Method for producing cell aggregates

Abstract

A method for manufacturing a cell mass adhesion-cultured on a cell culture substrate, the cell culture substrate having two regions (A) and (B), and the method having steps (1) to (3). (A) Island-shaped region having a cell-proliferating property and having an area of 0.001-1 mm2. (B) Region that is adjacent to region (A) and that does not have a cell-proliferating property. (1) Step for preparing a cell suspension containing mesenchymal cells and ectoderm-derived cells. (2) Step for bringing the cell suspension into contact with the cell culture substrate and bringing the above two types of cells randomly into contact with region (A), the total number of the above two types of cells on the surface of contact between the cell suspension and the cell culture substrate being 100 to 100,000 per cm2. (3) Step for culturing the cells adhered to region (A) and forming a cell mass including the above two types of cells.

Description

[Technical Field]

[0001] The present invention relates to a method for efficiently producing and stably transporting cell aggregates containing two or more types of cells, including mesenchymal cells and ectoderm-derived cells, wherein the cell aggregate has at least two regions: one region with a high content of ectoderm-derived cells and another region with a low content of the aforementioned cells (a high proportion of mesenchymal cells). [Background technology]

[0002] In recent years, there has been active research into regenerating damaged organs and other tissues by transplanting cells cultured outside the body into the human body. Besides using pluripotent stem cells such as iPS cells as cell sources for regenerative medicine, another reported method involves transplanting cell aggregates called organoids, which are composed of epithelial and mesenchymal cells. As a method for producing organ primordia, a method has been reported in which two types of cell aggregates, epithelial cell aggregates and mesenchymal cell aggregates, are arranged adjacent to each other in a collagen gel (see, for example, Patent Document 1). However, this method requires the individual fabrication of each organ primordia within the gel, which has the problem of poor mass production capabilities.

[0003] Another method for producing organ primordia is known, which involves using a U-shaped cell culture substrate that prevents cell adhesion and culturing a mixture of epithelial and mesenchymal cells to spontaneously form cell aggregates (see, for example, Patent Document 2). Although this method is excellent for mass production of organ primordia, the cell aggregates exist in a suspended state, making them prone to movement due to agitation of the culture medium during cultivation and transport, thus damaging the organ primordia. [Prior art documents] [Patent Documents]

[0004] [Patent Document 1] Patent No. 5932671 [Patent Document 2] Patent No. 6425319 [Overview of the project] [Problems that the invention aims to solve]

[0005] The object of the present invention is to provide a method for efficiently producing cell aggregates containing two or more types of cells, wherein the cell aggregates have at least two regions: a region with a high content of ectoderm-derived cells and a region with a low content of the aforementioned cells (a high proportion of mesenchymal cells) while the cells are adhered to a substrate, and which can be stably transported. [Means for solving the problem]

[0006] In light of the above points, the inventors conducted extensive research and, as a result, discovered that the above problems can be solved by adhering and culturing two or more types of cells on a cell culture substrate in which cell adhesion regions are patterned, thus completing the present invention. In other words, the present invention is <1> A method for producing a cell aggregate cultured on a cell culture substrate, wherein the cell culture substrate has two regions (A) and (B) as described below, and the method comprises the following steps (1) to (3). (A) Has cell proliferation properties and an area of ​​0.001-1 mm² 2 An island-like region. (B) A region adjacent to the region (A) above that does not have cell proliferation properties. (1) A step of preparing a cell suspension containing mesenchymal cells and ectoderm-derived cells. (2) A step of bringing the cell suspension prepared in step (1) into contact with the cell culture substrate and randomly adhering the mesenchymal cells and ectoderm-derived cells to the region (A), wherein the total number of mesenchymal cells and ectoderm-derived cells at the contact surface between the cell suspension and the cell culture substrate is 100 to 100,000 cells per unit area. (3) A step of culturing the cells attached to the region (A) to form a cell mass containing the mesenchymal cells and the ectoderm-derived cells. <2>The production method according to <1>, wherein the ratio of the number of mesenchymal cells to the number of cells derived from the ectoderm in the cell suspension is 1:10 to 10:1. <3>In the step (3), the cell mass includes a site where the content ratio of the mesenchymal cells is higher than that of the cells derived from the ectoderm and a site where the content ratio of the cells derived from the ectoderm is higher than that of the mesenchymal cells, and the ratio of the maximum cross-sectional area in the in-plane direction of the substrate of the site where the content ratio of the mesenchymal cells is higher than that of the cells derived from the ectoderm to the maximum cross-sectional area in the in-plane direction of the substrate of the site where the ratio of the cells derived from the ectoderm is higher than that of the mesenchymal cells is 2:1 to 1:20. The method for producing a cell mass according to <1> or <2>. <4>The method for producing a cell mass according to any one of claims <1> to <3>, further comprising the following step (4). (4) Culturing the cell mass formed in the step (3) to form an organ primordium. <5>The method for producing a cell mass according to <4>, wherein Versican is present in the organ primordium. <6>A hair follicle primordium differentiation induction kit comprising a cell culture substrate having two regions of the following (A) and (B). (A) A circular region having cell proliferation ability and an area of 0.001 to 1 mm 2 of the circle. (B) A region adjacent to the region (A) and having no cell proliferation ability. <7>The hair follicle primordium differentiation induction kit according to <6>, further comprising a medium in which mesenchymal cells and epithelial cells have proliferation ability. [Advantages of Invention]

[0007] The method for producing a cell mass of the present invention is a cell mass containing two or more types of cells including mesenchymal cells and cells derived from the ectoderm, and can efficiently produce a cell mass having at least two sites, namely, a site where the content of the cells derived from the ectoderm is high and a site where the content of the cells is low (where the content ratio of the mesenchymal cells is high), and can provide a method for producing a cell mass that can be stably transported. Furthermore, the method for producing the cell mass of the present invention forms a site with a high content ratio of ectoderm-derived cells and a site with a high content ratio of mesenchymal cells, thereby generating a tissue structure similar to that in vivo, and enabling the produced cell mass to differentiate into a mature state with human regenerative ability.

Brief Description of the Drawings

[0008] [Figure 1] Schematic diagram showing the method for producing the cell mass of the present invention. [Figure 2] Phase-contrast microscope image of the cell mass on the first day of culture in Example 1. [Figure 3] Phase-contrast microscope image of the cell mass on the sixth day of culture in Example 1. [Figure 4] Phase-contrast microscope image of the cell mass on the eleventh day of culture in Example 1. [Figure 5] Phase-contrast microscope image of the cell mass on the first day of culture in Example 3. [Figure 6] Fluorescence microscope image of the cell mass on the first day of culture in Example 3. [Figure 7] Phase-contrast microscope image of the cell mass on the seventh day of culture in Example 3. [Figure 8] [[ID=二十九]]Fluorescence microscope image of the cell mass on the seventh day of culture in Example 3. [Figure 9] Phase-contrast microscope image of the cell mass on the seventh day of culture in Example 4. [Figure 10] Fluorescence microscope image of the cell mass on the seventh day of culture in Example 4. [Figure 11] Phase-contrast microscope image of the cell mass on the seventh day of culture in Example 5. [Figure 12] Fluorescence microscope image of the cell mass on the seventh day of culture in Example 5. [Figure 13] Phase-contrast microscope image of the cell mass on the sixth day of culture in Comparative Example 4.

Modes for Carrying Out the Invention

[0009] The following describes in detail embodiments for carrying out the present invention (hereinafter simply referred to as "these embodiments"). These embodiments are illustrative for explaining the present invention and are not intended to limit the present invention to the following content. The present invention can be appropriately modified and implemented within the scope of its spirit.

[0010] In this specification, "cell aggregate" refers to a three-dimensional aggregate of cells formed by the gathering of multiple cells. "Organ primordium" refers to a cell aggregate formed from epithelial cells and mesenchymal cells, which, when transplanted into a living organism, can enable the regeneration of tissues or organs.

[0011] Furthermore, in this specification, "co-culture" refers to culturing two or more types of cells together.

[0012] Furthermore, in this specification, "stimulus responsiveness" refers to a change in structure or degree of hydrophilicity / hydrophobicity in response to external stimuli. Here, "external stimuli" in this specification refers to mechanical stimuli such as ultrasound, vibration, and convection; electromagnetic stimuli such as light, electricity, and magnetism; and thermodynamic stimuli such as heating and cooling, but excludes biological reactions such as enzymatic reactions.

[0013] Furthermore, in this specification, "temperature responsiveness" refers to a change in the degree of hydrophilicity / hydrophobicity due to temperature changes. In addition, the boundary temperature at which the degree of hydrophilicity / hydrophobicity changes is referred to as the "response temperature."

[0014] Furthermore, in this specification, "cell adhesion" refers to the ease with which cells adhere to a cell culture substrate at the temperature in which they are cultured. "Having cell adhesion" means that cells can adhere to the substrate or cell culture substrate directly or via a bio-derived substance at the culture temperature. "Not having cell adhesion" means that cells cannot adhere to the substrate or cell culture substrate at the culture temperature.

[0015] Furthermore, in this specification, "cell proliferation" refers to the ease with which cells proliferate at the culture temperature, and "having cell proliferation" means that cells adhere directly to the substrate or cell culture substrate, either directly or via a bio-derived substance, at the culture temperature and are capable of further proliferation. "Not having cell proliferation" means that cells cannot adhere to the substrate or cell culture substrate at the culture temperature, or that they adhere but cannot proliferate. Moreover, "high cell proliferation" means that a larger number of cells proliferate when compared over the same culture period.

[0016] Furthermore, in this specification, "bio-derived substance" refers to a substance present in the body of an organism, which may be a natural product, an artificially synthesized substance using genetic engineering technology, or a chemically synthesized substance based on the aforementioned bio-derived substance. There are no particular limitations on bio-derived substances, but examples include nucleic acids, proteins, and polysaccharides, which are the basic building blocks of living organisms, as well as their constituent elements such as nucleotides, nucleosides, amino acids, various sugars, lipids, vitamins, and hormones.

[0017] One aspect of the present invention relates to a method for producing a cell aggregate cultured on a cell culture substrate, wherein the cell culture substrate has two regions (A) and (B) as described below, and the method comprises the following steps (1) to (3). (A) Has cell proliferation properties and an area of ​​0.001-1 mm² 2 island-like regions (B) A region adjacent to the region (A) that does not have cell proliferation properties. (1) Steps to prepare a cell suspension containing mesenchymal cells and ectoderm-derived cells. (2) A step of bringing the cell suspension prepared in step (1) into contact with a cell culture substrate and randomly adhering mesenchymal cells and ectoderm-derived cells to the region (A), wherein the total number of mesenchymal cells and ectoderm-derived cells at the contact surface between the cell suspension and the cell culture substrate is 100 to 100,000 cells per unit area. (3) A step of culturing the cells attached to the region (A) above to form a cell mass containing mesenchymal cells and ectoderm-derived cells.

[0018] The cell culture substrate used in the present invention has two regions of the following (A) and (B). (A) An island-shaped region having cell growth ability and an area of 0.001 to 1 mm 2 ². (B) A region adjacent to the region (A) and having no cell growth ability.

[0019] The region (A) has cell growth ability. By having cell growth ability, the cell culture substrate of the present invention can culture cells to produce cell masses. If it does not have cell growth ability, cells cannot be cultured to form cell masses.

[0020] (A) The region is also an island-shaped region with an area of 0.001 to 1 mm 2 ². By being an island-shaped region with an area of 0.001 to 1 mm 2 ², when culturing two or more types of cells by adhesion, one type of cell can spontaneously aggregate to form a site with a high content ratio thereof. If the area is less than 0.001 mm 2 ², cells cannot be co-cultured uniformly in all regions (A). Also, if the area exceeds 1 mm 2 ², when culturing two or more types of cells by co-culture, all cells become a uniformly dispersed cell mass, and a cell mass having a site where one type of cell spontaneously aggregates and has a high content ratio thereof cannot be produced. Also, since it is suitable for forming cell masses having human regenerative ability such as organ primordia, an area of 0.005 to 0.5 mm 2 ² is preferable, an area of 0.01 to 0.5 mm 2 ² is more preferable, an area of 0.015 to 0.25 mm 2 ² is particularly preferable, and an area of 0.02 to 0.2 mm 2 ² is most preferable. Also, in the culture of hair follicle primordia, an area of 0.001 to 0.2 mm 2 ² is preferable, an area of 0.001 to 0.1 mm 2 ² is more preferable, an area of 0.001 to 0.05 mm 2 ² is particularly preferable, and an area of 0.005 to 0.05 mm 2 ² is most preferable.

[0021] Furthermore, since it is suitable for producing cell aggregates of uniform size and shape, the standard deviation / average area of ​​the area of ​​region (A) is preferably 80% or less, more preferably 50% or less, particularly preferably 20% or less, and most preferably 5% or less. The shape of the area (A) is not particularly limited and can be set as appropriate depending on the desired shape of the cell aggregate, but examples include circles, ellipses, polygons, and closed shapes consisting of irregular straight lines or curves. Furthermore, since it is suitable for producing cell aggregates with a shape close to a sphere, circles or ellipses and polygons are preferred, circles or ellipses and rectangles are more preferred, circles or ellipses and squares are particularly preferred, and circles or ellipses are most preferred.

[0022] Region (B) is adjacent to region (A) and does not have cell proliferation properties. Because it is adjacent to region (A) and does not have cell proliferation properties, when cells are cultured, cells can be made to proliferate only in region (A). If region (B) is not adjacent to region (A) or has cell proliferation properties, when cells are cultured, cells will spread to the area around region (A), making it impossible to produce cell aggregates in which one type of cell spontaneously aggregates and has a high proportion of that type. Furthermore, it is preferable that region (B) does not have cell proliferation properties as well as cell adhesion properties, as this is suitable for uniformizing the size and shape of the cell aggregates produced.

[0023] The shape of region (B) is not limited to being adjacent to region (A), but it is preferable that region (B) is adjacent to region (A) for a length of 20% or more of the boundary line between region (A) and region (B), more preferably 50% or more, particularly preferably 80% or more, and most preferably that region (B) is entirely surrounding region (A).

[0024] There are no particular limitations on the area ratio of region (A) to region (B), but it is preferable that the area of ​​region (A) be 10% or more of the total substrate, more preferably 30% or more, particularly preferably 50% or more, and most preferably 70% or more, as this is suitable for increasing the amount of cell aggregates that can be produced per unit area of ​​the culture substrate. Furthermore, it is preferable that the area of ​​region (B) be 20% or more of the total substrate, more preferably 40% or more, particularly preferably 60% or more, and most preferably 80% or more, as this is suitable for providing sufficient distance between multiple regions (A) and suppressing the fusion of cell aggregates in multiple regions (A) to form an uneven shape.

[0025] The cell culture substrate used in the present invention is suitable for forming cell aggregates of uniform size, and therefore preferably contains a layer of hydrophilic polymer on its surface, and region (A) is a region in which a part of the hydrophilic polymer layer has been decomposed or modified by plasma treatment, ultraviolet treatment, corona discharge treatment, or a combination thereof. By having a layer of hydrophilic polymer on its surface, the adsorption of proteins that contribute to substrate-cell adhesion in region (B) can be suppressed, making it possible to create a region that does not have cell adhesion or cell proliferation properties. Furthermore, by decomposing or modifying a part of the hydrophilic polymer layer, cell adhesion or cell proliferation properties can be imparted to region (A). Moreover, it is even more preferable that region (A) is a plasma-treated region in order to enhance the cell adhesion and cell proliferation properties of region (A) and to be suitable for forming cell aggregates in a short time.

[0026] Another method for producing the cell culture substrate used in the present invention is to coat only a portion of the cell culture substrate with a substance that promotes or inhibits cell adhesion using methods such as photolithography, inkjet printing, or screen printing.

[0027] The thickness of the hydrophilic polymer layer is preferably 10 nm or more, more preferably 50 nm or more, particularly preferably 100 nm or more, and most preferably 500 nm or more, as it is suitable for making region (B) a region that does not have cell adhesion or cell proliferation properties. Furthermore, as it is suitable for making region (A) a region that has cell adhesion and cell proliferation properties, the layer thickness is preferably 1000 nm or less, more preferably 500 nm or less, particularly preferably 100 nm or less, and most preferably 50 nm or less.

[0028] The method for forming the hydrophilic polymer layer can be carried out using at least one of the following: a method for forming chemical bonds and a method for physical interaction. Methods for forming chemical bonds include techniques for forming reactive functional groups such as ultraviolet irradiation, electron beam irradiation, gamma ray irradiation, plasma treatment, and corona treatment. Crosslinking reactions to the substrate surface using organic reactions with ions or radicals as reaction sources are also possible. Methods for physical interaction include coating, brush coating, dip coating, spin coating, bar coating, flow coating, spray coating, roll coating, air knife coating, blade coating, gravure coating, microgravure coating, and slot die coating, using a matrix with excellent compatibility with the target hydrophilic polymer as the coating material.

[0029] The hydrophilic polymers mentioned above are not particularly limited in type, but include those having polar groups such as hydroxyl groups, amino groups, and polyethylene glycol groups, and those having amphoteric structures such as betaine structures and phosphorylcholine groups. (B) Since it is suitable for making the region a region that does not have cell adhesion and cell proliferation properties, hydroxyl groups, phosphorylcholine groups, or polyethylene glycol groups are preferred, hydroxyl groups or phosphorylcholine groups are more preferred, and phosphorylcholine groups are particularly preferred. Commercial products such as BIOSURFINE-AWP (manufactured by Toyo Gosei Co., Ltd.) and Lipidure-CM5206 (manufactured by NOF Corporation) can be suitably used.

[0030] The hydrophilic polymer is suitable for suppressing the elution of hydrophilic polymers from the cell culture substrate and for suppressing the impact on quality due to the contamination of cell aggregates and cell masses with polymers. Therefore, it is preferably a random copolymer or block copolymer having both hydrophilic monomer units and hydrophobic / reactive monomer units, and more preferably a random copolymer having both hydrophilic monomer units and hydrophobic / reactive monomer units. Furthermore, as for the composition ratio of the copolymer, it is suitable for making region (B) a region that does not have cell adhesion and cell proliferation properties, so it is preferable that the hydrophilic monomer units are 30 wt% or more, more preferably 40 wt% or more, particularly preferably 50 wt% or more, and most preferably 60 wt% or more. Furthermore, in order to suppress the elution of hydrophilic polymers, it is preferable that the hydrophilic monomer units are 80 wt% or less, more preferably 50 wt% or less, particularly preferably 30 wt% or less, and most preferably 10 wt% or less.

[0031] The hydrophilic monomer units are not particularly limited other than being hydrophilic, but examples include those having an amino group such as 2-dimethylaminoethyl acrylate, 2-dimethylaminoethyl methacrylate, 2-diethylaminoethyl acrylate, 2-diethylaminoethyl methacrylate, and N-[3-(dimethylamino)propyl]acrylamide; those having a betaine such as N-(3-sulfopropyl)-N-methacroyloxyethyl-N,N-dimethylammonium betaine and N-methacryloyloxyethyl-N,N-dimethylammonium-α-N-methylcarboxybetaine; hydroxyethyl acrylate, hydroxyethyl methacrylate, N-(2-hydroxyethyl)acrylamide, polyethylene glycol monoacrylate, polyethylene glycol monomethacrylate, polypropylene glycol monoacrylate, polypropylene glycol monomethacrylate, methoxypolyethylene glycol monoacrylate, and methoxypolyethylene glycol monomethacrylate. Polyethylene glycol groups and methoxyethyl groups are found in acrylates, diethylene glycol monomethyl ether acrylate, diethylene glycol monomethyl ether methacrylate, diethylene glycol monoethyl ether acrylate, diethylene glycol monoethyl ether methacrylate, 2-methoxyethyl acrylate, 2-methoxyethyl methacrylate, 2-ethoxyethyl acrylate, 2-ethoxyethyl methacrylate, 3-butoxyethyl acrylate, 3-butoxyethyl methacrylate, 3-butoxyethyl acrylamide, furfuryl acrylate, furfuryl methacrylate, tetrahydrofurfuryl acrylate, tetrahydrofurfuryl methacrylate, etc.; acrylate groups are found in acrylates such as methoxymethyl acrylate, methoxymethyl methacrylate, 2-ethoxymethyl acrylate, 2-ethoxymethyl methacrylate, 3-butoxymethyl acrylate, 3-butoxymethyl methacrylate, 3-butoxymethyl acrylamide, etc.;Examples of phosphorylcholine compounds include those having a phosphorylcholine group, such as 2-methacryloyloxyethyl phosphorylcholine, 2-acryloyloxyethyl phosphorylcholine, 3-(meth)acryloyloxypropyl phosphorylcholine, 4-(meth)acryloyloxybutyl phosphorylcholine, 6-(meth)acryloyloxyhexyl phosphorylcholine, 10-(meth)acryloyloxydecyl phosphorylcholine, ω-(meth)acryloyl(poly)oxyethylene phosphorylcholine, 2-acrylamidoethyl phosphorylcholine, 3-acrylamidopropyl phosphorylcholine, 4-acrylamidobutyl phosphorylcholine, 6-acrylamidohexyl phosphorylcholine, 10-acrylamidodecyl phosphorylcholine, and ω-(meth)acrylamido(poly)oxyethylene phosphorylcholine.

[0032] The aforementioned hydrophobic monomer units are not particularly limited other than being hydrophobic, but examples include n-butyl acrylate, n-butyl methacrylate, isobutyl acrylate, isobutyl methacrylate, t-butyl acrylate, t-butyl methacrylate, n-hexyl acrylate, n-hexyl methacrylate, n-octyl acrylate, n-octyl methacrylate, n-decyl acrylate, n-decyl methacrylate, n-dodecyl acrylate, n-dodecyl methacrylate, n-tetradecyl acrylate, n-tetradecyl methacrylate, and the like. As the reactive monomer units, UV-reactive monomer units are preferred because they allow for the immobilization of hydrophilic polymers onto a substrate in a short time. Examples include 4-azidophenyl acrylate, 4-azidophenyl methacrylate, 2-((4-azidobenzoyl)oxy)ethyl acrylate, and 2-((4-azidobenzoyl)oxy)ethyl methacrylate.

[0033] The cell culture substrate used in the present invention may also be stimulus-responsive. The stimulus-responsiveness of the cell culture substrate allows for the detachment of cell aggregates from the substrate by external stimuli, enabling cell recovery while suppressing damage to the cells. The stimulus-responsiveness is not particularly limited as long as it allows for the detachment of cell aggregates by external stimuli, but examples include temperature responsiveness, light responsiveness, pH responsiveness, magnetic responsiveness, electric field responsiveness, and mechanical stimulus responsiveness. It is preferable that the stimulus be one or more of these, temperature responsiveness, light responsiveness, pH responsiveness, or mechanical stimulus responsiveness, more preferably one or more of these, particularly preferably one or more of these, and most preferably temperature responsiveness.

[0034] Furthermore, when culturing cells on a cell culture substrate, it is possible to culture the cells at a temperature close to body temperature. Therefore, while there are no particular limitations on the temperature responsiveness, a response temperature of 50°C or lower is preferred, more preferably 35°C or lower. Also, since it is suitable for suppressing cell detachment when performing operations such as changing the culture medium during cultivation, a temperature of 25°C or lower is particularly preferred, and 15°C or lower is most preferred. Moreover, since it is possible to form cell aggregates by cooling operations at a temperature that does not damage the cells, the lower limit of the response temperature is preferably 0°C or higher, more preferably 1°C or higher, more preferably 3°C or higher, and most preferably 4°C or higher. As a method for imparting the temperature responsiveness to the cell culture substrate, a method of providing a layer of temperature-responsive polymer on the surface of the substrate is preferred because it is excellent in terms of mass productivity of the cell culture substrate. There are no particular limitations on the type of polymer, but block copolymers immobilized on the substrate by physical actions such as hydrophobic interactions, copolymers immobilized on the substrate via reactive groups such as azide groups, and polymers immobilized on the substrate by coating monomers on the substrate and performing electron beam polymerization or radical polymerization on the substrate can be suitably used.

[0035] There are no particular limitations on the type of temperature-responsive polymer, but examples of monomer units for imparting temperature responsiveness include (meth)acrylamide compounds such as acrylamide and methacrylamide; N,N-diethylacrylamide, N-ethylacrylamide, Nn-propylacrylamide, Nn-propylmethacrylamide, N-isopropylacrylamide, N-isopropylmethacrylamide, N-cyclopropylacrylamide, N-cyclopropylmethacrylamide, Nt-butylacrylamide, N-ethoxyethylacrylamide, N-ethoxyethylmethacrylamide, N-tetrahydramide N-alkyl-substituted (meth)acrylamide derivatives such as flufurfuryl acrylamide and N-tetrahydrofurfuryl methacrylamide; N,N-dialkyl-substituted (meth)acrylamide derivatives such as N,N-dimethyl(meth)acrylamide, N,N-ethylmethylacrylamide, and N,N-diethylacrylamide; 1-(1-oxo-2-propenyl)-pyrrolidine, 1-(1-oxo-2-propenyl)-piperidine, 4-(1-oxo-2-propenyl)-morpholine, 1-(1-oxo-2-methyl-2-propenyl)-pyrrolidine, 1-(1-oxo-2-methyl-2-propenyl)- Examples of suitable materials include (meth)acrylamide derivatives having cyclic groups such as piperidine and 4-(1-oxo-2-methyl-2-propenyl)-morpholine; vinyl ethers such as methyl vinyl ether; and proline derivatives such as N-proline methyl ester acrylamide. Since these are suitable for setting the response temperature to 0-50°C, N,N-diethylacrylamide, Nn-propylacrylamide, N-isopropylacrylamide, Nn-propylmethacrylamide, N-ethoxyethylacrylamide, N-tetrahydrofurfurylacrylamide, and N-tetrahydrofurfurylmethacrylamide are preferred, with Nn-propylacrylamide and N-isopropylacrylamide being more preferred, and N-isopropylacrylamide being particularly preferred. Furthermore, when using a medium at room temperature during culture medium exchange, Nn-propylacrylamide and N-proline methyl ester acrylamide are preferred as they are suitable for setting the response temperature of the block copolymer to a temperature lower than room temperature.

[0036] In the present invention, the ratio of temperature-responsive constituent units contained in the temperature-responsive polymer is preferably 70 wt% or more, more preferably 80 wt% or more, particularly preferably 90 wt% or more, and most preferably 92 wt% or more, as it is suitable for rapidly performing the process of forming cell aggregates from cell aggregates.

[0037] The temperature-responsive polymer is preferably a random copolymer or block copolymer having both temperature-responsive monomer units and hydrophobic monomer units, and more preferably a block copolymer having both hydrophilic monomer units and hydrophobic monomer units, as it is suitable for suppressing the elution of the temperature-responsive polymer from the cell culture substrate and for suppressing the impact on quality due to the contamination of cell aggregates and cell clumps with the polymer. The hydrophobic monomer units can preferably be the same as those used in the case of the hydrophilic polymer described above.

[0038] In the present invention, temperature-responsive polymers may also include monomer units for controlling the response temperature. Examples include hydrophilic or hydrophobic monomer units, and are not particularly limited, but include those having amino groups such as 2-dimethylaminoethyl acrylate, 2-dimethylaminoethyl methacrylate, 2-diethylaminoethyl acrylate, 2-diethylaminoethyl methacrylate, and N-[3-(dimethylamino)propyl]acrylamide; those having betaines such as N-(3-sulfopropyl)-N-methacroyloxyethyl-N,N-dimethylammonium betaine and N-methacryloyloxyethyl-N,N-dimethylammonium-α-N-methylcarboxybetaine; hydroxyethyl acrylate, hydroxyethyl methacrylate, N-(2-hydroxyethyl)acrylamide, polyethylene glycol monoacrylate, polyethylene glycol monomethacrylate, polypropylene glycol monoacrylate, polypropylene glycol monomethacrylate, methoxypolyethylene glycol monoacrylate, and methoxypolyethylene glycol monomethacrylate. Polyethylene glycol groups and methoxyethyl groups are found in acrylates, diethylene glycol monomethyl ether acrylate, diethylene glycol monomethyl ether methacrylate, diethylene glycol monoethyl ether acrylate, diethylene glycol monoethyl ether methacrylate, 2-methoxyethyl acrylate, 2-methoxyethyl methacrylate, 2-ethoxyethyl acrylate, 2-ethoxyethyl methacrylate, 3-butoxyethyl acrylate, 3-butoxyethyl methacrylate, 3-butoxyethyl acrylamide, furfuryl acrylate, furfuryl methacrylate, tetrahydrofurfuryl acrylate, tetrahydrofurfuryl methacrylate, etc.; acrylate groups are found in acrylates such as methoxymethyl acrylate, methoxymethyl methacrylate, 2-ethoxymethyl acrylate, 2-ethoxymethyl methacrylate, 3-butoxymethyl acrylate, 3-butoxymethyl methacrylate, 3-butoxymethyl acrylamide, etc.;Examples of phosphorylcholine compounds include those having a phosphorylcholine group, such as 2-methacryloyloxyethyl phosphorylcholine, 2-acryloyloxyethyl phosphorylcholine, 3-(meth)acryloyloxypropyl phosphorylcholine, 4-(meth)acryloyloxybutyl phosphorylcholine, 6-(meth)acryloyloxyhexyl phosphorylcholine, 10-(meth)acryloyloxydecyl phosphorylcholine, ω-(meth)acryloyl(poly)oxyethylene phosphorylcholine, 2-acrylamidoethyl phosphorylcholine, 3-acrylamidopropyl phosphorylcholine, 4-acrylamidobutyl phosphorylcholine, 6-acrylamidohexyl phosphorylcholine, 10-acrylamidodecyl phosphorylcholine, and ω-(meth)acrylamido(poly)oxyethylene phosphorylcholine.

[0039] There are no particular restrictions on the molecular weight of the temperature-responsive polymer in the present invention, but it is preferable that the number average molecular weight be 1000 to 1,000,000, more preferably 2000 to 500,000, particularly preferably 50000 to 300,000, and most preferably 10,000 to 200,000, as this is suitable for increasing the strength of the temperature-responsive polymer.

[0040] In the present invention, since it is suitable for suppressing the incorporation of temperature-responsive polymers into cell aggregates, it is preferable that the component with a number average molecular weight of 5000 or less contained in the temperature-responsive polymer is 50% or less, more preferably 30% or less, particularly preferably 10% or less, and most preferably 5% or less. Furthermore, it is preferable that the component with a number average molecular weight of 10000 or less is 50% or less, more preferably 30% or less, particularly preferably 10% or less, and most preferably 5% or less. Moreover, it is preferable that the component with a number average molecular weight of 30000 or less is 50% or less, more preferably 30% or less, particularly preferably 10% or less, and most preferably 5% or less. The content of components with a specific molecular weight or less contained in the block copolymer can be measured by gel permeation chromatography.

[0041] The hydrophilic / temperature-responsive polymer in the present invention may optionally contain chain transfer agents, polymerization initiators, polymerization inhibitors, etc. There are no particular restrictions on the chain transfer agent, and commonly used ones can be suitably used. Examples include dithiobenzoate, trithiocarbonate, 4-cyano-4-[(dodecylsulfonylthiocarbonyl)sulfonyl]pentanoic acid, 2-cyanopropan-2-yl N-methyl-N-(pyridine-4-yl)carbamodithioate, and methylmethyl(4-pyridinyl)carbamodithioate 2-propionate. There are no particular restrictions on the polymerization initiator, and commonly used ones can be suitably used. Examples include azobisisobutyronitrile, 1,1'-azobis(cyclohexanecarbonile), di-tert-butyl peroxide, tert-butyl hydroperoxide, hydrogen peroxide, potassium peroxodisulfate, benzoyl peroxide, triethylborane, and diethylzinc. Furthermore, there are no particular restrictions on the polymerization inhibitor, and commonly used ones can be suitably used, but examples include hydroquinone, p-methoxyphenol, triphenylfeldazyl, 2,2,6,6-tetramethylpiperidinyl-1-oxyl, and 4-hydroxy-2,2,6,6-tetramethylpiperidinyl-1-oxyl.

[0042] The method for synthesizing hydrophilic / temperature-responsive polymers in the present invention is not particularly limited, but the living radical polymerization technology described in "Radical Polymerization Handbook," pp. 161-225 (2010), published by NTS Corporation, can be used.

[0043] In the present invention, since it is suitable for enhancing cell proliferation, the thickness of the layer made of temperature-responsive polymer is preferably 1000 nm or less, more preferably 200 nm or less, particularly preferably 100 nm or less, and most preferably 50 nm or less. Furthermore, since it is suitable for rapidly performing the process of forming cell aggregates from cell aggregates, the thickness of the layer made of temperature-responsive polymer is preferably 5 nm or more, more preferably 20 nm or more, particularly preferably 30 nm or more, and most preferably 35 nm or more.

[0044] In the present invention, the cell culture substrate may contain a bio-derived substance on its surface as needed. The bio-derived substance is not particularly limited, but examples include Matrigel, laminin, fibronectin, vitronectin, collagen, and the like.

[0045] These bio-derived substances may be natural products, artificially synthesized using genetic engineering techniques, or fragments obtained by cleaving with restriction enzymes, or synthetic proteins or peptides based on these bio-derived substances.

[0046] In the present invention, as the Matrigel, commercially available products such as Matrigel (manufactured by Corning Incorporated) or Geltrex (manufactured by Thermo Fisher Scientific) can be suitably used due to their availability.

[0047] The type of laminin is not particularly limited, but for example, laminin 511, laminin 521, or laminin 511-E8 fragment, which have been reported to show high activity against α6β1 integrin expressed on the surface of human iPS cells, can be used. The laminin may be a natural product, artificially synthesized by genetic engineering or the like, or a synthetic protein or synthetic peptide based on the laminin. Due to its availability, iMatrix-511 (manufactured by Nippi Corporation) can be preferably used as a commercially available product.

[0048] The aforementioned vitronectin may be a natural product, artificially synthesized using genetic engineering technology, or a synthetic protein or peptide based on the vitronectin. Due to their availability, commercially available products such as vitronectin derived from human plasma (manufactured by Wako Pure Chemical Industries, Ltd.), synthemax (manufactured by Corning Incorporated), and Vitronectin (VTN-N) (manufactured by Thermo Fisher Scientific) can be suitably used.

[0049] The fibronectin may be a natural product, artificially synthesized using genetic engineering technology, or a synthetic protein or peptide based on the fibronectin. Due to their availability, commercially available products such as fibronectin solution, human plasma-derived fibronectin (manufactured by Wako Pure Chemical Industries, Ltd.), and Retronectin (manufactured by Takara Bio Inc.) can be suitably used.

[0050] The type of collagen is not particularly limited, but for example, type I collagen or type IV collagen can be used. The collagen may be a natural product, artificially synthesized using genetic engineering technology, or a synthetic peptide based on the collagen. Due to their availability, commercially available products such as Collagen I, Human (manufactured by Corning Incorporated) and Collagen IV, Human (manufactured by Corning Incorporated) can be suitably used.

[0051] In the present invention, it is preferable that bio-derived substances are immobilized on the cell culture substrate by non-covalent bonds rather than covalent bonds, in order to suppress the denaturation of bio-derived substances and enhance cell proliferation. Here, in the present invention, "non-covalent bonds" refer to bonding forces other than covalent bonds that originate from intermolecular forces, such as electrostatic interactions, water-insoluble interactions, hydrogen bonds, π-π interactions, dipole-dipole interactions, London dispersion forces, and other van der Waals interactions. The immobilization of bio-derived substances on block copolymers may be by a single bonding force or a combination of multiple bonding forces.

[0052] In the present invention, the method for immobilizing bio-derived substances is not particularly limited, but for example, a method of immobilizing by applying a solution of bio-derived substances to a cell culture substrate for a predetermined time, or a method of immobilizing by adsorbing bio-derived substances onto a cell culture substrate by adding bio-derived substances to the culture medium when culturing cells, can be suitably used.

[0053] In the present invention, the material of the substrate is not particularly limited, but in addition to materials such as glass and polystyrene that are normally used in cell culture, materials that can generally be given shape, such as polymer compounds such as polycarbonate, polyethylene terephthalate, polyvinylidene fluoride, polytetrafluoroethylene, polyethylene, polypropylene, and polymethyl methacrylate, as well as ceramics and metals, can be used. For ease of culture operations, it is preferable that the material of the substrate contains at least one of glass, polystyrene, polycarbonate, polyethylene terephthalate, polyethylene, and polypropylene, and it is even more preferable that it contains at least one of glass, polystyrene, polycarbonate, polyethylene terephthalate, and polyethylene. Polystyrene, polycarbonate, polyethylene terephthalate, and polyethylene are particularly preferred because they are suitable for increasing flexibility. Furthermore, polystyrene, polycarbonate, and polyethylene terephthalate are most preferred because they are suitable for giving cell proliferation properties by patterning by hydrophilization treatment described later.

[0054] There are no particular restrictions on the shape of the substrate; it may be a flat shape such as a plate or film, or it may be a fiber, porous particle, porous membrane, or hollow fiber. It may also be a container commonly used for cell culture (such as a Petri dish, flask, plate, or bag). For ease of culture operations, a flat shape such as a plate or film, or a flat porous membrane is preferred. Furthermore, if necessary, a structure for separating each cell aggregate may be provided on the substrate, such as by providing a partition plate.

[0055] In the present invention, it is preferable that the substrate is a porous substrate, and that the pore diameter of the porous substrate is smaller than that of a cell, as it is suitable for rapidly performing the process of forming cell aggregates from cell aggregates, and furthermore, even when producing large cell aggregates, it is possible to distribute nutrients evenly throughout the inside of the cell aggregates. Also, as it is suitable for rapidly performing the process of forming cell aggregates from cell aggregates, the pore diameter is preferably 0.01 to 8 μm, more preferably 0.01 to 3 μm, particularly preferably 0.01 to 1 μm, and most preferably 0.1 to 1 μm. Here, in the present invention, the "pore diameter" of the porous substrate refers to the average value of the diameter of the pores in the porous substrate along the in-plane direction of the porous substrate, and can be calculated by measuring the diameter of 20 or more pores in a laser microscope image, scanning electron microscope image, or transmission electron microscope image of the porous substrate and determining the average value.

[0056] The porous substrate is also suitable for rapidly performing the process of forming cell aggregates from cell aggregates, and therefore, it is preferable that the porosity is 0.01 to 60%, more preferably 0.01 to 20%, particularly preferably 0.01 to 4%, and most preferably 0.01 to 1.5%. Here, in the present invention, the "porosity" of the porous substrate is the value obtained by dividing the total area of ​​the pores by the substrate area for one main surface of the porous substrate, and indicates the extent of voids present on the substrate surface as an area ratio. This can be measured by observing a square region with sides at least 200 times the pore diameter of the porous substrate in a laser microscope image, scanning electron microscope image, or transmission electron microscope image of the porous substrate.

[0057] The cell culture substrate of the present invention may be sterilized. There are no particular limitations on the sterilization method, but autoclaving, UV sterilization, gamma ray sterilization, ethylene oxide gas sterilization, etc., can be used. Autoclaving, UV sterilization, and ethylene oxide gas sterilization are preferred because they are suitable for suppressing the denaturation of the block copolymer, UV sterilization or ethylene oxide gas sterilization are more preferred because they are suitable for suppressing deformation of the substrate, and ethylene oxide gas sterilization is particularly preferred because it is excellent in terms of mass productivity.

[0058] The cells used in the method for producing cell aggregates of the present invention are not particularly limited as long as they can adhere to a cell culture substrate. For example, in addition to various cell lines such as Chinese hamster ovary-derived CHO cells, mouse connective tissue L929, human fetal kidney-derived HEK293 cells, and human cervical cancer-derived HeLa cells, other cells that can be used include epithelial cells and endothelial cells that make up various tissues and organs in the body, contractile skeletal muscle cells, smooth muscle cells, cardiomyocytes, neurons, glial cells, fibroblasts that make up the nervous system, hepatocytes, non-parenchymal hepatocytes, and adipocytes that are involved in the metabolism of the body, as well as cells with differentiation potential, such as mesenchymal stem cells, bone marrow cells, and Muse cells, which are stem cells present in various tissues, and also pluripotent stem cells (pluripotent stem cells) such as ES cells and iPS cells, and cells differentiated from them. It is preferable to use epithelial cells and mesenchymal cells because they are suitable for forming organ primordia.

[0059] The present invention provides a method for producing cell aggregates, comprising the following steps (1) to (3). (1) A step of preparing a cell suspension containing mesenchymal cells and ectoderm-derived cells. (2) A step of bringing the cell suspension prepared in step (1) into contact with a cell culture substrate and randomly adhering mesenchymal cells and ectoderm-derived cells to region (A), wherein the total number of mesenchymal cells and ectoderm-derived cells at the contact surface between the cell suspension and the cell culture substrate is 100 to 100,000 cells per unit area. (3) A step of culturing the cells attached to the region (A) to form a cell mass containing mesenchymal cells and ectoderm-derived cells.

[0060] Furthermore, the method for producing cell aggregates of the present invention may also include the following step (4). (4) A step of culturing the cell aggregate formed in step (3) above to form an organoid.

[0061] In step (1) above, a cell suspension containing two types of cells, mesenchymal cells and ectoderm-derived cells, is prepared. There are no particular limitations on the method of preparing the cell suspension; two or more types of cells that have been cultured separately in advance are detached and collected as single cells using enzymes or the like, and mixed in a culture medium in a desired ratio. The mixing ratio of the two or more types of cells is preferably in the range of 0.5 to 1.5 times, more preferably in the range of 0.8 to 1.2 times, particularly preferably in the range of 0.9 to 1.1 times, and most preferably in the range of 0.95 to 1.05 times, based on the cell ratio when all cells are mixed in a uniform ratio, as this is suitable for forming regions with a high proportion of one type of cell. The ratio of the number of mesenchymal cells to ectoderm-derived cells in the cell suspension may be 1:10 to 10:1, may be 1:5 to 5:1, and is preferably 1:1. When the ratio of mesenchymal cells to ectoderm-derived cells in the cell suspension falls within these ranges, it becomes more suitable for forming regions with a high proportion of either mesenchymal cells or ectoderm-derived cells.

[0062] In step (2) above, the cell suspension prepared in step (1) above is brought into contact with a cell culture substrate, and mesenchymal cells and ectoderm-derived cells are randomly adhered to area (A). Here, the total number of mesenchymal cells and ectoderm-derived cells at the contact surface between the cell suspension and the cell culture substrate is 100 to 100,000 cells per unit area. By adhering the cells, the cell aggregate is fixed to the cell culture substrate, which reduces damage to the cell aggregate due to shaking during culture work and transport. If the cells are not adhered, the cell aggregate is easily damaged by collision with the walls of the cell culture substrate or other cell aggregates during culture work and transport. In order to adhere a sufficient amount of cells to area (A) and to easily form areas with a high proportion of one type of cell, the number of seeded cells per unit area of ​​the cell culture substrate (total number of mesenchymal cells and ectoderm-derived cells at the contact surface between the cell suspension and the cell culture substrate) is set to 1000 cells / cm 2 The above is preferable, with 3000 cells / cm². 2 The above is even more preferable, with 5000 cells / cm². 2 The above is particularly preferred, with 10,000 cells / cm². 2 The above is the most preferable. Furthermore, in order to suppress the aggregation of cells in a suspended state, the number of seeded cells per unit area of ​​the cell culture substrate (the total number of mesenchymal cells and ectoderm-derived cells at the contact surface between the cell suspension and the cell culture substrate) should be 100,000 cells / cm². 2 The following is preferable: 80,000 cells / cm 2 The following is even more preferable: 50,000 cells / cm 2 The following is particularly preferred: 20,000 cells / cm 2 The following is the most preferable.

[0063] In step (3) above, cells adhered to region (A) are cultured to form a cell aggregate containing mesenchymal cells and ectoderm-derived cells. Furthermore, by using the cell culture substrate of the present invention, a cell aggregate is formed having at least two regions: one region with a high proportion of ectoderm-derived cells and another region with a low proportion of the aforementioned cells (a high proportion of mesenchymal cells). By culturing two or more types of cells in a region having the aforementioned area, only one type of cell can spontaneously aggregate, forming a region with a high proportion of that one type of cell.

[0064] In step (3) above, the cell aggregate may include regions where the proportion of ectoderm-derived cells is higher than that of mesenchymal cells (also simply referred to as regions where the proportion of ectoderm-derived cells is higher), as this is suitable for organoid formation. Furthermore, the cell aggregate may also include regions where the proportion of mesenchymal cells is higher than that of ectoderm-derived cells (also simply referred to as regions where the proportion of mesenchymal cells is higher). When the cell aggregate includes regions where the proportion of mesenchymal cells is higher and regions where the proportion of ectoderm-derived cells is higher, the ratio of the maximum cross-sectional area in the substrate plane of the region where the proportion of mesenchymal cells is higher to the maximum cross-sectional area in the substrate plane of the region where the proportion of ectoderm-derived cells is higher is preferably 2:1 to 1:20, more preferably 2:1 to 1:10, particularly preferably 1:1 to 1:5, and most preferably 1:1 to 1:2, as this is more suitable for organoid formation.

[0065] In step (4) above, the cell aggregate formed in step (3) above is cultured to form an organoid. The organoid may express Versican protein, which is a marker for dermal papilla cells.

[0066] The cell aggregate preferably has at least two parts: a part consisting of cells adhered to the (A) region and a substantially spherical part adhered to the adhered cells, wherein the diameter of the substantially spherical part is smaller than the diameter of the (A) region. The shape of the cell aggregate facilitates differentiation into organoids such as hair follicle primordia.

[0067] There are no particular restrictions on the type of culture medium used in this invention, and it can be appropriately selected depending on the cells to be cultured. For example, when using two types of cells, mesenchymal cells and epithelial cells, a culture medium consisting of DMEM (manufactured by Takara Bio Inc.) and keratinocyte proliferation medium (manufactured by Takara Bio Inc.) in a 1:1 ratio is preferred.

[0068] When two or more types of cells (e.g., mesenchymal cells and ectoderm-derived cells) are co-cultured, a high proportion of one type of cell (e.g., ectoderm-derived cells) means that, after performing a treatment that allows fluorescent labeling only for specific cells (immunostaining), and then observing the cell aggregate using a phase-contrast microscope and a fluorescence microscope, the proportion of cells of one type of cell (e.g., ectoderm-derived cells) to the total number of cells in a cross-section of the cell aggregate in a certain region is 50% or more. When two or more types of cells (e.g., mesenchymal cells and ectoderm-derived cells) are co-cultured, a low proportion of one type of cell (e.g., ectoderm-derived cells) means that the above proportion is less than 50%.

[0069] Another aspect of the present invention relates to a kit for inducing differentiation of hair follicle primordia, comprising a cell culture substrate having the following two regions (A) and (B). The cell culture substrate included in this kit can be the same substrate used for cell culture substrates used in methods for producing cell aggregates. (A) Has cell proliferation properties and an area of ​​0.001-1 mm² 2 A circular area. (B) A region adjacent to the region (A) above that does not have cell proliferation properties.

[0070] The hair follicle primordium differentiation induction kit may further include a culture medium that promotes the proliferation of mesenchymal and epithelial cells. The culture medium that promotes the proliferation of mesenchymal and epithelial cells may be the same culture medium used in the method for producing cell aggregates (for example, a culture medium prepared by mixing DMEM (manufactured by Takara Bio Inc.) and keratinocyte proliferation medium (manufactured by Takara Bio Inc.) in a 1:1 ratio). [Examples]

[0071] The present invention will be described in detail below with reference to embodiments for carrying it out. However, these are merely examples to illustrate the present invention and are not intended to limit it to the following. Furthermore, the present invention can be implemented with appropriate modifications within the scope of its essence. Unless otherwise specified, commercially available reagents were used. [Example 1] A dish (manufactured by Sumitomo Bakelite Co., Ltd., product name: PrimeSurface) with a hydrophilic polymer surface and a diameter of 35 mm was covered with a metal mask (manufactured by Mitani Micronics Co., Ltd.) having multiple circular holes with a diameter of 0.2 mm. Plasma treatment (under a gas pressure of 20 Pa, conductive current of 20 mA, irradiation time of 30 seconds) was performed from above the metal mask using a plasma irradiation device (manufactured by Vacuum Device Co., Ltd., product name: Plasma Ion Bombardier PIB-20), thereby creating a cell culture substrate with areas of cell adhesion and cell proliferation.

[0072] Mesenchymal cells (bone marrow-derived mesenchymal stem cells) and epithelial cells (human gingival epithelial cells) cultured in polystyrene flasks were detached with 0.5×TrypLE-EDTA solution, centrifuged, and redispersed in a culture medium consisting of a 1:1 mixture of DMEM (Takara Bio Inc.) and keratinocyte growth medium (Takara Bio Inc.). The number of cells was counted, and cell suspensions were prepared by mixing mesenchymal cells and epithelial cells in a 1:1 ratio.

[0073] The cell suspension is added to the patterned cell culture substrate, at a rate of 4500 cells / cm³. 2 Cells were seeded. They were cultured for 12 days at 37°C and a CO2 concentration of 5%. Phase-contrast microscopy revealed that on day 1 of culture, cells were randomly adhered within region (A), but on day 4 of culture, epithelial cells gathered and formed cell aggregates with a high proportion of epithelial cells. Furthermore, on day 11 of culture, the cell aggregates had at least two regions: a region consisting of cells adhered to region (A), and a roughly spherical region adhered to the adhered cells.

[0074] When the cell culture substrate containing the cell aggregate described above was shaken for one hour using a shaker, all the cells remained fixed in an adherent state, and no damage to the cell aggregate was observed.

[0075] [Example 2] Instead of using a metal mask with multiple circular holes of 0.2 mm in diameter as in Example 1, a metal mask with multiple circular holes of 0.5 mm in diameter was used. Also, the cell seeding density was 4500 cells / cm² as in Example 1. 2 Instead, a cell seeding density of 15,000 cells / cm² 2 Cell culture was performed. On day 1 of culture, cells were randomly adhered within region (A), but on day 6 of culture, epithelial cells gathered and formed cell aggregates with a high proportion of epithelial cells.

[0076] When the cell culture substrate containing the cell aggregate described above was shaken for one hour using a shaker, all the cells remained fixed in an adherent state, and no damage to the cell aggregate was observed.

[0077] [Example 3] Human dermal papilla cells, fluorescently stained with Vybrant DiO Cell-Labeling Solution, were used as mesenchymal cells, and the culture was carried out in the same manner as in Example 1.

[0078] Cultured cells were observed using phase-contrast and fluorescence microscopy. On day 1 of culture, mesenchymal and epithelial cells were randomly present in region (A). On day 7 of culture, it was confirmed that cell aggregates were formed, containing areas with a high proportion of mesenchymal cells (low proportion of epithelial cells) and areas with a low proportion of mesenchymal cells (high proportion of epithelial cells). Images of the cell aggregates were acquired using phase-contrast and fluorescence microscopy from a direction perpendicular to the cell culture substrate. In these images, the ratio of the maximum cross-sectional area in the substrate plane of areas with a high proportion of mesenchymal cells (low proportion of epithelial cells) to the maximum cross-sectional area in the substrate plane of areas with a low proportion of mesenchymal cells (high proportion of epithelial cells) was 1:8.5.

[0079] [Example 4] Human dermal papilla cells were used as mesenchymal cells, and culture was carried out in the same manner as in Example 1. After that, Versican was fluorescently stained using Human Versican Isoform V0 Antibody (manufactured by R&D SYSTEMS) and Donkey anti-Goat IgG (H+L) Cross-Adsorbed Secondary Antibody, Alexa Fluor 488 (manufactured by Thermo Fisher).

[0080] Cultured cells were observed using phase-contrast and fluorescence microscopy. On day 1 of culture, mesenchymal and epithelial cells were randomly present in region (A). On day 7 of culture, cell aggregates were formed, containing areas with a high proportion of mesenchymal cells (low proportion of epithelial cells) and areas with a low proportion of mesenchymal cells (high proportion of epithelial cells). Furthermore, cultured cells were observed using fluorescence microscopy and confirmed to express Versican. Images of the cell aggregates were acquired using phase-contrast and fluorescence microscopy from a direction perpendicular to the cell culture substrate. In these images, the ratio of the maximum cross-sectional area in the substrate plane of areas with a high proportion of mesenchymal cells (low proportion of epithelial cells) to the maximum cross-sectional area in the substrate plane of areas with a low proportion of mesenchymal cells (high proportion of epithelial cells) was 1:4.9.

[0081] [Example 5] Instead of using a metal mask with multiple circular holes of 0.2 mm in diameter as in Example 1, a metal mask with multiple circular holes of 0.1 mm in diameter was used to create a cell culture substrate with areas for cell adhesion and cell proliferation. Human dermal papilla cells were used as mesenchymal cells, and the culture was carried out in the same manner as in Example 4.

[0082] Cultured cells were observed using phase-contrast and fluorescence microscopy. On day 1 of culture, mesenchymal and epithelial cells were randomly present in region (A). On day 7 of culture, cell aggregates were formed, containing areas with a high proportion of mesenchymal cells (low proportion of epithelial cells) and areas with a low proportion of mesenchymal cells (high proportion of epithelial cells). Furthermore, cultured cells were observed using fluorescence microscopy and confirmed to express Versican. Images of the cell aggregates were acquired using phase-contrast and fluorescence microscopy from a direction perpendicular to the cell culture substrate. In these images, the ratio of the maximum cross-sectional area in the substrate plane of areas with a high proportion of mesenchymal cells (low proportion of epithelial cells) to the maximum cross-sectional area in the substrate plane of areas with a low proportion of mesenchymal cells (high proportion of epithelial cells) was 1.5:1.

[0083] [Comparative Example 1] In this example, instead of using mesenchymal cells as in Example 1, only epithelial cells were used for culturing, and the rest of the culturing process was the same as in Example 1. Even after 12 days of continuous culturing, only cell aggregates with a uniform distribution of all cells were formed, and no cell aggregates with a high proportion of one cell type were formed.

[0084] [Comparative Example 2] In this example, instead of using epithelial cells as in Example 1, only mesenchymal cells were used for culturing, and the rest of the culturing process was the same as in Example 1. Even after 12 days of continuous culturing, only cell aggregates with a uniform distribution of all cells were formed, and no cell aggregates with a high proportion of one cell type were formed.

[0085] [Comparative Example 3] Instead of using a metal mask with multiple circular holes of 0.2 mm in diameter as in Example 1, a metal mask with multiple circular holes of 1.5 mm in diameter was used. Also, the cell seeding density was 4500 cells / cm² as in Example 1. 2 Instead, a cell seeding density of 15,000 cells / cm² 2The cells were cultured. Even after continuing the culture for 12 days, only cell aggregates with a uniform distribution of all cells were formed, and no cell aggregates with a high proportion of one type of cell were formed.

[0086] [Comparative Example 4] A cell-non-adherent U-bottom container (Sumitomo Bakelite Co., Ltd., product name: PrimeSurface 96-well plate) was used as the cell culture substrate, and culture was carried out at a cell seeding density of 2400 cells / well, with the rest of the procedure being the same as in Example 1. On the fourth day of culture, it was confirmed that epithelial cells had aggregated and formed cell clumps with a high proportion of epithelial cells.

[0087] The cell aggregates were all collected in a dish (manufactured by Sumitomo Bakelite Co., Ltd., product name: PrimeSurface) with a diameter of 35 mm and a surface coated with a hydrophilic polymer. When the cell culture substrate was shaken in a shaker for 1 hour, the cell aggregates were damaged and did not retain their original shape.

[0088] [Comparative Example 5] Cell seeding density in Example 1: 4500 cells / cm² 2 Instead, a cell seeding density of 150,000 cells / cm² 2 Cell culture was carried out. On the 7th day of culture, the cells were suspended and had formed cell clumps. When the cell culture substrate was shaken in a shaker for 1 hour, the cell clumps were damaged and did not maintain their original shape.

[0089] [Table 1] [Explanation of Symbols]

[0090] A (A) area B (B) area 1 Epithelial cells 2. Mesenchymal cells 3 cell clusters 4. A cell mass having at least two regions: a region with a high proportion of epithelial cells (a low proportion of mesenchymal cells) and a region with a low proportion of the aforementioned cells (a high proportion of mesenchymal cells).

Claims

1. A method for producing a cell aggregate cultured on a cell culture substrate, wherein the cell culture substrate has two regions (A) and (B) as described below, and the method comprises the following steps (1) to (3). (A) Has cell proliferation properties and an area of ​​0.001 to 0.5 mm ２ An island-like region. (B) A region adjacent to the region (A) that does not have cell proliferation properties. (1) A step of preparing a cell suspension containing mesenchymal cells and ectoderm-derived cells. (2) A step of bringing the cell suspension prepared in step (1) into contact with the cell culture substrate and randomly adhering the mesenchymal cells and the ectoderm-derived cells to the region (A), wherein the total number of mesenchymal cells and ectoderm-derived cells at the contact surface between the cell suspension and the cell culture substrate is 1,000 to 20,000 cells per unit area. (3) A step of culturing cells attached to the region (A) to form a cell mass containing the mesenchymal cells and the ectoderm-derived cells, wherein the cell mass includes a region in which the proportion of mesenchymal cells is greater than that of the ectoderm-derived cells and a region in which the proportion of ectoderm-derived cells is greater than that of the mesenchymal cells, and the ratio of the maximum cross-sectional area in the direction of the substrate plane of the region in which the proportion of mesenchymal cells is greater than that of the ectoderm-derived cells is 2:1 to 1:

20.

2. The method for producing a cell aggregate according to claim 1, wherein the ratio of the number of mesenchymal cells to the number of ectoderm-derived cells in the cell suspension is 1:10 to 10:

1.

3. A method for producing a cell aggregate according to claim 1 or 2, further comprising the following step (4). (4) A step of culturing the cell aggregate formed in step (3) above to form an organoid.

4. The method for producing a cell aggregate according to claim 3, characterized in that Versican is present in the organoid.

5. A hair follicle primordium differentiation induction kit comprising a cell culture substrate having the following two regions (A) and (B), A hair follicle primordium differentiation induction kit, wherein the hair follicle primordium includes a region where the proportion of mesenchymal cells is greater than that of ectoderm-derived cells, and a region where the proportion of ectoderm-derived cells is greater than that of mesenchymal cells, and the ratio of the maximum cross-sectional area in the substrate plane of the region where the proportion of mesenchymal cells is greater than that of ectoderm-derived cells to the maximum cross-sectional area in the substrate plane of the region where the proportion of ectoderm-derived cells is greater than that of mesenchymal cells is 2:1 to 1:

20. (A) Has cell proliferation properties and an area of ​​0.001 to 0.5 mm ２ A circular area. (B) A region adjacent to the region (A) that does not have cell proliferation properties.

6. A hair follicle primordium differentiation induction kit according to claim 5, further comprising a culture medium that promotes the proliferation of mesenchymal and epithelial cells.

Images