification, unless otherwise defined, examples of a "linear or branched alkyl group having 1 to 5 carbon atoms" include a methyl group, an ethyl group, an n-propyl group, an isopropyl group, an n-butyl group, an isobutyl group, an s-butyl group, a t-butyl group, an n-pentyl group, a 1-methylbutyl group, a 2-methylbutyl group, a 3-methylbutyl group, a 1,1-dimethylpropyl group, a 1,2-dimethylpropyl group, a 2,2-dimethylpropyl group, and a 1-ethylpropyl group.

[0018] R a1 and R b are preferably each independently selected from a hydrogen atom and a methyl group. U a1 and U a2 are each preferably independently selected from a hydrogen atom, a methyl group, an ethyl group, a n-propyl group, an isopropyl group, and a n-butyl group, more preferably a methyl group or an ethyl group, and most preferably a methyl group.

[0019] In this specification, unless otherwise defined, examples of the "straight-chain or branched alkylene group having 1 to 5 carbon atoms" include a methylene group, an ethylene group, a propylene group, a trimethylene group, a tetramethylene group, a 1-methylpropylene group, a 2-methylpropylene group, a dimethylethylene group, an ethylethylene group, a pentamethylene group, a 1-methyl-tetramethylene group, a 2-methyl-tetramethylene group, a 1,1-dimethyl-trimethylene group, a 1,2-dimethyl-trimethylene group, a 2,2-dimethyl-trimethylene group, and a 1-ethyl-trimethylene group. a2 is preferably selected from an ethylene group and a propylene group.

[0020] Therefore, the cationic monomer represented by the above formula (I) includes 2-N,N-dimethylaminoethyl methacrylate, N,N-dimethylaminomethyl methacrylate, etc., and 2-N,N-dimethylaminoethyl methacrylate is preferred. The anionic monomer represented by the above formula (II) includes acrylic acid, methacrylic acid, etc., and methacrylic acid is preferred.

[0021] The molar ratio of the repeating unit represented by formula (Ia) / the repeating unit represented by formula (IIa) in the polymer is 100 / 0 to 50 / 50, preferably 98 / 2 to 50 / 50, more preferably 98 / 2 to 60 / 40, and particularly preferably 98 / 2 to 70 / 30. When the molar ratio of formula (IIa) is 50 or less, a decrease in cell adhesive strength due to the anionic nature of the polymer can be suppressed.

[0022] The polymer may be a polymer obtained by polymerizing a monomer having two or more carbon-carbon unsaturated bonds together with the monomer represented by formula (I) / formula (II). The monomer having two or more carbon-carbon unsaturated bonds is specifically a monomer having two or more carbon-carbon double bonds, such as a polyfunctional acrylate compound, a polyfunctional acrylamide compound, a polyfunctional polyester, or an isoprene compound.

[0023] Preferred specific examples include monomers represented by the following formulas (III) to (V). [ka] [ka] [ka]

[0024] In the formula, R c and R d each independently represents a hydrogen atom or a linear or branched alkyl group having 1 to 5 carbon atoms; R e represents a linear or branched alkylene group having 1 to 5 carbon atoms, and n represents a number from 1 to 50.

[0025] R c and R d are preferably each independently selected from a hydrogen atom and a methyl group. R e is preferably selected from methylene, ethylene and propylene groups, most preferably ethylene groups. Although n is a number from 1 to 50, n is preferably a number from 1 to 30, and more preferably, n is a number from 1 to 10.

[0026] Among these, the monomer represented by formula (III) is preferable. Examples of the monomer represented by formula (III) include ethylene glycol di(meth)acrylate, diethylene glycol di(meth)acrylate, triethylene glycol di(meth)acrylate, polyethylene glycol di(meth)acrylate, and polypropylene glycol di(meth)acrylate, and ethylene glycol dimethacrylate is preferable. In the present invention, (meth)acrylate means acrylate or methacrylate. For example, (meth)acrylic acid means acrylic acid and methacrylic acid.

[0027] The molar ratio of the monomers represented by formulas (III) to (V) to the entire polymer is preferably 0 to 50%, and more preferably 2 to 25%. When the molar ratio of the formulae (III) to (V) is 50% or less, gelation of the solid content during the production due to high molecular weight caused by excessive crosslinking can be suppressed, and the production can be facilitated.

[0028] The molar ratio of the repeating units in the polymer can be measured by an appropriate analytical instrument such as NMR, but may also be a value roughly calculated from the amounts of monomers charged. For example, the difference between the mol% value of the monomer represented by formula (II) relative to the entire polymer and the mol% value of the monomer represented by formula (II) relative to the total monomer charge amount in the preparation step is 0 to 10 mol%. The polymer of the present application has a small difference between the monomer charge ratio and the actual measured value of the produced polymer by the production method described later, which is 0 to 10 mol%, and more preferably 0 to 8 mol%.

[0029] The weight average molecular weight (Mw) of the polymer is preferably from 20,000 to 1,000,000, and more preferably from 50,000 to 800,000. The ratio (Mw / Mn) of the weight average molecular weight (Mw) to the number average molecular weight (Mn) of the polymer is 1.01 to 10.00, preferably 1.2 to 8.0, preferably 1.4 to 6.0, preferably 1.5 to 5.0, and preferably 1.6 to 4.5. The number average molecular weight (Mn) and the number average molecular weight (Mn) can be determined, for example, by gel filtration chromatography described in the examples.

[0030] By using the polymer of the present application as a base film for cell culture, it is possible to form a cell aggregate after seeding cells, forming a monolayer, and then peeling the cells off. The cell aggregate refers to a structure formed as a result of cell aggregation, and is not limited to a spherical or ring shape. There is an advantage in that the size and shape of the cell aggregate can be adjusted depending on the size and shape of the base film (cell aggregates of any size and shape can be produced). The entire disclosures of WO 2020 / 040247, WO 2021 / 167042, and WO 2023 / 282253 are incorporated by reference into this application.

[0031] The cell culture vessel according to the manufacturing method of the present invention can be manufactured, for example, by applying a base film forming agent containing the polymer to the culture surface of a commercially available cell culture vessel. The base film forming agent contains a solvent in addition to the polymer. If necessary, other substances can be added within a range that does not impair the performance of the resulting base film. Examples of other substances include pH adjusters, crosslinking agents, preservatives, surfactants, primers that increase adhesion to the vessel or substrate, antifungal agents and sugars, cell adhesive substances, etc.

[0032] The solvent contained in the undercoat film forming agent is not limited as long as it can dissolve the polymer, but is preferably an aqueous solution that contains water. The aqueous solution may be water, a salt-containing aqueous solution such as physiological saline or a phosphate buffer solution, or a mixed solvent of water or a salt-containing aqueous solution with an alcohol. The alcohol may be an alcohol having 2 to 6 carbon atoms, such as ethanol, propanol, isopropanol, 1-butanol, 2-butanol, isobutanol, t-butanol, 1-pentanol, 2-pentanol, 3-pentanol, 1-heptanol, 2-heptanol, 2,2-dimethyl-1-propanol (=neopentyl alcohol), 2-methyl-1-propanol, 2-methyl-1-butanol, 2-methyl-2-butanol (=t-amyl alcohol), 3-methyl-1-butanol, 3-methyl-3-pentanol, cyclopentanol, 1-hexanol, etc. Examples of suitable solvents include cyclohexanol, 2-hexanol, 3-hexanol, 2,3-dimethyl-2-butanol, 3,3-dimethyl-1-butanol, 3,3-dimethyl-2-butanol, 2-ethyl-1-butanol, 2-methyl-1-pentanol, 2-methyl-2-pentanol, 2-methyl-3-pentanol, 3-methyl-1-pentanol, 3-methyl-2-pentanol, 3-methyl-3-pentanol, 4-methyl-1-pentanol, 4-methyl-2-pentanol, 4-methyl-3-pentanol, and cyclohexanol. These solvents may be used alone or in combination. The water content in the aqueous solution is, for example, 50% by mass to 100% by mass, 80% by mass to 100% by mass, or 90% by mass to 100% by mass.

[0033] The method for applying the undercoat film-forming agent is not particularly limited, and may be, for example, an inkjet method, a screen printing method, a slit coating method, a roll-to-roll method, or the like. Preferably, the application is performed by a printing technique such as an inkjet method or screen printing.

[0034] Another application method is, for example, adding the base film forming agent to a cell culture vessel, optionally protecting the non-formed portion of the base film, and leaving it to stand for a predetermined time. The addition can be performed, for example, by adding the base film forming agent in an amount of 0.5 to 1 times the total volume of the vessel using a syringe or the like. The leaving is performed by appropriately selecting the time and temperature depending on the material of the vessel and the type of base film forming agent for cell culture, and is performed, for example, for 1 minute to 24 hours, preferably 5 minutes to 3 hours, at 10 to 80°C. In this way, the cell culture vessel according to the present invention can be produced.

[0035] The undercoat film obtained by such a method may be washed with at least one solvent selected from water and an aqueous solution containing an electrolyte in order to remove remaining impurities, unfixed polymers, etc. Washing is preferably performed by washing with running water or ultrasonic washing. The aqueous solution containing water and an electrolyte may be heated, for example, to a temperature in the range of 40°C to 95°C. The aqueous solution containing an electrolyte is preferably PBS, saline (containing only sodium chloride), Dulbecco's phosphate buffered saline, Tris buffered saline, HEPES buffered saline, and Veronal buffered saline, and PBS is particularly preferred. After fixing, the coating film remains firmly fixed to the substrate without being dissolved even when washed with water, PBS, alcohol, etc.

[0036] The undercoat film obtained by such a method may be subjected to a drying step. The drying step is carried out in air or vacuum, preferably at a temperature within the range of −200° C. to 200° C. The drying step removes the solvent in the undercoat film forming agent, thereby completely fixing the agent to the substrate. The undercoat film can be formed by drying at room temperature (10°C to 35°C, preferably 20°C to 30°C, e.g. 25°C), but in order to form the undercoat film more quickly, it may be dried at, for example, 40°C to 80°C. If the drying temperature is less than -200°C, an uncommon refrigerant must be used, which is not versatile, and drying takes a long time due to solvent sublimation, making it inefficient. If the drying temperature exceeds 200°C, thermal decomposition of the polymer occurs. The drying temperature is more preferably 10°C to 180°C, and more preferably 20°C to 150°C.

[0037] Furthermore, the cell culture vessel provided with the undercoat film obtained by such a method may be subjected to a sterilization step, if necessary. Sterilization may be appropriately selected from known sterilization means such as heating method (high pressure steam method, dry heat method), irradiation method (radiation method), gas method (ethylene oxide gas method, hydrogen peroxide gas plasma method) depending on the material of the cell culture vessel, etc., but radiation method is preferred. Radiation method refers to a sterilization method by irradiation with gamma rays, X-rays, or electron beams, preferably gamma rays or X-rays, more preferably gamma rays.

[0038] The maximum and minimum thicknesses of the undercoat film are in the range of 1 to 1000 nm, and preferably in the range of 5 to 500 nm.

[0039] The cell culture vessel according to the present invention can be produced by applying the base film forming agent to the culture surface of, for example, a commercially available cell culture vessel and drying it. Here, the "(culture) surface" refers to the surface that comes into contact with the contents, such as cells or cell culture medium.

[0040] There are no particular limitations on the material, shape, etc. of the cell culture vessel, but examples include dishes such as Petri dishes, tissue culture dishes, and multi-dishes that are commonly used for cell culture; flasks such as cell culture flasks, spinner flasks, and multi-tier flasks; bags such as plastic bags, Teflon (registered trademark) bags, and culture bags; plates such as microplates, microwell plates, multi-plates, and multi-well plates; chamber slides, tubes, trays, and bottles such as roller bottles.

[0041] The cell culture vessel according to the present invention is preferably manufactured using a vessel whose culture surface has cell adhesion suppression ability. Alternatively, the culture surface may be treated to suppress cell adhesion before the formation of the base film. The vessel having cell adhesion suppression ability may be a commercially available cell culture dish that has been subjected to a cell low adhesion treatment, a cell culture vessel having cell adhesion suppression ability, etc., and for example, the cell culture vessel described in JP-A-2008-61609 can be used, but is not limited thereto. In addition, for example, a cell culture vessel manufactured through a process of applying a known coating film forming composition having cell adhesion suppression ability may be used. For example, the coating film forming composition described in WO 2014 / 196650 (the entire disclosure of which is incorporated herein by reference) can be used as the coating film forming composition.

[0042] (cell) In step (i), cells are seeded in the cell culture vessel provided with the base film. The cells to be seeded can be appropriately selected depending on the intended treatment, so long as they have the ability to form cell aggregates. The cell in the present invention is the most basic unit constituting an animal or a plant, and has as its elements a cytoplasm and various organelles inside the cell membrane. In this case, the nucleus containing DNA may or may not be contained inside the cell. For example, the animal-derived cells in the present invention include germ cells such as sperm and eggs, somatic cells constituting an organism, stem cells (pluripotent stem cells, etc.), progenitor cells, cancer cells isolated from an organism, cells (cell lines) isolated from an organism that have acquired immortalization ability and are stably maintained outside the body, cells isolated from an organism that have been artificially genetically modified, cells isolated from an organism that have been artificially replaced with a nucleus, etc. Examples of somatic cells that constitute a living body include, but are not limited to, fibroblasts, bone marrow cells, B lymphocytes, T lymphocytes, neutrophils, red blood cells, platelets, macrophages, monocytes, bone cells, bone marrow cells, pericytes, dendritic cells, keratinocytes, adipocytes, mesenchymal cells, epithelial cells, epidermal cells, endothelial cells, vascular endothelial cells, hepatic parenchymal cells, chondrocytes, cumulus cells, nervous system cells, glial cells, neurons, oligodendrocytes, microglia, astrocytes, cardiac cells, esophageal cells, muscle cells (e.g., smooth muscle cells or skeletal muscle cells), pancreatic beta cells, melanocytes, hematopoietic progenitor cells (e.g., CD34-positive cells derived from umbilical cord blood), and mononuclear cells. The somatic cells include cells collected from any tissue, such as skin, kidney, spleen, adrenal gland, liver, lung, ovary, pancreas, uterus, stomach, colon, small intestine, large intestine, bladder, prostate, testis, thymus, muscle, connective tissue, bone, cartilage, vascular tissue, blood (including umbilical cord blood), bone marrow, heart, cardiac muscle, eye, brain, or neural tissue, etc. Furthermore, the somatic cells include cells induced to differentiate from stem cells or progenitor cells.

[0043] Stem cells are cells that have the ability to replicate themselves and differentiate into cells of multiple lineages. Examples of stem cells include, but are not limited to, embryonic stem cells (ES cells), embryonic tumor cells, embryonic germline stem cells, induced pluripotent stem cells (iPS cells), neural stem cells, hematopoietic stem cells, mesenchymal stem cells, hepatic stem cells, pancreatic stem cells, muscle stem cells, germline stem cells, intestinal stem cells, cancer stem cells, and hair follicle stem cells. Among the above stem cells, pluripotent stem cells include ES cells, embryonic germline stem cells, and iPS cells. Progenitor cells are cells that are in the middle stage of differentiation from the above stem cells into specific somatic cells or germ cells. Cancer cells are cells that are derived from somatic cells and have acquired infinite proliferation capabilities. Cell lines are cells that have acquired infinite proliferation capabilities through artificial manipulation outside the body. Among these, fibroblasts, stem cells, and among stem cells, pluripotent stem cells are more preferable.

[0044] The seeding of cells is not particularly limited, and can be carried out by a known method using, for example, an appropriate medium depending on the type of cells.

[0045] Process (ii) In step (ii), the seeded cells are cultured to form a cell monolayer on the base film. The cell culture is also not particularly limited, and can be performed by a known method using an appropriate medium under an appropriate environment (temperature, pH, osmotic pressure, oxygen partial pressure, carbon dioxide partial pressure, etc.) according to the type of seeded cells, for example, under an environment of 37°C and 5% CO2 concentration. In the production method of the present invention, a cell culture vessel equipped with a base film containing the polymer is used, and a monolayer of the seeded cells is first formed on the base film.

[0046] Process (iii) (Contrast media) In step (iii), a contrast agent is added onto the cell monolayer formed in step (ii). The contrast agent is not particularly limited as long as it is detectable by a known imaging diagnostic system and does not substantially affect cell survival and proliferation (low cytotoxicity), but is preferably detectable by an X-ray imaging diagnostic system. Such contrast agents are metal elements or inorganic compounds containing metal elements, and examples thereof include metal elements selected from the group consisting of cadmium, cesium, cobalt, copper, gallium, lead, manganese, molybdenum, niobium, rubidium, ruthenium, scandium, technetium, titanium, gold, tantalum, iridium, platinum, tungsten, rhodium, palladium, strontium, samarium, thallium, holmium, scandium, zirconium, yttrium, silver, iron, gadolinium, chromium, zinc, barium, magnesium, and calcium (including each isotope), as well as inorganic compounds such as nitrides, carbides, oxides, borides, sulfates, and carbonates of the metal elements. Among these, titanium, tungsten, or inorganic compounds thereof are preferred.

[0047] When the cell monolayer formed on the base membrane in step (ii) is continued to be cultured, the cell monolayer usually starts to detach from the edge and simultaneously starts to aggregate. Therefore, the contrast agent is added in step (iii) after the cell monolayer is formed and before detachment occurs. The time from the formation of the cell monolayer to detachment varies depending on the type of cell, culture environment, etc., but is usually within about 1 to 24 hours, and preferably about 1 to 6 hours.

[0048] The amount of contrast agent added is appropriately selected depending on the purpose of treatment, the type of cells and contrast agent, and the visibility of the image. 2 ) of contrast agent added (μg) per 2 ) is, for example, 100 μg / cm 2 More than 500μg / cm 2 It is preferable that the concentration is 700 μg / cm or more. 2 More preferably, it is equal to or greater than this.

[0049] Process (iv) In step (iv), the cell monolayer to which the contrast agent has been added is continued to be cultured to form a cell aggregate. As the culture is continued, detachment occurs from the edge of the cell monolayer and aggregation begins at the same time. At this time, the contrast agent added in step (iii) precipitates on the surface of the cell monolayer, and aggregation of the cell monolayer occurs while remaining attached, forming a cell aggregate carrying the contrast agent. The cell culture is preferably carried out according to the culture conditions in step (ii).

[0050] <Cell aggregates containing contrast agent> The cell aggregate containing the contrast agent of the present invention can be obtained by the above-mentioned manufacturing method. The details of the cells, contrast agent, etc. are as described above. Since the cell aggregate containing the contrast agent of the present invention can be detected by an imaging diagnostic system using X-rays, etc., the transplanted cell aggregate can be observed non-invasively, and is extremely useful in various therapeutic methods involving cell transplantation. EXAMPLES

[0051] The present invention will be described in more detail below with reference to examples and comparative examples, but the present invention is not limited to the following examples.

[0052] <Method for measuring weight average molecular weight> The weight average molecular weights shown in the following synthesis examples are the results obtained by gel filtration chromatography (hereinafter abbreviated as GFC). (Measurement conditions) Equipment: HLC-8320GPC (Tosoh Corporation) GFC column: TSKgel G 6000 + 3000 PWXL-CP ·Flow rate: 1.0mL / min Eluent: Salt-containing water / organic mixed solvent Column temperature: 40℃ Detector: RI Injection concentration: polymer solids 0.05% by mass ·Injection volume: 100μL Calibration curve: Cubic approximation curve Standard sample: Polyethylene oxide (Agilent) x 10 types

[0053] <Synthesis Example 1> 24.00g of 2-(dimethylamino)ethyl methacrylate (Tokyo Chemical Industry Co., Ltd.), 1.46g of methacrylic acid (Tokyo Chemical Industry Co., Ltd.), 5.09g of ethylene glycol dimethacrylate (Tokyo Chemical Industry Co., Ltd.), 0.31g of dimethyl 1,1'-azobis(1-cyclohexanecarboxylate) (VE-073, Fujifilm Wako Pure Chemical Industries, Ltd.), and 111.09g of 2-propanol were mixed and polymerized dropwise into 166.62g of 2-propanol at reflux temperature to synthesize a polymer. The reaction product was reprecipitated in hexane, a poor solvent, and the precipitate was collected by filtration and dried under reduced pressure. The weight average molecular weight of this polymer determined by GFC was 228,000 (hereinafter referred to as "Synthetic Example Polymer 1").

[0054] <Preparation Example 1> To 0.005 g of the polymer obtained in Synthesis Example 1 above, 33.3 g of pure water was added and thoroughly stirred to prepare an undercoat film forming agent 1.

[0055] <Example 1> Production of cell aggregates containing contrast agents Human bone marrow derived mesenchymal stem cells were seeded on a culture well (flat-bottom 96-well) whose entire culture surface had been coated with the base film forming agent 1 obtained in Preparation Example 1, and cultured for about 1 hour to form a cell monolayer without gaps on the bottom of the well. Next, tungsten carbide (WC) powder was added as a contrast agent at 1 to 1000 μg / cm. 2 When WC was added at a density of 500 μg / cm, the black WC powder settled and was adsorbed to the upper surface of the cell monolayer. As the culture was continued, the cell monolayer detached from the outer edge with the WC adsorbed, and at the same time aggregated, and about one day after seeding, spherical cell aggregates were formed carrying almost all of the added WC and were suspended in the culture medium. Here, the blackness of the cell aggregates increased with increasing WC addition density, indicating that the amount of WC carried could be controlled by the addition density (see Figure 2A). When the cell aggregates were observed under X-ray fluoroscopy, it was found that when the WC addition density to the cell monolayer was 500 μg / cm, the blackness of the cell aggregates increased. 2The above was now visible as a fluoroscopic image (see Figure 2B).

[0056] Assessment of cell aggregate activity The cell metabolic activity of the cell aggregates containing the contrast agent obtained above was evaluated by MTT assay. As a result, the WC-added density that could be visually confirmed under X-ray fluoroscopy was 500 μg / cm 2 Even in these cases, it was confirmed that cell metabolic activity was maintained at 90% or more compared to when no contrast agent was added (see Figure 3). Furthermore, it was found that when WC-supported cell aggregates were cultured statically on a culture dish, they attached to the surface within an hour, just like WC-free cell aggregates, and cells leached out over several days (see Figure 4).

[0057] <Example 2> Production of cell aggregates containing fibrous contrast agents A ring-shaped culture groove (chamber) was created by attaching a 2 mm thick silicone disk (diameter: 6 cm) to the center of a culture dish (diameter: 10 cm). The base film forming agent 1 obtained in Preparation Example 1 was applied to the bottom surface of the chamber, and then 2.0 × 10 human bone marrow derived mesenchymal stem cells were added. 5 cells / cm 2 The cells were seeded at a density of 1 mg / cm. The cells formed a cell monolayer sheet on the bottom surface of the chamber within 2 hours after seeding. Then, tungsten carbide (WC) powder was added as a contrast agent at a density of 1 mg / cm. 2 After about one day of culture, the cell monolayer sheet detached from the periphery and simultaneously aggregated, contracting toward the inside of the culture dish to form a ring-shaped cell aggregate wrapped around the silicone disk. The ring-shaped cell aggregate was cut at one point to obtain a fibrous cell aggregate with a length of 18 cm, determined by the circumference of the silicone disk (see Figure 5A).

[0058] Transplantation of cell aggregates Immediately after the WC-bearing fibrous cell aggregate obtained above was transplanted into a porcine aortic aneurysm model via a catheter, the fluoroscopic image of the black fibrous cell aggregate was visible within the aneurysm model (see Figure 5B).

[0059] <Example 3> Production of cell aggregates containing contrast agents 40 μL of the aqueous solution (100 μg / mL) of the polymer obtained in Synthesis Example 1 was dropped into each well of a culture well plate (flat-bottom 96-well plate), left to stand for 5 minutes, and then aspirated. A suspension of human bone marrow-derived mesenchymal stem cells was added to the wells at 3.1 × 10 5 cells / cm 2 After seeding at a density of 100-1000 μg / cm, the cells were cultured for about 1 hour, forming a cell monolayer without gaps on the bottom of the well. Titanium powder (average particle size: 45 μm) was then added at 100-1000 μg / cm 2 When titanium powder was added at a density of 100 μg / cm, the black titanium powder settled and was adsorbed to the upper surface of the cell monolayer. As culture was continued, the cell monolayer detached from the outer edge with the titanium adsorbed, and simultaneously aggregated, and approximately one day after seeding, spherical cell aggregates were formed carrying almost all of the added titanium powder and were suspended in the culture medium. Here, the blackness of the cell aggregates increased with increasing titanium particle addition density, indicating that the amount of titanium carried could be controlled by the addition density (see Figure 6). Observation of the cell aggregates under X-ray fluoroscopy showed that when titanium was added to the cell monolayer at a density of approximately 500 μg / cm, the blackness of the cell aggregates increased. 2 The above was visible as a fluoroscopic image.

[0060] Assessment of cell aggregate activity It was confirmed by observing tissue sections (HE staining) that the cell aggregates obtained above carried titanium. It was also confirmed by immunohistochemical staining that they maintained the mesenchymal stem cell markers CD44 and CD105 (maintained stemness).

[0061] <Example 4> Production of cell aggregates containing fibrous contrast agents A ring-shaped culture groove (chamber) was created by attaching a 2 mm thick silicone disk (diameter: 6 cm) to the center of a culture dish (diameter: 10 cm). 5 mL of an aqueous solution of PDMAEMA-co-PMA (100 μg / mL) was dropped onto the bottom of the chamber and left to stand for 5 minutes, after which it was aspirated. 3.8 × 10 human bone marrow derived mesenchymal stem cells were cultured. 6cells / cm 2 The cells were seeded at a density of 100 μg / cm or more. Within 2 hours of seeding, the cells formed a cell monolayer sheet on the bottom of the chamber. Titanium powder (average particle size: 45 μm) was then added as a contrast agent at a density of 500 μg / cm. 2 When titanium powder was added at a density of 180 nm, the black titanium powder settled and was adsorbed to the upper surface of the cell monolayer. After about one day of culture, the cell monolayer sheet detached from the periphery and simultaneously aggregated, contracting toward the inside of the culture dish to form a ring-shaped cell aggregate wrapped around the silicone disk. By cutting the ring-shaped cell aggregate at one point, a fibrous cell aggregate with a length of 18 cm, determined by the circumference of the silicone disk, was obtained (see Figure 7). [Industrial Applicability]

[0062] The method for producing a cell aggregate containing a contrast agent of the present invention utilizes the process in which cells seeded in a cell culture vessel equipped with a base film containing a specific polymer form a cell aggregate through monolayer formation. The present inventors have succeeded in producing a cell aggregate containing a contrast agent that can be detected by an imaging diagnostic system using X-rays or the like while maintaining cell metabolic activity, simply by adding a contrast agent during the several hours in which the cell monolayer is formed. The cell aggregate containing the contrast agent thus obtained can be detected by an imaging diagnostic system using X-rays or the like, and therefore the transplanted cell aggregate can be observed non-invasively, which is extremely useful in various therapies involving cell transplantation. In particular, in the manufacturing method of the present invention, when a base film containing a specific polymer is applied to the culture surface, the application area can be appropriately selected, thereby making it possible to give the cell aggregate containing the contrast agent a desired shape, for example a fiber-like shape, and the visibility of the resulting cell aggregate containing the contrast agent under X-ray fluoroscopy after transplantation was also confirmed.

Claims

1. (i) A compound represented by the following formula (Ia): 【Chemistry 1】 [In the formula, U ａ１ and U ａ２ each independently represents a hydrogen atom or a linear or branched alkyl group having 1 to 5 carbon atoms; R a１ represents a hydrogen atom or a linear or branched alkyl group having 1 to 5 carbon atoms; R a２ represents a linear or branched alkylene group having 1 to 5 carbon atoms; (ii) culturing the seeded cells to form a cell monolayer on the base membrane; (iii) adding an imaging agent onto the cell monolayer; and (iv) Continuing the culture to form cell aggregates A method for producing a cell aggregate containing a contrast agent, comprising:

2. The polymer may further comprise formula (IIa): 【Chemistry 2】 [In the formula, R ｂ represents a hydrogen atom or a linear or branched alkyl group having 1 to 5 carbon atoms.

3. The method according to claim 1 , wherein the polymer further comprises a crosslinked structure.

4. The contrast agent has a loading density of 500 μg / cm 2 The method for producing a semiconductor device according to claim 1 ,

5. The method of claim 1 , wherein the contrast agent is detectable by an X-ray imaging diagnostic system.

6. The method of claim 1 , wherein the contrast agent is titanium, tungsten, or an inorganic compound thereof.

7. A cell aggregate containing a contrast agent, obtained by the method according to claim 1.

8. The cell aggregate according to claim 7 , wherein the contrast agent is titanium, tungsten, or an inorganic compound thereof.

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