WO2023176949A1

WO2023176949A1 - Cell culture container having high cell utilization efficiency

Info

Publication number: WO2023176949A1
Application number: PCT/JP2023/010495
Authority: WO
Inventors: 康平鈴木; 佳臣広井; 淳二福田
Original assignee: 日産化学株式会社; 国立大学法人横浜国立大学
Priority date: 2022-03-17
Filing date: 2023-03-17
Publication date: 2023-09-21

Abstract

A container for use in the production of a cell structure, the container being provided with a plurality of depressed parts and partitioning walls that partitions the plurality of depressed parts, in which the shape of a top part of each of the partitioning walls is a convex shape, and the depth of each of the depressed parts is more than 1.0 time the equivalent diameter of each of the depressed parts at the center in the depth direction of each of the depressed parts.

Description

Cell culture container with high cell utilization efficiency

The present invention relates to a container for manufacturing a cell structure, a method for manufacturing a container for manufacturing a cell structure, a cell structure, and a method for manufacturing a cell structure.

Cell structures or cell aggregates (also referred to as spheroids or cell aggregates) are cell aggregates in which cells self-assemble and become three-dimensional aggregates.Since a living body-like structure is constructed, cell functions It has been reported that this can be maintained for a long period of time and physiological functions are improved. Therefore, expectations are increasing for the use of cell aggregates in drug discovery research, cell therapy, and regenerative therapy.

Along with this, the development of tissue engineering technology that can easily and quickly produce uniform cell structures or cell aggregates in large quantities has become an important issue for the practical application of regenerative medicine and the efficiency of drug discovery testing. In conventional methods that utilize the random aggregation phenomenon of floating cells using culture dishes with low cell adhesion (e.g., multi-well plates), only one spheroid is formed per well, making it difficult to operate and mass-produce. There was an issue of not being good at it.

Therefore, in order to mass culture spheroids, a technique has been proposed in which spheroids are formed in a microscopic space (see, for example, Patent Document 1).

International Publication No. 2012/036011 pamphlet

In the technology of Patent Document 1, when a flat surface is formed on the substrate surface between recesses that are close to each other in a culture substrate, when spheroid culture is performed using the culture substrate, in addition to spheroids, single cells are formed. This was done in view of the problem that many layer-cultured cells and spheroids of non-uniform size are formed (see paragraph [0006] of Patent Document 1). In the technique of Patent Document 1, a plurality of depressions forming compartments in which the cultured material is cultured are formed on the surface of the culture substrate, and the surface of the culture substrate between the depressions that are close to each other is non-transparent. Culture substrates that are flat surfaces have been proposed.
However, even with the technique described in International Publication No. 2012/036011 pamphlet, spheroids (cell structures) of non-uniform size may be generated.

The present invention was made in view of the above-mentioned circumstances, and an object of the present invention is to provide a container for producing cell structures that can produce cell structures of uniform size without reducing culture efficiency. do. The present invention also provides a method for manufacturing the cell structure manufacturing container, a cell structure manufactured using the cell structure manufacturing container, and a cell structure manufacturing method using the cell structure manufacturing container. The purpose is to provide a method.

The present inventors have found that a container for producing cell structures in which the partition wall has a specific shape and the depth of the concave portion is deep to a certain extent relative to the equivalent diameter can solve the above-mentioned problems, and the present invention completed.

That is, the present invention includes the following aspects.
[1] It has a plurality of recesses and a partition wall that partitions the plurality of recesses,
The top of the partition wall has a convex shape,
The depth of the recess is more than 1.0 times the equivalent diameter of the recess at the center in the depth direction of the recess.
Container for producing cell structures.
[2] The cell according to [1], wherein the width of the partition wall that partitions two adjacent recesses is 0.1 mm to 0.5 mm when measured at the center in the depth direction of the recess. Container for manufacturing structures.
[3] The cell structure manufacturing container according to [1] or [2], which is made of a gas-permeable elastic material.
[4] The container for producing a cell structure according to [3], wherein the gas-permeable elastic material is silicone.
[5] The cell structure production according to any one of [1] to [4], wherein the depth of the recess is 1.5 times or more the equivalent diameter of the recess at the center in the depth direction of the recess. container.
[6] The container for producing a cell structure according to any one of [1] to [5], wherein the central part of the recess excluding the bottom and the opening has a non-tapered shape.
[7] The cell structure manufacturing container according to [6], wherein the length in the depth direction of the recess in the non-tapered central portion is 0.5 times or more the depth of the recess.
[8] From [1] to [7], the shape of the top of the partition wall in a cross section including the center line of each of the two adjacent recesses is a convex arc or a shape having a corner at the top. ] The container for producing a cell structure according to any one of the above.
[9] The width of the partition wall that partitions two adjacent recesses is 0.1 times or more and 0.5 times or less the equivalent diameter of the recess, when measured at the center in the depth direction of the recess. The container for producing a cell structure according to any one of [1] to [8].
[10] The plurality of recesses are arranged such that the distances between the center point of the opening of the specific recess and the center points of each of the openings of two or more recesses arranged adjacent to the specific recess are equal to each other. The cell structure manufacturing container according to any one of [1] to [9], wherein the recessed portion is arranged.
[11] A location corresponding to the center of the equilateral triangle in the partition wall surrounded by three mutually adjacent recesses arranged to form an equilateral triangle when the center points of the openings are connected is located in the partition wall. The container for producing a cell structure according to [10], which is the most expensive.
[12] The cell structure manufacturing container according to any one of [1] to [11], wherein the bottoms of the plurality of recesses have an arc shape or an inverted triangular shape in cross section.
[13] The container for producing a cell structure according to any one of [1] to [12], wherein the equivalent diameter of the recess is 0.3 mm to 1.5 mm.
[14] The container for producing a cell structure according to any one of [1] to [13], wherein the depth of the recess is 0.5 mm to 3.0 mm.
[15] The container for producing a cell structure according to any one of [1] to [14], which has 5 to 2000 recesses.
[16] The container for producing a cell structure according to any one of [1] to [15], which has a cylindrical outer shape.
[17] The cell structure manufacturing container according to any one of [1] to [16], which can be housed in a recessed part of a multiwell plate.
[18] The cell structure manufacturing container according to any one of [1] to [17], wherein at least a portion of the surface of the cell structure manufacturing container is provided with a coating film having the ability to suppress cell adhesion.
[19] A method for manufacturing a cell structure manufacturing container according to any one of [1] to [18], comprising:
A method for manufacturing a cell structure manufacturing container, the method comprising molding the cell structure manufacturing container using a mold.
[20] A cell structure produced using the cell structure production container according to any one of [1] to [18].
[21] A method for producing a cell structure, comprising the step of adding a medium in which cells are dispersed to a culture space of the container for producing a cell structure according to any one of [1] to [18].

According to the present invention, it is possible to provide a container for producing cell structures that can produce cell structures of uniform size without reducing culture efficiency. Further, according to the present invention, there is provided a method for manufacturing a container for manufacturing a cell structure, a cell structure manufactured using the container for manufacturing a cell structure, and a cell structure using the container for manufacturing a cell structure. A manufacturing method can be provided.

FIG. 1 is a perspective view of an example of a cell structure manufacturing container. FIG. 2 is a top view of the cell structure manufacturing container of FIG. 1. FIG. 3A is a cross-sectional view taken along line A-A' of the cell structure manufacturing container shown in FIG. FIG. 3B is an enlarged view of area A in FIG. 3A. FIG. 4 is a cross-sectional view for explaining one embodiment of the bottom of the recess of the cell structure manufacturing container. FIG. 5 is a sectional view taken along the line B-B' of the cell structure manufacturing container shown in FIG. FIG. 6 is a photograph of the observation results of Example 1. FIG. 7A is a photograph of the observation results of Comparative Example 1 (Part 1). FIG. 7B is a photograph of the observation results of Comparative Example 1 (Part 2). FIG. 8A is a photograph of the observation results of Example 2 (Part 1). FIG. 8B is a photograph of the observation results of Example 2 (Part 2). FIG. 9 is a photograph of the observation results of Example 1 after the medium was replaced. FIG. 10 is a photograph of the observation results of Comparative Example 2 after the medium was replaced. FIG. 11A is a photograph of the bright field observation results of the hair follicle primordium formation test. FIG. 11B is a photograph of the fluorescence observation results of the hair follicle primordium formation test. FIG. 11C is a photograph in which the photograph in FIG. 11A and the photograph in FIG. 11B are superimposed. FIG. 12 is a photograph of the observation results of a hair regeneration test using the patch method. FIG. 13A is a photograph of the observation results of Example 3 (Part 1). FIG. 13B is a photograph of the observation results of Example 3 (Part 2). FIG. 14 is a photograph of the observation results of Example 4.

(Container for producing cell structures)
The cell structure manufacturing container of the present invention has a plurality of recesses and a partition wall that partitions the plurality of recesses.
The top of the partition wall has a convex shape.
The depth of the recess is more than 1.0 times the equivalent diameter of the recess at the center in the depth direction of the recess.

The present inventors have conducted extensive studies in order to provide a container for producing cell structures that can produce cell structures of uniform size without reducing culture efficiency.
First, as described in paragraph [0006] of International Publication No. 2012/036011 pamphlet, when a flat surface is formed on the substrate surface between the recesses that are close to each other in the culture substrate, the culture substrate is When cell structures are cultured using this method, in addition to uniformly sized cell structures formed within the depression, many monolayer cultured cells and non-uniformly sized cell structures are also formed. I was sure that. The inventors of the present invention considered that the cause lies in the shape of the upper surface (top) of the partition wall. Therefore, the present inventors made the top of the partition wall convex. By doing so, even if the compartment wall is thick, cells or cell structures are less likely to exist on the top of the compartment wall, and more uniform cell structures can be formed compared to when the top of the compartment wall is flat. Do you get it.
By the way, in culturing cells, the medium is usually replaced multiple times during the culturing period. The present inventors discovered that when the surface of the culture substrate is made into a non-flat surface (convex shape) like the culture substrate of the technique of International Publication No. 2012/036011 pamphlet, when the culture medium is replaced, the growing It has been found that spheroids (cell structures) tend to separate from depressions (concavities). As a result, the efficiency of culture decreases. Furthermore, if the detached and growing cell structures coalesce and return to the depression (recess), the uniformity of the size of the produced cell structures will be impaired. In addition, in the technology of International Publication No. 2012/036011 pamphlet, for example, the depth of the recess 20 is designed to be 200 ± 20 μm, and the diameter of the opening of the recess 20 is designed to be 500 ± 20 μm. (See paragraph [0025] of International Publication No. 2012/036011 pamphlet).
Therefore, even if the apex is made convex, the depth of the recess is made deeper (the depth of the recess is larger than the equivalent diameter of the recess), thereby improving the cell structure during culture medium exchange. It was found that the body was difficult to separate from the recess. As a result, it became possible to produce cell structures of uniform size without reducing culture efficiency, and the present invention was completed.

Hereinafter, embodiments of the cell structure manufacturing container will be described using the drawings.
FIG. 1 is a perspective view of an example of a cell structure manufacturing container 100. The outer shape of the cell structure manufacturing container 100 is cylindrical with circular upper and lower surfaces, and the diameters of the upper and lower surfaces are, for example, 5 mm to 50 mm. The height is, for example, 5 mm to 50 mm.
The bottom surface 1 of the cell structure manufacturing container 100 is usually flat-bottomed.
The cell structure manufacturing container has an external shape that can be accommodated in, for example, a recessed part of a multiwell plate. The number of wells in a multiwell plate is, for example, 6 to 96, more specifically 6, 12, 24, 48, or 96. A multiwell plate, also called a cell culture multiplate, is a plate having a plurality of wells used for cell culture, tissue culture, etc., and the volume of each well is about 0.1 mL to 20 mL.
Since the container for cell structure production can be accommodated in the recessed portion of the multiwell plate, a plurality of containers for cell structure production can be handled as one, which increases the ease of handling.

The cell structure manufacturing container 100 has an outer peripheral wall 2 and a large opening 3 on the top surface, and the space inside the cell structure manufacturing container 100 surrounded by the outer peripheral wall 2 and the opening 3 is This is a culture space 4 that can accommodate a culture medium.

FIG. 2 is a top view of the cell structure manufacturing container 100 of FIG. 1.
In the culture space 4 surrounded by the outer peripheral wall 2, a plurality of recesses 10 and a partition wall 11 that partitions the plurality of recesses 10 are formed. The number of recesses 10 in the cell structure manufacturing container 100 in FIG. 2 is nineteen. In FIG. 2, the plurality of recesses 10 are circular when viewed from the top side.
The number of recesses in the cell structure manufacturing container can be appropriately selected depending on the size of the cell structure manufacturing container itself and the size of the plurality of recesses, and is not particularly limited, but for example, 5 recesses. ~2000 pieces.

FIG. 3A is a sectional view taken along the line AA' of the cell structure manufacturing container 100 of FIG. 2.
In the cross-sectional view of FIG. 3A, five recesses 10, partition walls 11 that partition them, and an outer peripheral wall 2 higher than the partition walls 11 at the outermost side can be confirmed.
The cross section of FIG. 3A can be said to be a cross section that includes the respective center lines of the two adjacent recesses 10. Here, the center line means a line passing through the center of the opening surface 10a of the recess 10 and extending in the depth direction of the recess 10. When the shape of the aperture surface 10a is a circle, the center is the center of the circle, but when the shape of the aperture surface 10a is other than a circle, the center means the center of gravity.
In FIG. 3A, the shape of the top portion 11a of the partition wall 11 (in other words, the top portion 11a of the partition wall 11 in a cross section including the center line of each of the two adjacent recesses 10) is a convex shape. Specifically, it is arcuate. The shape of the top portion 11a in FIG. 3A is not particularly limited as long as it is a convex shape, for example, and may have a corner portion at the top, for example.
Since the top portion 11a of the partition wall 11 in FIG. 3A has a convex shape, it becomes difficult for cells to stay on the top portion 11a. As a result, cells are not cultured in a monolayer or structured in the apex 11a, so the cell structures obtained when cells are cultured are more uniform in size than in the case where the apex of the partition wall is flat. Sex increases.

The depth of the recess 10 is not particularly limited, but is, for example, 0.5 mm to 3.0 mm. In FIG. 3A, the depth (d) of the recess 10 is 2.0 mm.
The equivalent diameter of the recess 10 is not particularly limited, but is, for example, 0.3 mm to 1.5 mm. In FIG. 3A, the equivalent diameter (r) of the recess 10 is, for example, 1.0 mm.
Here, the depth (d) of the recess 10 refers to the length (d in FIG. 3A) from the lowest point of the bottom 10b of the recess to the opening surface 10a of the recess.
Further, the equivalent diameter (r) of the recess 10 is the equivalent diameter at the center 10c of the recess 10 in the depth direction. The equivalent diameter means the diameter of a circle when the cross-sectional area of the cross section of the recess that is perpendicular to the depth direction of the recess and passes through the center of the recess in the depth direction is the area of the circle. If the cross section is a circle, the diameter of the circle is the equivalent diameter (r). If the cross section is other than a circle, calculate the area of the cross section and use the formula for the area of a circle ((r'/2) ² × π) (r' is the diameter of the circle) to find the equivalent r'. It is the diameter.

Since the depth (d) of the recess is more than 1.0 times the equivalent diameter (r) of the recess at the center of the recess in the depth direction, even if the top is made convex, cells cannot be The structure becomes difficult to separate from the recess.
The depth (d) of the recess is preferably 1.5 times or more, more preferably 1.7 times or more, the equivalent diameter (r). The larger d/r is, the more difficult it becomes for the cell structure to separate from the recess when replacing the medium.
The upper limit of d/r is not particularly limited, but if it is large, the recesses will easily trap air bubbles, and the culture medium in the recess will not be replaced sufficiently when changing the culture medium, so the depth of the recess (d) is preferably 5.0 times or less than the equivalent diameter (r), more preferably 3.5 times or less, particularly preferably 2.5 times or less.

The width (w) of the partition wall 11 that partitions two adjacent recesses 10 is not particularly limited, but is 0.1 of the equivalent diameter (r) of the recess when measured at the center 10c of the recess in the depth direction. It is preferable that it is 0.5 times or more.
The width (w) of the partition wall 11 that partitions two adjacent recesses 10 is not particularly limited, but is, for example, 0.1 mm to 0.5 mm when measured at the center 10c of the recess in the depth direction. .

In FIG. 3A, the bottom portions 10b of the plurality of recesses 10 have an arcuate shape in cross section. The arc shape may be a semicircle (or a hemisphere when viewed stereoscopically), an ellipse, or the like. Moreover, the shape of the bottom of the plurality of recesses may be flat, or may have a corner at the deepest part.
The shape having the corner at the deepest part is, for example, a shape as shown in FIG. 4, which is an inverted triangular shape in cross section (for example, an inverted conical shape when viewed stereoscopically). In FIG. 4, the depth (d') of the bottom portion 10b is, for example, 1.0 mm.

FIG. 3B is an enlarged view of area A in FIG. 3A.
In FIG. 3B, the shape of the central portion 10e of the recessed portion 10 excluding the bottom portion 10b and the opening portion 10d is a non-tapered shape. A non-tapered shape means a shape in which the wall constituting the recess is formed in a straight line in the depth direction and has no taper angle. In other words, for example, if the side surface is cylindrical with a straight line in the height direction, the top and bottom surfaces are the same shape, and the side surface is perpendicular to the top and bottom surfaces, the side surface has a taper angle. Since there is no cylindrical shape, such a cylindrical shape is non-tapered. Further, the cylindrical shape and the rectangular parallelepiped shape are non-tapered.
It is preferable that the length (l) in the depth direction of the concave portion of the non-tapered central portion 10e is 0.5 times or more the depth (d) of the concave portion. Note that the length (l) is less than 1.0 times the depth (d) of the recess, and preferably 0.75 times or less.

In FIG. 2, for the plurality of recesses, the distance (L) between the center point of the opening of a specific recess and the center point of each of the openings of two or more recesses arranged adjacent to the specific recess is They are arranged so that they are equal to each other. Hereinafter, such an arrangement will be referred to as a honeycomb arrangement.
By adopting a honeycomb arrangement, it is possible to reduce the portions where the width of the partition wall is thick, and as a result, it becomes difficult for cells to remain at the top of the partition wall.

FIG. 5 is a BB' cross-sectional view of the cell structure manufacturing container 100 of FIG. 2.
As shown in FIGS. 2 and 5, it corresponds to the center of an equilateral triangle in a partition wall 11 surrounded by three mutually adjacent recesses 10 arranged so as to form an equilateral triangle when the center points of the openings are connected. It is preferable that the apex point 11b is the highest in the partition wall 11. In the honeycomb arrangement, the width of the partition wall 11 is relatively thick at the apex portion 11b, but since it is the highest there, it becomes difficult for cells to remain in the apex portion 11b and its surroundings. As a result, the size uniformity of cell structures obtained when cells are cultured is further increased.
Note that the apex portion 11b in FIG. 5 is higher than the apex 11c of the partition wall 11 in the cross section including the respective center lines of the two adjacent recesses 10 in FIGS. 2, 3b, and 5.

Examples of materials for the container for cell structure production include silicone (e.g., polydimethylsiloxane (PDMS)), polystyrene (PS), cycloolefin polymer (COP), polyacrylonitrile (PAN), and polyester polymer alloy (PEPA). , polysulfone (PSF), polyethylene terephthalate (PET), polymethyl methacrylate (PMMA), polyvinyl alcohol (PVA), polyurethane (PU), ethylene vinyl alcohol (EVAL), polyethylene (PE), polyester, polypropylene (PP), polyfluoride Vinylidene chloride (PVDF), polyethersulfone (PES), polycarbonate (PC), polyvinyl chloride (PVC), polytetrafluoroethylene (PTFE), ultra-high molecular weight polyethylene (UHPE), acrylonitrile-butadiene-styrene resin (ABS) , Teflon (registered trademark), glass, etc.
Among these, polystyrene (PS) and polydimethylsiloxane (PDMS) are more preferred from the viewpoint of moldability and transparency for easy observation of cell culture.

Furthermore, it is preferable that the cell structure manufacturing container is made of a gas-permeable elastic material. This is because, depending on the type of cells, containers with gas permeability are more suitable for culturing. Examples of the gas-permeable elastic material include silicone. Examples of silicone include polydimethylsiloxane (PDMS).

Preferably, at least a portion of the surface of the cell structure manufacturing container is provided with a coating film having the ability to inhibit cell adhesion.
At least a portion of the surface includes, for example, a surface of a recess.
The coating film having the ability to suppress cell adhesion is not particularly limited, but examples include coating films containing the following copolymers.

The copolymer includes a repeating unit containing an organic group represented by the following formula (a) and a repeating unit containing an organic group represented by the following formula (b).
The copolymer is, for example, a copolymer described in WO2014/196652 pamphlet. The contents of WO2014/196652 pamphlet are incorporated herein to the same extent as if expressly set forth in their entirety.

(In the formula, U ^a1 , U ^a2 , U ^b1 , U ^b2 and U ^b3 each independently represent a hydrogen atom or a straight chain or branched alkyl group having 1 to 5 carbon atoms, and An ⁻ is a halogen (Represents an anion selected from the group consisting of compound ions, inorganic acid ions, hydroxide ions, and isothiocyanate ions.)

There are no particular restrictions on the copolymer as long as it contains a repeating unit containing an organic group represented by the above formula (a) and a repeating unit containing an organic group represented by the above formula (b). None. The copolymer is preferably obtained by radical polymerization of a monomer containing an organic group represented by the above formula (a) and a monomer containing an organic group represented by the above formula (b), Those subjected to polycondensation or polyaddition reactions can also be used. Examples of copolymers include vinyl polymers reacted with olefins, polyamides, polyesters, polycarbonates, polyurethanes, etc. Among these, especially vinyl polymers reacted with olefins or (meth)acrylate compounds polymerized. (Meth)acrylic polymers are preferred. In addition, in this invention, a (meth)acrylate compound means both an acrylate compound and a methacrylate compound. For example (meth)acrylic acid means acrylic acid and methacrylic acid.

Preferably, the monomers containing organic groups represented by the above formulas (a) and (b) are monomers represented by the following formulas (A) and (B), respectively.

(In the formula, T ^a , T ^b , U ^a1 , U ^a2 , U ^b1 , U ^b2 and U ^b3 each independently represent a hydrogen atom or a straight chain or branched alkyl group having 1 to 5 carbon atoms, Q ^a and Q ^b each independently represent a single bond, an ester bond, or an amide bond, and R ^a and R ^b each independently represent a carbon atom number of 1 to 10 that may be substituted with a halogen atom. represents a linear or branched alkylene group, An ⁻ represents an anion selected from the group consisting of a halide ion, an inorganic acid ion, a hydroxide ion, and an isothiocyanate ion, and m represents an integer from 0 to 6. represent.)

Therefore, the repeating units derived from the monomers represented by formulas (A) and (B) are represented by the following formulas (a1) and (b1), respectively.

(In the formula, T ^a , T ^b , U ^a1 , U ^a2 , U ^b1 , U ^b2 and U ^b3 , Q ^a and Q ^b , R ^a and R ^b , An ⁻ and m have the same meanings as above.)

In the present invention, examples of "straight chain or branched alkyl group having 1 to 5 carbon atoms" include methyl group, ethyl group, n-propyl group, isopropyl group, n-butyl group, isobutyl group, s-butyl group, t-butyl group, n-pentyl group, 1-methylbutyl group, 2-methylbutyl group, 3-methylbutyl group, 1,1-dimethylpropyl group, 1,2-dimethylpropyl group, 2,2-dimethylpropyl group or 1 -Ethylpropyl group.

In the present invention, "ester bond" means -C(=O)-O- or -OC(=O)-, and "amide bond" means -NHC(=O)- or -C( =O) means NH-.

In the present invention, "a straight chain or branched alkylene group having 1 to 10 carbon atoms which may be substituted with a halogen atom" refers to a straight chain or branched alkylene group having 1 to 10 carbon atoms, or a halogen atom or more. means a straight chain or branched alkylene group having 1 to 10 carbon atoms substituted with . Here, "a linear or branched alkylene group having 1 to 10 carbon atoms" is a divalent organic group in which one hydrogen atom is removed from the above alkyl group, such as a methylene group, ethylene group, propylene group. , trimethylene group, tetramethylene group, 1-methylpropylene group, 2-methylpropylene group, dimethylethylene group, ethylethylene group, pentamethylene group, 1-methyl-tetramethylene group, 2-methyl-tetramethylene group, 1, Examples include 1-dimethyl-trimethylene group, 1,2-dimethyl-trimethylene group, 2,2-dimethyl-trimethylene group, 1-ethyl-trimethylene group, hexamethylene group, octamethylene group and decamethylene group. Among these, ethylene group, propylene group, octamethylene group and decamethylene group are preferable, and linear or branched alkylene groups having 1 to 5 carbon atoms, such as ethylene group, propylene group, trimethylene group and tetramethylene group, are more preferable. Preferably, ethylene or propylene groups are particularly preferred. "A linear or branched alkylene group having 1 to 10 carbon atoms substituted with one or more halogen atoms" refers to an alkylene group in which one or more arbitrary hydrogen atoms are replaced with a halogen atom. Particularly preferred are ethylene groups or propylene groups in which some or all of the hydrogen atoms are replaced with halogen atoms.

In the present invention, examples of the "halogen atom" include a fluorine atom, a chlorine atom, a bromine atom, and an iodine atom.
In the present invention, "halide ion" means an anion of a halogen atom, and includes fluoride ion, chloride ion, bromide ion, and iodide ion, and preferably chloride ion.
In the present invention, "inorganic acid ion" means carbonate ion, sulfate ion, phosphate ion, hydrogen phosphate ion, dihydrogen phosphate ion, nitrate ion, perchlorate ion, or borate ion.
Preferred examples of the An ^- are halide ions, sulfate ions, phosphate ions, hydroxide ions, and isothiocyanate ions, and particularly preferred are halide ions.

In formulas (A) and (B), T ^a and T ^b are preferably each independently a hydrogen atom, a methyl group, or an ethyl group, and more preferably each independently a hydrogen atom or a methyl group. It is.

In formulas (a), (b), formulas (A) and (B), U ^a1 , U ^a2 , U ^b1 , U ^b2 and U ^b3 are preferably each independently a hydrogen atom, a methyl group or It is an ethyl group. In formula (a) and formula (A), U ^a1 and U ^a2 are more preferably hydrogen atoms. In formulas (b) and (B), U ^b1 , U ^b2 (and U ^b3 ) are more preferably a methyl group or an ethyl group, particularly preferably a methyl group.

In formulas (A) and (B), Q ^a and Q ^b preferably each independently represent an ester bond (-C(=O)-O- or -O-C(=O)-) or an amide bond. A bond (-NHC(=O)- or -C(=O)NH-), more preferably each independently -C(=O)-O- or -C(=O)NH- -C(=O)-O- is particularly preferred.

In formulas (A) and (B), R ^a and R ^b are preferably each independently a straight chain or branched alkylene group having 1 to 3 carbon atoms, which may be substituted with a halogen atom, More preferably, each independently is an ethylene group or a propylene group, or an ethylene group or a propylene group substituted with one chlorine atom, and particularly preferably an ethylene group or a propylene group.

In formulas (A) and (B), m preferably represents an integer of 0 to 3, more preferably an integer of 1 or 2, and particularly preferably 1.

Specific examples of the above formula (A) include vinylphosphonic acid, acid phosphooxyethyl (meth)acrylate, 3-chloro-2-acid phosphooxypropyl (meth)acrylate, acid phosphooxypropyl (meth)acrylate, and acid phosphooxypropyl (meth)acrylate. Examples include oxymethyl (meth)acrylate, acid phosphooxypolyoxyethylene glycol mono(meth)acrylate, acid phosphooxypolyoxypropylene glycol mono(meth)acrylate, among which vinylphosphonic acid, acid phosphooxyethyl methacrylate ( =2-(methacryloyloxy)ethyl phosphate) is preferably used.

The structural formulas of vinylphosphonic acid, acid phosphooxyethyl methacrylate (=2-(methacryloyloxy)ethyl phosphate), and acid phosphooxypolyoxyethylene glycol monomethacrylate are the following formulas (A-1) to (A-3), respectively. ).

At the time of synthesis, in addition to these compounds, a (meth)acrylate compound having two functional groups, as represented by the general formula (C) or (D) described below, may be used in combination.

Specific examples of the above formula (B) include dimethylaminoethyl (meth)acrylate, diethylaminoethyl (meth)acrylate, dimethylaminopropyl (meth)acrylate, 2-(t-butylamino)ethyl (meth)acrylate, methacroyl Examples include choline chloride, among which dimethylaminoethyl (meth)acrylate, methacroylcholine chloride, or 2-(t-butylamino)ethyl (meth)acrylate is preferably used.

Dimethylaminoethyl acrylate (=2-(dimethylamino)ethyl acrylate), dimethylaminoethyl methacrylate (=2-(dimethylamino)ethyl methacrylate), methacroylcholine chloride and 2-(t-butylamino)ethyl methacrylate ( The structural formulas of =2-(t-butylamino)ethyl methacrylate are represented by the following formulas (B-1) to (B-4), respectively.

The proportion of the repeating unit containing the organic group represented by formula (a) (or the repeating unit represented by formula (a1)) in the copolymer is 20 mol% to 80 mol%, preferably 30 mol%. It is mol% to 70 mol%, more preferably 40 mol% to 60 mol%. Note that the copolymer may contain a repeating unit containing two or more types of organic groups represented by formula (a) (or a repeating unit represented by formula (a1)).

The proportion of the repeating unit containing an organic group represented by formula (b) (or the repeating unit represented by formula (b1)) in the above copolymer is the proportion of the repeating unit containing the organic group represented by formula (a) ( Alternatively, it may be the entire remainder after subtracting the ratio of formula (a1)), or it may be the remainder after subtracting the total ratio of the above formula (a) (or formula (a1)) and the third component described below. . Note that the copolymer may contain a repeating unit containing two or more types of organic groups represented by formula (b) (or a repeating unit represented by formula (b1)).

Furthermore, the copolymer may be copolymerized with an arbitrary third component. For example, a (meth)acrylate compound having two or more functional groups may be copolymerized as the third component, and a portion of the polymer may be partially three-dimensionally crosslinked. Examples of such a third component include a bifunctional monomer represented by the following formula (C) or (D).

(In the formula, T ^c , T ^d and U ^d each independently represent a hydrogen atom or a straight chain or branched alkyl group having 1 to 5 carbon atoms, and R ^c and R ^d each independently, Represents a straight chain or branched alkylene group having 1 to 10 carbon atoms that may be substituted with a halogen atom.)
That is, the copolymer preferably contains a crosslinked structure derived from such a difunctional monomer.

In formulas (C) and (D), T ^c and T ^d are preferably each independently a hydrogen atom, a methyl group, or an ethyl group, and more preferably each independently a hydrogen atom or a methyl group. It is.

In formulas (C) and (D), U ^d is preferably a hydrogen atom, a methyl group or an ethyl group, more preferably a hydrogen atom.

In formulas (C) and (D), R ^c and R ^d are preferably each independently a straight chain or branched alkylene group having 1 to 3 carbon atoms which may be substituted with a halogen atom, More preferably, each independently is an ethylene group or a propylene group, or an ethylene group or a propylene group substituted with one chlorine atom, and particularly preferably an ethylene group or a propylene group.

Preferable examples of the bifunctional monomer represented by formula (C) include ethylene glycol di(meth)acrylate, triethylene glycol di(meth)acrylate, and propylene glycol di(meth)acrylate. The bifunctional monomer represented by formula (D) is preferably bis(methacryloyloxymethyl) phosphate, bis[2-(methacryloyloxy)ethyl] phosphate, bis[2-(methacryloyloxy)propyl phosphate ] etc.

The optional third component may be a trifunctional monomer. Examples of such a trifunctional monomer as the third component include phosphinylidine triacrylate tris(oxy-2,1-ethanediyl).

Among these, ethylene glycol di(meth)acrylate represented by the following formula (C-1) and bis[2-(methacryloyloxy)ethyl] phosphate represented by the following formula (D-1) are particularly preferred. .

The copolymer may contain one or more of these third components. Among the above, the bifunctional monomer represented by formula (D) is preferred, and the difunctional monomer represented by formula (D-1) is particularly preferred.
The proportion of the third component (for example, a crosslinked structure derived from a bifunctional monomer represented by the above formula (C) or (D)) in the copolymer is 0 mol% to 50 mol%.

The method for producing the copolymer is not particularly limited, but includes, for example, the method for producing the copolymer described in WO2014/196652 pamphlet.

The method of coating the copolymer onto the container for cell structure production is not particularly limited, and includes, for example, the coating process described in WO2014/196652 pamphlet.

Further, the coating film may be a coating film obtained from the composition for forming a coating film described in WO2021/167037 pamphlet. The contents of pamphlet WO2021/167037 are incorporated herein to the same extent as if expressly set forth in their entirety.
The composition for forming a coating film described in the WO2021/167037 pamphlet will be described below. In this specification, it is referred to as a second aspect of the composition for forming a coating film.

The coating film forming composition of the second aspect has the following formula (1):

(In the formula,
R ¹ represents a hydrogen atom or a methyl group, R ² represents an alkylene group having 1 to 6 carbon atoms, and n represents an integer of 1 to 30)
A compound represented by the following formula (2):

(In the formula,
^R3 represents a hydrogen atom or a methyl group, ^R4 represents a monovalent organic group having cationic properties)
A compound represented by and the following formula (3):

(In the formula,
R ⁵ each independently represents a hydrogen atom or a straight chain or branched alkyl group having 1 to 6 carbon atoms, and R ⁶ represents a straight chain having 1 to 10 carbon atoms which may be substituted with a halogen atom. or represents a branched alkylene group, m represents an integer from 1 to 30)
A monomer mixture containing a compound represented by the formula (3), which is obtained by polymerizing a monomer mixture in which the proportion of the compound represented by the formula (3) to the total mass of the monomer mixture is 2 to 40% by mass. Contains a copolymer and a solvent.

In the compound of formula (1) which is a monomer component of the copolymer according to the second embodiment, R ¹ represents a hydrogen atom or a methyl group, R ² represents an alkylene group having 1 to 6 carbon atoms, It preferably represents a straight chain or branched alkylene group having 1 to 6 carbon atoms, more preferably a straight chain or branched alkylene group having 2 to 5 carbon atoms, particularly preferably an ethylene group or a propylene group, and n is , represents an integer of 1 to 30, preferably represents an integer of 1 to 20, more preferably represents an integer of 2 to 10, particularly preferably represents an integer of 3 to 6.

Specific examples of the compound of formula (1) above include acid phosphooxyethyl (meth)acrylate, 3-chloro-2-acid phosphooxypropyl (meth)acrylate, acid phosphooxypropyl (meth)acrylate, acid phosphooxymethyl (meth)acrylate, acid phosphooxypolyoxyethylene glycol mono(meth)acrylate, acid phosphooxypolyoxypropylene glycol mono(meth)acrylate, etc. Among these, acid phosphooxyethyl methacrylate (= phosphoric acid 2-( (methacryloyloxy)ethyl), acid phosphooxypolyoxyethylene glycol monomethacrylate and acid phosphooxypolyoxypropylene glycol monomethacrylate are preferably used.

The structural formulas of acid phosphooxyethyl methacrylate (=2-(methacryloyloxy)ethyl phosphate), acid phosphooxypolyoxyethylene glycol monomethacrylate, and acid phosphooxypolyoxypropylene glycol monomethacrylate are the following formulas (1-1): ～Represented by formula (1-3).

In the compound of formula (2) which is a monomer component of the copolymer according to the second aspect, R ³ represents a hydrogen atom or a methyl group, R ⁴ represents a monovalent organic group having cationic properties, Typically, it is a monovalent group having a primary amine, secondary amine, tertiary amine or quaternary ammonium structure.
Examples of monovalent groups having a primary amine, secondary amine, tertiary amine or quaternary ammonium structure include the formulas: -R ^4a -NH ₂ , -R ^4a -NHR, -R ^4a -NRR', respectively. , -R ^4a -N ⁺ RR'R'' [where R ^4a is an alkylene group having 1 to 6 carbon atoms, which may be interrupted by an ester bond, amide bond, ether bond or phosphodiester bond; R, R' and R'' are each independently a straight chain or branched alkyl group having 1 to 6 carbon atoms, an aryl group having 6 to 10 carbon atoms, or an aralkyl group having 7 to 16 carbon atoms. means a group represented by ]. The monovalent group having a primary amine, secondary amine or tertiary amine structure in the second embodiment may be quaternized or chlorinated, and similarly the monovalent group having a quaternary ammonium structure is May be chlorinated.

Specific examples of the compound of formula (2) above include dimethylaminoethyl (meth)acrylate, diethylaminoethyl (meth)acrylate, dimethylaminopropyl (meth)acrylate, 2-(t-butylamino)ethyl (meth)acrylate, Examples include (meth)acryloylcholine chloride, 2-(meth)acryloyloxyethylphosphorylcholine (MPC), and the like.

The structural formulas of 2-(dimethylamino)ethyl methacrylate, methacryloylcholine chloride, and 2-methacryloyloxyethylphosphorylcholine (MPC) are represented by the following formulas (2-1) to (2-3), respectively.

The compound of formula (3), which is a monomer component of the copolymer according to the second aspect, is a bifunctional monomer having crosslinking properties. In the compound of formula (3), R ⁵ each independently represents a hydrogen atom or a linear or branched alkyl group having 1 to 6 carbon atoms, preferably a hydrogen atom or a methyl group, and R ⁶ is represents a straight chain or branched alkylene group having 1 to 10 carbon atoms which may be substituted with a halogen atom, preferably a straight chain or branched alkylene group having 1 to 6 carbon atoms which may be substituted with a halogen atom. represents a group, more preferably represents a straight chain or branched alkylene group having 1 to 6 carbon atoms which may be substituted with a chlorine atom, particularly preferably represents an ethylene group, a propylene group or a trimethylene group, m is Represents an integer from 1 to 30, preferably represents an integer from 1 to 20, more preferably represents an integer from 1 to 10, still more preferably represents an integer from 1 to 6, particularly preferably represents an integer from 1 to 4. .

Specific examples of the compound of formula (3) above include poly(ethylene glycol) di(meth)acrylate, poly(trimethylene glycol) di(meth)acrylate, poly(propylene glycol) di(meth)acrylate, etc. .

The structural formulas of poly(ethylene glycol) dimethacrylate, poly(trimethylene glycol) dimethacrylate, and poly(propylene glycol) dimethacrylate are represented by the following formulas (3-1) to (3-3), respectively.

The copolymer according to the second aspect is obtained by polymerizing a monomer mixture containing the compounds of the above formulas (1), (2), and (3) as monomer components. Such a monomer mixture may contain an ethylenically unsaturated monomer, a polysaccharide, or a derivative thereof as an optional fourth component, as long as it does not impair the ability to suppress the adhesion of biological substances. Examples of ethylenically unsaturated monomers include one or two selected from the group consisting of (meth)acrylic acid and its esters; vinyl acetate; vinylpyrrolidone; ethylene; vinyl alcohol; and hydrophilic functional derivatives thereof. More than one ethylenically unsaturated monomer can be mentioned. Examples of polysaccharides or derivatives thereof include cellulosic polymers such as hydroxyalkylcellulose (eg, hydroxyethylcellulose or hydroxypropylcellulose), starch, dextran, and curdlan. The copolymer according to the second aspect is preferably obtained by polymerizing a monomer mixture containing only the compounds of formulas (1), (2) and (3) above as monomer components.

The copolymer according to the second aspect is a monomer mixture containing compounds of the above formulas (1), (2), and (3) as monomer components, and the total mass of the monomer components contained in the monomer mixture. It can be obtained by polymerizing a monomer mixture characterized in that the proportion of the compound of the formula (3) is 2 to 40% by mass. Based on the total mass of monomer components contained in the monomer mixture, the compound of formula (3), which is a difunctional monomer, is added in an amount of 2 to 40% by mass, preferably 3 to 35% by mass, more preferably 5 to 35% by mass. By including the copolymer in the range of 30% by mass, the coating film forming composition of the second embodiment, which includes the copolymer obtained from the monomer mixture, has an excellent ability to suppress the adhesion of biological substances. Note that the monomer mixture may contain two or more types of compounds of formula (3).

In the monomer mixture, the proportion of the compound of formula (1) to the total mass of monomer components is 10 to 95% by mass, preferably 10 to 80% by mass, and more preferably 45 to 75% by mass. Note that the monomer mixture may contain two or more types of compounds of formula (1).

In the monomer mixture, the proportion of the compound of formula (2) to the total mass of monomer components is 3 to 90% by mass, preferably 10 to 70% by mass, and more preferably 15 to 35% by mass. Note that the monomer mixture may contain two or more types of compounds of formula (2).

The copolymer according to the second embodiment can be synthesized by methods such as radical polymerization, anionic polymerization, and cationic polymerization, which are common methods for synthesizing (meth)acrylic polymers. Various methods such as solution polymerization, suspension polymerization, emulsion polymerization, and bulk polymerization are possible. In one embodiment, a monomer mixture containing the compounds of formulas (1), (2), and (3) above is reacted (polymerized) in a solvent at a total concentration of monomer components of 0.01 to 20% by mass. It can be prepared by a manufacturing method including steps.

The solvent in the polymerization reaction may be water, a phosphate buffer, an alcohol such as ethanol, or a mixed solvent of these, but preferably contains water or ethanol. Furthermore, it is preferable that water or ethanol is contained in an amount of 10% by mass or more and 100% by mass or less. Furthermore, it is preferable that water or ethanol is contained in an amount of 50% by mass or more and 100% by mass or less. Furthermore, it is preferable that water or ethanol is contained in an amount of 80% by mass or more and 100% by mass or less. Furthermore, it is preferable that water or ethanol is contained in an amount of 90% by mass or more and 100% by mass or less. Preferably, the total amount of water and ethanol is 100% by mass.

In order to proceed with the polymerization reaction efficiently, it is desirable to use a polymerization initiator. Examples of polymerization initiators include "thermal radical polymerization initiators" and "photoradical polymerization initiators."
Examples of "thermal radical polymerization initiators" include 2,2'-azobis(isobutyronitrile), 2,2'-azobis(2-methylbutyronitrile), 2,2'-azobis(2,4 -dimethylvaleronitrile) (Fuji Film Wako Pure Chemical Industries, Ltd. product name: V-65, 10-hour half-life temperature: 51°C), 4,4'-azobis(4-cyanovaleric acid), 2,2'- Azobis(4-methoxy-2,4-dimethylvaleronitrile), 1,1'-azobis(cyclohexane-1-carbonitrile), 1-[(1-cyano-1-methylethyl)azo]formamide, 2,2 '-Azobis[2-(2-imidazolin-2-yl)propane] dihydrochloride (Fujifilm Wako Pure Chemical Industries, Ltd.; VA-044, 10-hour half-life temperature; 44°C), 2,2'-azobis [2-(2-imidazolin-2-yl)propane] (Fujifilm Wako Pure Chemical Industries, Ltd.; VA-061, 10-hour half-life temperature: 61°C), 2,2'-azobis(2-methylpropionamidine) ) dihydrochloride, 2,2'-azo(2-methyl-N-(2-hydroxyethyl)propionamide (Fujifilm Wako Pure Chemical Industries, Ltd.) Product name: VA-086, 10 hour half-life temperature: 86°C ), benzoyl peroxide (BPO), 2,2'-azobis(N-(2-carboxyethyl)-2-methylpropionamidine) n-hydrate (Fujifilm Wako Pure Chemical Industries, Ltd. product name: VA- 057, 10-hour half-life temperature; 57°C), 4,4'-azobis(4-cyanopentanoic acid) (Fujifilm Wako Pure Chemical Industries, Ltd. product name: VA-501), 2,2'-azobis [2-(2-imidazolin-2-yl)propane] disulfate dihydrate (Fujifilm Wako Pure Chemical Industries, Ltd. product name: VA-046B, 10-hour half-life temperature: 46°C), 2,2'- Azobis(2-amidinopropane) dihydrochloride (Fujifilm Wako Pure Chemical Industries, Ltd. product name: V-50, 10-hour half-life temperature: 56°C), peroxodisulfate, t-butyl hydroperoxide, dimethyl 1,1' -Azobis(1-cyclohexanecarboxylate) (Fuji Film Wako Pure Chemical Industries, Ltd. product name; VE-073) and the like.

Examples of "photoradical polymerization initiators" include acetophenone, chloroacetophenone, hydroxyacetophenone, 2-aminoacetophenone, dialkylaminoacetophenone, 2,2-diethoxyacetophenone, 2,2-dimethoxy-2'-phenylacetophenone (BASF BASF product name: Irgacure 651), 2-hydroxy-2-methyl-1-phenylpropanone (BASF product name: Irgacure 1173), 1-[4-(2-hydroxyethoxy)-phenyl]-2-hydroxymethylpropanone (BASF company product name: Irgacure 2959), 2-hydroxy-1-{4-[4-(2-hydroxy-2-methylpropionyl)benzyl]phenyl}-2-methyl-1-propan-1-one (BASF company Product name: Irgacure 127), 2-methyl-1-[4-(methylthio)phenyl]-2-morpholinopropan-1-one (BASF product name: Irgacure 907), 2-benzyl-2-(dimethylamino)-4 - Acetophenones such as morpholinobutyrophenone (BASF product name: Irgacure 369); benzoin, benzoin methyl ether, benzoin ethyl ether, benzoin isopropyl ether, benzoin isobutyl ether, 1-hydroxycyclohexylphenyl ketone (BASF product name: Irgacure 184) Benzoins such as benzophenone, benzoylbenzoic acid, methyl benzoylbenzoate, methyl-o-benzoylbenzoate, 4-phenylbenzophenone, hydroxybenzophenone, hydroxypropylbenzophenone, acrylic benzophenone, 4,4'-bis(dimethylamino)benzophenone, Benzophenones such as 4,4'-dichlorobenzophenone; Thioxanthone such as thioxanthone, 2-chlorothioxanthone, 2-methylthioxanthone, diethylthioxanthone, and dimethylthioxanthone; 2,4,6-trimethylbenzoyl-diphenylphosphine oxide (BASF product) Acyl phosphine oxides such as Irgacure TPO), bis(2,4,6-trimethylbenzoyl)-phenylphosphine oxide (BASF product name Irgacure 819); oxyphenylacetic acid, 2-[2-oxo-2-phenylacetoxy ethoxy]ethyl ester and oxyphenylacetic acid, mixture of 2-(2-hydroxyethoxy)ethyl ester (BASF product name; Irgacure 754), methyl benzoylformate (BASF product name; IrgacureMBF), α-acyl oxime ester, benzyl- (o-ethoxycarbonyl)-α-monoxime, glyoxy ester, 2-ethyl anthraquinone, camphorquinone, tetramethylthiuram sulfide, azobisisobutyronitrile, benzoyl peroxide, dialkyl peroxide, tert-butyl peroxy pivalate, etc. Can be mentioned.

Considering solubility in water, ionic balance, and interaction with monomers, 2,2'-azo(2-methyl-N-(2-hydroxyethyl)propionamide, 2,2'-azobis(N- (2-carboxyethyl)-2-methylpropionamidine) n-hydrate, 4,4'-azobis(4-cyanopentanoic acid), 2,2'-azobis[2-(2-imidazoline-2 -yl)propane] dihydrochloride, 2,2'-azobis[2-(2-imidazolin-2-yl)propane]disulfate dihydrate, 2,2'-azobis[2-(2-imidazolin-2-yl) yl)propane], 2,2'-azobis(2-amidinopropane) dihydrochloride and peroxodisulfuric acid.
When considering solubility in organic solvents, ion balance, and interaction with monomers, 2,2'-azobis(2,4-dimethylvaleronitrile) or 2,2'-azobis(isobutyronitrile) is used. This is desirable.

The amount of the polymerization initiator added is 0.05% by mass to 10% by mass based on the total mass of monomer components used for polymerization.

The reaction conditions are to heat the reaction vessel to 50 to 200°C in an oil bath or the like and stir for 1 to 48 hours, more preferably at 80 to 150°C for 5 to 30 hours, so that the polymerization reaction progresses and the second embodiment is achieved. A copolymer is obtained. The reaction atmosphere is preferably a nitrogen atmosphere.

As a reaction procedure, all reactants may be placed in a reaction solvent at room temperature and then heated to the above temperature for polymerization, or all or a portion of the reactant mixture may be added to a preheated solvent. It may be added dropwise.

The weight average molecular weight of the copolymer according to the second aspect may range from several thousand to several million, preferably from 5,000 to 5,000,000, more preferably from 10,000 to 2,000, It is 000. Further, it may be a random copolymer, a block copolymer, or a graft copolymer, and preferably a random copolymer.
Furthermore, since the copolymer produced in this manner contains a difunctional monomer, it is considered to be a three-dimensional polymer, and is dissolved or dispersed in a solution containing water or alcohol.

The composition for forming a coating film according to the second aspect may be prepared by isolating/purifying the copolymer thus obtained and then diluting it to a predetermined concentration with a desired solvent. good.
Furthermore, the composition for forming a coating film according to the second aspect may be prepared from the reaction solution (ie, copolymer-containing varnish) obtained after the polymerization reaction.

Examples of the solvent contained in the coating film forming composition of the second embodiment include water, phosphate buffered saline (PBS), and alcohol. Examples of the alcohol include alcohols 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, 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- Examples include methyl-3-pentanol and cyclohexanol, which may be used alone or as a mixed solvent in combination, but from the viewpoint of dissolving the copolymer, water, PBS, ethanol and propanol are preferred. preferable.

The concentration of solids in the composition for forming a coating film according to the second aspect is preferably 0.01 to 50% by mass in order to form a uniform coating film. The concentration of the copolymer in the composition for forming a coating film is preferably 0.01 to 5% by mass, more preferably 0.01 to 4% by mass, particularly preferably 0.01 to 3% by mass. be. If the concentration of the copolymer is 0.01% by mass or less, the concentration of the copolymer in the resulting coating film-forming composition will be too low to form a coating film with a sufficient thickness, and if it is 5% by mass or more If so, the storage stability of the coating film forming composition may deteriorate, and precipitation or gelation of dissolved substances may occur.

Furthermore, in addition to the above-mentioned copolymer and solvent, other substances can be added to the coating film forming composition of the second embodiment, if necessary, within a range that does not impair the performance of the resulting coating film. Other substances include preservatives, surfactants, primers that increase adhesion to the substrate, fungicides, sugars, and the like.

In order to adjust the ion balance of the copolymer in the coating film forming composition according to the second embodiment, when obtaining the coating film according to the second embodiment, the pH in the coating film forming composition is further adjusted. It may also include a step of adjusting in advance. The pH adjustment can be carried out, for example, by adding a pH adjuster to a composition containing the above copolymer and a solvent, and adjusting the pH of the composition to 2 to 13.5, preferably 2 to 8.5, more preferably 3 to 8. Alternatively, it may be carried out by setting it to preferably 8.5 to 13.5, more preferably 10 to 13.5. The type and amount of the pH adjuster that can be used are appropriately selected depending on the concentration of the copolymer, the abundance ratio of anions and cations, and the like.
Examples of pH adjusters include organic amines such as ammonia, diethanolamine, pyridine, N-methyl-D-glucamine, and tris(hydroxymethyl)aminomethane; alkali metal hydroxides such as potassium hydroxide and sodium hydroxide; chloride Alkali metal halides such as potassium and sodium chloride; inorganic acids or alkali metal salts thereof such as sulfuric acid, phosphoric acid, hydrochloric acid, and carbonic acid; quaternary ammonium cations such as choline; or mixtures thereof (e.g., phosphate buffered saline) (buffer solutions such as). Among these, ammonia, diethanolamine, sodium hydroxide, choline, N-methyl-D-glucamine, and tris(hydroxymethyl)aminomethane are preferred, and ammonia, diethanolamine, sodium hydroxide, and choline are particularly preferred.

Further, the coating film may be a coating film obtained from the composition for forming a coating film described in the WO2022/259998 pamphlet. It will be incorporated into.
The composition for forming a coating film described in the WO2022/259998 pamphlet will be described below. In this specification, it is referred to as a third aspect of the composition for forming a coating film.

The coating film forming composition of the third aspect is used for suppressing the adhesion of biological substances.
The third aspect of the composition for forming a coating film contains at least a copolymer and, if necessary, other components such as a solvent.
Note that the composition for forming a coating film according to the third aspect can form a coating film that is difficult to dissolve in phosphate buffered saline, but the composition for forming a coating film according to the third aspect can be used for biological As long as it is used to suppress the adhesion of substances, it is not particularly limited, and is not limited to forming a coating film in contact with phosphate buffered saline.

The copolymer according to the third aspect is water-insoluble. Here, "water-soluble" means that 1.0 g or more can be dissolved in 100 g of water at 25°C. "Water-insoluble" means that it does not fall under "water-soluble", that is, the solubility in 100 g of water at 25° C. is less than 1.0 g.
The copolymer according to the third aspect has a repeating unit (A) represented by the following formula (A) and a repeating unit (B) represented by the following formula (B).
The molar ratio (A:B) of repeating units (A) and repeating units (B) in the copolymer according to the third embodiment is 89:11 to 50:50.

(In the formula, R ¹ to R ³ each independently represent a hydrogen atom or an alkyl group having 1 to 5 carbon atoms, and X ¹ and X ² each independently represent a single bond, an ester bond, an ether (Represents an alkylene group having 1 to 5 carbon atoms that may be interrupted by a bond, amide bond, or oxygen atom.)

The copolymer according to the third aspect may have two or more types of repeating units (A).
The copolymer according to the third aspect may have two or more types of repeating units (B).
The copolymer according to the third aspect preferably has one type of repeating unit (A) and one type of repeating unit (B).

Examples of the alkyl group having 1 to 5 carbon atoms include methyl group, ethyl group, n-propyl group, isopropyl group, n-butyl group, isobutyl group, s-butyl group, t-butyl group, n-pentyl group, 1 -methylbutyl group, 2-methylbutyl group, 3-methylbutyl group, 1,1-dimethylpropyl group, 1,2-dimethylpropyl group, 2,2-dimethylpropyl group, 1-ethylpropyl group and the like.
R ¹ to R ³ are each independently preferably a hydrogen atom, a methyl group, or an ethyl group.

The above "ester bond" means -C(=O)-O- or -OC(=O)-, "ether bond" means -O-, and "amide bond" means - It means NHC(=O)- or -C(=O)NH-.

The alkylene group having 1 to 5 carbon atoms may be interrupted by an oxygen atom.
Examples of alkylene groups having 1 to 5 carbon atoms include methylene group, ethylene group, propylene group, trimethylene group, tetramethylene group, 1-methylpropylene group, 2-methylpropylene group, dimethylethylene group, ethylethylene group, pentamethylene group. group, 1-methyl-tetramethylene group, 2-methyl-tetramethylene group, 1,1-dimethyl-trimethylene group, 1,2-dimethyl-trimethylene group, 2,2-dimethyl-trimethylene group, 1-ethyl-trimethylene group Examples include groups. The above X ¹ and X ² are preferably a methylene group, an ethylene group, or a propylene group.
The phrase "may be interrupted by an oxygen atom" means that one or more carbon-carbon bonds of an alkylene group having 1 to 5 carbon atoms are bonded via an ether bond.

The copolymer according to the third aspect is preferably a copolymer in which R ¹ and R ² are hydrogen atoms, R ³ is a methyl group, and X ¹ and X ² are single bonds.

The molar ratio (A:B) of the repeating unit (A) and the repeating unit (B) is 89:11 to 50:50.
When the total number of moles of repeating units (A) and repeating units (B) in the copolymer according to the third aspect is 100, the molar ratio of repeating units (A) and repeating units (B) ( A:B) can be expressed as (100-m):m. In that case, the range of m is 11-50. The lower limit of m may be 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30. The upper limit of m may be 49, 48, 47, 46, 45, 44, 43, 42, 41, 40, 38, 37, 36, or 35. The range of m is, for example, 12-49, 12-48, 15-48, 20-49, 20-45, 22-49, or 22-45.

The total mol% of repeating units (A) and repeating units (B) in all repeating units in the copolymer according to the third aspect is not particularly limited, but is preferably 90 mol% or more, and 95 mol% or more. is more preferable, 99.5 mol% or more is even more preferable, and 100% is particularly preferable.

In the third aspect, in order to obtain a coating film that is difficult to dissolve in phosphate buffered saline, the molar ratio of the repeating unit (A) and repeating unit (B) in the copolymer is set within a specific range. . Therefore, in the third embodiment, a coating film that is difficult to dissolve in phosphate buffered saline can be obtained without crosslinking the copolymer. Therefore, the copolymer does not need to have a photosensitive group for crosslinking the copolymer. That is, it is preferable that the copolymer does not have a photosensitive group. Examples of the photosensitive group include an azide group.
In a third aspect, the copolymer does not need to have photosensitive groups to crosslink the copolymer. Therefore, when forming a coating film, there is no need to perform light irradiation for crosslinking the copolymer. Therefore, the process for forming the coating film can be simplified.

The viscosity average degree of polymerization (hereinafter sometimes simply referred to as "degree of polymerization") of the copolymer according to the third aspect is not particularly limited, but from the viewpoint of suitably obtaining the effects of the third aspect, from 200 to 3 ,000 is preferred, 200 to 2,500 is more preferred, and 200 to 2,000 is particularly preferred.
The viscosity average degree of polymerization is measured with the copolymer completely saponified.
The "viscosity average degree of polymerization" of polyvinyl alcohol obtained by complete saponification is determined by the following formula from the intrinsic viscosity [η] (g/dL) measured at 30°C using an Ostwald viscometer using ion-exchanged water as a solvent. This is the calculated value.
log(P)=1.613×log([η]×10 ⁴ /8.29)
Here, P indicates the viscosity average degree of polymerization.
The viscosity average degree of polymerization can be determined according to JIS K 6726.

The method for producing the copolymer according to the third aspect is not particularly limited, but for example, a compound represented by the following formula (C) is polymerized to produce a homopolymer, and the obtained homopolymer is publicly known. A method for obtaining a copolymer by partial hydrolysis using a saponification reaction may be mentioned.

(In the formula, R ¹ , R ³ , and X ¹ have the same meanings as above.)

Further, as a method for producing the copolymer according to the third aspect, for example, a compound represented by the following formula (C) and a compound represented by the following formula (D) are copolymerized to form a copolymer. One method is to obtain union.

(In the formula, R ¹ to R ³ , X ¹ and X ² have the same meanings as above.)

The copolymer according to the third aspect may be a random copolymer or a block copolymer.
As the copolymer according to the third aspect, commercially available products may be used. A specific example of a commercially available copolymer is polyvinyl acetate (trade name: JMR-10L (registered trademark), manufactured by Nippon Vinyl Poval Co., Ltd.).

The content of the copolymer in the film-forming component in the coating film-forming composition of the third aspect is not particularly limited, but is preferably 80% by mass or more, more preferably 90% by mass or more, and 95% by mass or more. is particularly preferred. Note that the film-forming component refers to a component obtained by excluding the solvent component from all components of the composition.

The content of the copolymer in the coating film forming composition of the third aspect is not particularly limited, but from the viewpoint of easily forming a coating film with a desired thickness, it is preferably 0.1 to 10% by mass, and 0.1 to 10% by mass is preferable. .3 to 8% by weight is more preferable, and 0.5 to 5% by weight is particularly preferable. Further, the content of the copolymer in the composition for forming a coating film may be 0.02 to 2% by mass, or 0.05 to 1% by mass.

Examples of the solvent according to the third aspect include water, phosphate buffered saline (PBS), alcohol, and water-soluble organic solvents (excluding alcohol).

Examples of the alcohol include alcohols having 2 to 6 carbon atoms.
Examples of the alcohol include 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, 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-pen Tanol, 3-methyl-2-pentanol, 3-methyl-3-pentanol, 4-methyl-1-pentanol, 4-methyl-2-pentanol, 4-methyl-3-pentanol and cyclohexanol Can be mentioned. These can be used alone or in combination of two or more.

A water-soluble organic solvent refers to an organic solvent that can be mixed with water and alcohol in any proportion and does not separate after mixing.
Examples of water-soluble organic solvents include ethylene glycol monomethyl ether, ethylene glycol monoethyl ether, methyl cellosolve acetate, ethyl cellosolve acetate, diethylene glycol monomethyl ether, diethylene glycol monoethyl ether, propylene glycol, propylene glycol monomethyl ether, and propylene glycol monoethyl ether. , propylene glycol monomethyl ether acetate, and propylene glycol propyl ether acetate. These can be used alone or in combination of two or more.

In the coating film forming composition of the third aspect, water, phosphate buffered saline (PBS), alcohol, or a water-soluble organic solvent may be used alone as a solvent.
In the coating film forming composition of the third aspect, a combination of two or more of water, phosphate buffered saline (PBS), alcohol, and a water-soluble organic solvent may be used as the solvent.
From the viewpoint of solubility of the copolymer, the solvent is preferably selected from water, alcohol, water-soluble organic solvents, and combinations of two or more thereof, and water, ethanol, water-soluble organic solvents, and two or more thereof. More preferably, it is selected from a combination of.

The following combinations of solvents are preferred.
・Water and alcohol ・Water, alcohol and water-soluble organic solvents ・Alcohol and water-soluble organic solvents

As the combination of solvents, the following combinations are more preferable.
・Water and ethanol ・Water, ethanol and propylene glycol monomethyl ether ・Ethanol and propylene glycol monomethyl ether

In the coating film forming composition of the third aspect, the water:alcohol mass ratio is, for example, 1:99 to 70:30, and 1:99 to 50:50.
The mass ratio (A:B:C) of water:alcohol:water-soluble organic solvent in the composition for forming a coating film of the third aspect is, for example, 5 to 30:65 to 92:1 to 30 (A+B+C is 100).
In the coating film forming composition of the third aspect, the mixing ratio (mass ratio) of alcohol:water-soluble organic solvent is, for example, 30:70 to 97:3.

The content of the solvent in the coating film forming composition of the third aspect is not particularly limited, but from the viewpoint of easily forming a coating film with a desired thickness, it is preferably 90% by mass or more, and more preferably 92% by mass or more. It is preferably 95% by mass or more, particularly preferably 95% by mass or more.

The coating film forming composition of the third aspect can also contain other components as necessary.
Other components include, for example, pH adjusters, preservatives, surfactants, fungicides, sugars, and the like.

The method for manufacturing a cell structure manufacturing container is not particularly limited, but the following method for manufacturing a cell structure manufacturing container of the present invention is preferred.

(Method for manufacturing a container for manufacturing cell structures)
The method for manufacturing a cell structure manufacturing container of the present invention includes molding the cell structure manufacturing container of the present invention using a mold.

As another method for producing a cell structure production container, there is a method of producing a recessed part (recessed part) by laser irradiation, for example, as described in International Publication No. 2012/036011 pamphlet. However, in this case, if the depth of the recessed portion (recessed portion) is increased, the diameter of the opening will also become larger. Therefore, it is difficult to make the depth of the recess more than 1.0 times the equivalent diameter of the recess at the center in the depth direction of the recess.
On the other hand, when molding is performed using a mold, a mold with a convex portion corresponding to the desired depth of the concave portion is used, and molding including the concave portion can be performed. The diameter may be more than 1.0 times the equivalent diameter of the recess at the center in the depth direction of the recess.

(cell structure)
The cell structure of the present invention is produced using the cell structure production container of the present invention.
The cell structure of the present invention is preferably produced by the following method for producing a cell structure of the present invention.
Note that the cell structure refers to a structure formed as a result of aggregation of cells, and the shape is not limited to a spherical shape, a ring shape, or the like.

(Method for manufacturing cell structures)
The cell structure production method uses the cell structure production container of the present invention.
The method for manufacturing a cell structure includes, for example, at least the following step (1).
The method for producing a cell structure further includes, for example, the following steps (2) to (4).
Step (1): Adding a medium in which cells are dispersed to the culture space of the cell structure manufacturing container of the present invention. Step (2): Cultivating the cells in the culture space to form a cell structure. Step (3): Step of exchanging the medium in the culture space Step (4): Step of maturing the cell structures in the culture space

The method for manufacturing a cell structure may further include the following steps (5) and (6).
Step (5): Floating the cell structure in the medium Step (6): Recovering the cell structure

Each step will be explained below.

<Step (1)>
Step (1) is a step of adding a medium in which cells are dispersed to the culture space of the cell structure manufacturing container of the present invention.
Step (1) is a step of preparing to culture cells, and for example, the following total number of cells are dispersed in a medium and added to a cell structure manufacturing container.
The lower limit of the total number of cells is, for example, equal to or greater than the number (n) of recesses present in the cell structure manufacturing container.
The upper limit of the total number of cells is, for example, less than or equal to the value obtained by dividing the volume (V) of the recesses in the cell structure manufacturing container by the volume (V) of the cells to be seeded, multiplied by the number of recesses (n). do. When expressed using a mathematical formula using symbols, it can be expressed as the upper limit of the total number of cells=V/v×n. Here, it is assumed that the volumes (V) of the plurality of recesses are the same. If different, use the average value.
Adjust the medium depending on the cells to be cultured. The type of cells may be one type, or two or more types of cells may be used.
In step (1), before adding the culture medium in which cells are dispersed to the culture space of the cell structure production container of the present invention, the culture medium is added to the culture space of the cell structure production container of the present invention in order to remove bubbles. You may also add only 100% of the total amount and perform centrifugation. By doing so, it is possible to prevent the recess from trapping air bubbles.

The concentration of cells in the medium in which the cells are dispersed is not particularly limited.

A cell is the most basic unit constituting an animal or plant, and its elements include cytoplasm and various organelles inside the cell membrane. At this time, the nucleus containing DNA may or may not be included inside the cell. For example, animal-derived cells in the present invention include reproductive cells such as sperm and eggs, somatic cells constituting a living body, stem cells (pluripotent stem cells, etc.), progenitor cells, cancer cells isolated from a living body, and Cells (cell lines) that have acquired immortality and are stably maintained outside the body, cells that have been isolated from a living body and have undergone artificial genetic modification, and cells that have been isolated from a living body and have had their nuclei artificially exchanged. etc. are included.
Examples of somatic cells constituting a living body include, but are not limited to, fibroblasts, bone marrow cells, B lymphocytes, T lymphocytes, neutrophils, red blood cells, platelets, macrophages, monocytes, and bone cells. cells, bone marrow cells, pericytes, dendritic cells, keratinocytes, adipocytes, mesenchymal cells, epithelial cells, epidermal cells, endothelial cells, vascular endothelial cells, hepatocytes, chondrocytes, cumulus cells, nervous system cells, Glial cells, neurons, oligodendrocytes, microglia, astrocytes, cardiac cells, esophageal cells, muscle cells (e.g. smooth or skeletal muscle cells), pancreatic beta cells, melanocytes, hematopoietic progenitor cells (e.g. CD34-positive cells derived from umbilical cord blood), mononuclear cells, etc. The somatic cells include, for example, 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, blood vessel. Includes cells taken from any tissue, such as tissue, blood (including umbilical cord blood), bone marrow, heart, heart muscle, eye, brain, nervous tissue, hair, etc. Furthermore, the somatic cells include cells induced to differentiate from stem cells or progenitor cells.

Stem cells are cells that have both the ability to replicate themselves and the ability to differentiate into cells of multiple other lineages. Examples include, but are not limited to, embryonic stem cells (ES cells). , embryonic tumor cells, embryonic germ stem cells, induced pluripotent stem cells (iPS cells), neural stem cells, hematopoietic stem cells, mesenchymal stem cells, liver stem cells, pancreatic stem cells, muscle stem cells, germ stem cells, intestinal stem cells, cancer stem cells, Contains hair follicle stem cells. Among the above-mentioned stem cells, examples of pluripotent stem cells include ES cells, embryonic germ stem cells, and iPS cells.
Progenitor cells are cells that are in the process of differentiating from the stem cells into specific somatic cells or reproductive cells.
Cancer cells are cells that are derived from somatic cells and have acquired unlimited proliferation potential.
A cell line is a cell that has acquired unlimited proliferation ability through artificial manipulation outside the body.
Among these, fibroblasts, stem cells, and pluripotent stem cells are more preferred.

The medium can be selected as appropriate depending on the type of cells to be used. For example, if the purpose is to culture mammalian cells, a medium commonly used for culturing mammalian cells can be used as the medium. can do.
Examples of media for mammalian cells include Dulbecco's Modified Eagle's Medium (DMEM), Ham's Nutrient Mixture F12, DMEM/F12 medium, and McCoy's 5A medium. s 5A medium), Eagle's Minimum Essential Medium; EMEM, αMEM medium (alpha Modified Eagle's Minimum Essential Medium; αMEM), MEM Medium (Minimum Essential Medium), RPMI1640 medium, Iscove's modified Dulbecco medium ( Iscove's Modified Dulbecco's Medium; IMDM), MCDB131 medium, William medium E, IPL41 medium, Fischer's medium, StemPro34 (Invitrogen), X-VIVO 10 (Cambrex), X-VIVO 15 ( HPGM (manufactured by Cambrex), StemSpan H3000 (manufactured by Stem Cell Technology), StemSpanSFEM (manufactured by Stem Cell Technology), Stemline II (manufactured by Sigma-Aldrich), QBSF-60 (manufactured by Quality Biological), Examples include StemProhESCSFM (manufactured by Invitrogen), mTeSR1 or 2 medium (manufactured by Stem Cell Technology), Sf-900II (manufactured by Invitrogen), Opti-Pro (manufactured by Invitrogen), HuMedia-KG2 (manufactured by Kurashiki Boseki), and the like.

Those skilled in the art may freely add sodium, potassium, calcium, magnesium, phosphorus, chlorine, various amino acids, various vitamins, antibiotics, serum, fatty acids, sugar, etc. to the above medium according to the purpose. When culturing mammalian cells, those skilled in the art can also add one or more combinations of other chemical components or biological components depending on the purpose.

Components that can be added to media for mammalian cells include fetal bovine serum, human serum, horse serum, insulin, transferrin, lactoferrin, cholesterol, ethanolamine, sodium selenite, monothioglycerol, 2-mercaptoethanol, bovine serum. Examples include albumin, sodium pyruvate, polyethylene glycol, various vitamins, various amino acids, agar, agarose, collagen, methylcellulose, various cytokines, various hormones, various growth factors, various extracellular matrices, and various cell adhesion molecules.

Examples of cytokines that can be added to the medium include interleukin-1 (IL-1), interleukin-2 (IL-2), interleukin-3 (IL-3), interleukin-4 (IL-4), Interleukin-5 (IL-5), interleukin-6 (IL-6), interleukin-7 (IL-7), interleukin-8 (IL-8), interleukin-9 (IL-9), Interleukin-10 (IL-10), interleukin-11 (IL-11), interleukin-12 (IL-12), interleukin-13 (IL-13), interleukin-14 (IL-14), Interleukin-15 (IL-15), interleukin-18 (IL-18), interleukin-21 (IL-21), interferon-α (IFN-α), interferon-β (IFN-β), interferon- γ (IFN-γ), granulocyte colony stimulating factor (G-CSF), monocyte colony stimulating factor (M-CSF), granulocyte-macrophage colony stimulating factor (GM-CSF), stem cell factor (SCF), flk2/ Examples include, but are not limited to, flt3 ligand (FL), leukemia cell inhibitory factor (LIF), oncostatin M (OM), erythropoietin (EPO), thrombopoietin (TPO), and the like.

Hormones that may be added to the medium include melatonin, serotonin, thyroxine, triiodothyronine, epinephrine, norepinephrine, dopamine, anti-Mullerian hormone, adiponectin, adrenocorticotropic hormone, angiotensinogen and angiotensin, antidiuretic hormone, atrial Natriuretic peptide, calcitonin, cholecystokinin, corticotropin-releasing hormone, erythropoietin, follicle-stimulating hormone, gastrin, ghrelin, glucagon, gonadotropin-releasing hormone, growth hormone-releasing hormone, human chorionic gonadotropin, human placental lactogen, growth hormone, inhibin , insulin, insulin-like growth factor, leptin, luteinizing hormone, melanocyte-stimulating hormone, oxytocin, parathyroid hormone, prolactin, secretin, somatostatin, thrombopoietin, thyroid-stimulating hormone, thyrotropin-releasing hormone, cortisol, aldosterone, testosterone, dehydro Epiandrosterone, androstenedione, dihydrotestosterone, estradiol, estrone, estriol, progesterone, calcitriol, calcidiol, prostaglandins, leukotrienes, prostacyclin, thromboxane, prolactin-releasing hormone, lipotropin, brain natriuretic peptide, nerve These include, but are not limited to, peptide Y, histamine, endothelin, pancreatic polypeptide, renin, and enkephalin.

Growth factors that can be added to the medium include transforming growth factor-α (TGF-α), transforming growth factor-β (TGF-β), macrophage inflammatory protein-1α (MIP-1α), epidermal growth factor ( EGF), fibroblast growth factor-1, 2, 3, 4, 5, 6, 7, 8, or 9 (FGF-1, 2, 3, 4, 5, 6, 7, 8, 9), nerve Cell growth factor (NGF), hepatocyte growth factor (HGF), leukemia inhibitory factor (LIF), protease nexin I, protease nexin II, platelet-derived growth factor (PDGF), cholinergic differentiation factor (CDF), chemokine , Notch ligand (such as Delta1), Wnt protein, angiopoietin-like protein 2, 3, 5 or 7 (Angpt2, 3, 5, 7), insulin-like growth factor (IGF), insulin-like growth factor binding protein (IGFBP), play Examples include, but are not limited to, otrophin (Pleiotrophin) and the like.

It is also possible to add those cytokines and growth factors whose amino acid sequences have been artificially modified using genetic recombination technology. Examples include IL-6/soluble IL-6 receptor complex or Hyper IL-6 (a fusion protein of IL-6 and soluble IL-6 receptor).

Examples of various extracellular matrices and various cell adhesion molecules include collagens I to XIX, fibronectin, laminin-1 to 12, nitogen, tenascin, thrombospondin, von Willebrand factor, osteopontin, fibrinogen, and various elastins. , various proteoglycans, various cadherins, desmocollins, desmogleins, various integrins, E-selectin, P-selectin, L-selectin, immunoglobulin superfamily, Matrigel, poly-D-lysine, poly-L-lysine, chitin, chitosan, Examples include sepharose, hyaluronic acid, alginate gel, various hydrogels, and cut fragments thereof.

Examples of antibiotics that may be added to the medium include sulfa preparations, penicillin, pheneticillin, methicillin, oxacillin, cloxacillin, dicloxacillin, flucloxacillin, nafcillin, ampicillin, penicillin, amoxicillin, cyclacillin, carbenicillin, ticarcillin, piperacillin, azlocillin, Mecdulocilin, mecillinam, andinocillin, cephalosporins and their derivatives, oxolinic acid, amifloxacin, temafloxacin, nalidixic acid, pyromidic acid, ciprofloxan, cinoxacin, norfloxacin, perfloxacin, rosaxacin, ofloxacin, enoxacin, pipemidic acid, sulbactam, clavuric acid , β-bromopenicillanic acid, β-chloropenicillanic acid, 6-acetylmethylene-penicillanic acid, cefoxazole, sultanepicillin, adinocillin and formaldehyde foodrate ester of sulbactam, tazobactam, aztreonam, sulfazetin, isosulfazetin, nocardicin , methyl phenylacetamidophosphonate, chlortetracycline, oxytetracycline, tetracycline, demeclocycline, doxycycline, methacycline, and minocycline.

Note that, as described above, serum and/or a serum substitute may be added to the medium.
The concentration of serum added to the medium may be appropriately set depending on the cell type, culture conditions, culture purpose, etc., but in one embodiment, the concentration of serum (and/or serum substitute) in the medium is 15% by weight. % or less, 10% by weight or less, 9% by weight or less, 8% by weight or less, 7% by weight or less, 6% by weight or less, 5% by weight or less, 4% by weight or less, 3% by weight or less, 2% by weight or less, 1% by weight % or less, 0.9% by weight or less, 0.8% by weight or less, 0.7% by weight or less, 0.6% by weight or less, 0.5% by weight or less, 0.4% by weight or less, or 0.3% by weight % or less. Incidentally, the concentration of serum may be kept constant during the culture period, or may be increased or decreased when changing the medium, as necessary.

<Step (2)>
Step (2) is a step of culturing cells in the culture space to form a cell structure.
In step (2), for example, cells are cultured for 12 hours or more in a culture space of a cell structure production container to form a cell structure.
When a medium is added to the culture space of the cell structure manufacturing container, the cells dispersed in the medium are taken into the recesses and cultured in each recess. It is preferable that a certain number of cells be taken into each recess, and it is preferable that one cell structure be formed in the space formed by the bottom of the recess. In order to culture cell structures at high density, it is preferable that cell structures be formed in all the recesses, so it is preferable that at least one cell exists in each recess. From the viewpoint of production efficiency, it is preferable to reduce the initial number of cells as much as possible while still being able to recover as many cell structures as possible, so the smaller the number of cells present in each recess, the better. Therefore, it is preferred that one or more cells exist in each recess.

<Step (3)>
Step (3) is a step of exchanging the medium in the culture space.
In exchanging the medium, a certain amount of the medium in the culture space of the cell structure manufacturing container is sucked out, and then the same amount of fresh medium is injected. The medium is preferably replaced at least once during cell culture.
The fixed amount may be, for example, 20% to 80% by mass of the medium, or 20% to 50% by mass.
Examples of the medium include the medium exemplified in step (1).

<Step (4)>
Step (4) is a step of maturing the cell structure within the culture space.
In step (4), steps (2) to (3) are performed multiple times to mature the cell structure. "Maturation" means, for example, increasing the size of a cell structure to a desired size, forming an organoid structure, or guiding it to a target tissue by inducing differentiation.
In the case of inducing differentiation, it is preferable to form a cell structure in the space formed by the bottom of the recess and then exchange the culture medium with a differentiation-inducing medium for differentiation.

<Step (5)>
Step (5) is a step of suspending the cell structure in a medium.
In step (5), for example, after growing the cell structures to a desired size, the medium in the culture space is stirred to suspend the cell structures cultured in each recess in the medium. For example, it is carried out by stirring the medium. Specifically, agitation of the medium involves (i) shaking the cell structure production container to agitate the medium, and (ii) aspirating and discharging the medium from the culture space (pipetting operation) to remove the medium. (iii) placing a stirring blade in the culture space to stir the medium; and (iv) placing a stirring bar in the culture space to stir the medium. Two or more of these may be combined.

<Step (6)>
Step (6) is a step of collecting the cell structure.
In step (6), for example, the medium in which the cell structures are floating in the culture space of the cell structure production container is sucked up using a suction machine, and the cell structures suspended in the medium are recovered.

Hereinafter, the present invention will be explained in more detail based on Synthesis Examples, Examples, Test Examples, etc., but the present invention is not limited thereto.

<Method for measuring weight average molecular weight>
The weight average molecular weight of the copolymer shown in the following synthesis example is the result of measurement by Gel Filtration Chromatography (hereinafter abbreviated as GFC). The measurement conditions are as follows.
(GFC measurement conditions)
・Device: Prominence (manufactured by Shimadzu Corporation)
・GFC column: TSKgel GMPWXL (7.8mm I.D. x 30cm) x 2-3 ・Flow rate: 1.0ml/min
・Eluent: Aqueous solution containing ionic substances or mixed solution of ethanol ・Column temperature: 40℃
・Detector: RI
・Injection concentration: Polymer solid content 0.05-0.5% by mass
・Injection volume: 100μL
・Calibration curve: Cubic approximation curve ・Standard sample: Polyethylene oxide (manufactured by Agilent) x 10 types

<Synthesis example 1>
Phosmer M (product name, manufactured by Unichemical Co., Ltd., nonvolatile content in drying method at 100° C. for 1 hour: 91.8% by mass) was used.
Phosmer M is a mixture of acid phosphooxyethyl methacrylate (44.2% by mass), bis[2-(methacryloyloxy)ethyl] phosphate (28.6% by mass), and other substances (27.2% by mass). It is.
Add 5.00 g of Phosmer M to 7.97 g of ethanol and 23.92 g of pure water, stir and dissolve, and add 3.82 g of 2-(dimethylamino)ethyl methacrylate (manufactured by Tokyo Kasei Kogyo Co., Ltd.) while keeping the temperature below 20°C. , and 0.04 g of 2,2'-azobis(N-(2-carboxyethyl)-2-methylpropionamidine) n-hydrate (product name: VA-057, manufactured by Wako Pure Chemical Industries, Ltd.) , were added in order while keeping the temperature below 20°C. A mixed solution containing all of the above materials, which had been sufficiently stirred to become homogeneous, was introduced into the dropping funnel. On the other hand, 47.84 g of pure water was separately placed in a three-necked flask equipped with a cooling tube, nitrogen was flowed into the flask, and the temperature was raised to the reflux temperature while stirring. While maintaining this state, the dropping funnel into which the mixture had been introduced was set in a three-necked flask, and the mixture was dropped into the boiling liquid over a period of 1 hour. After the dropwise addition, 88.60 g of a copolymer-containing varnish with a solid content of about 9.70% by mass was obtained by heating and stirring while maintaining the above environment for 24 hours. The weight average molecular weight of the resulting transparent liquid measured by GFC was approximately 280,000.

<Synthesis example 2>
Acid phosphooxypolypropylene glycol monomethacrylate (average number of moles of propylene oxide added 5) (product name: PPM-5P, manufactured by Toho Chemical Industry Co., Ltd., absolute mass % (purity) 97.3 mass %) 7.5 g, polyethylene Glycol dimethacrylate (average number of moles added of polyethylene glycol: 4) (Product name: Bremmer (registered trademark) PDE-200, manufactured by NOF Corporation) 2.0 g (% by mass based on the entire copolymer: 21.2% by mass) ), 2-(dimethylamino)ethyl methacrylate (manufactured by Mitsubishi Gas Chemical Co., Ltd.) 1.9 g, and dimethyl-1,1'-azobis(1-cyclohexanecarboxylate) (product name: VE-073, Fujifilm) A mixed solution was prepared by adding 31.1 g of ion-exchanged water and 20.8 g of ethanol (manufactured by Kanto Chemical Co., Ltd.) to 58 mg (manufactured by Wako Pure Chemical Industries, Ltd.) and stirring uniformly. Separately, 31.2 g of ion-exchanged water and 20.8 g of ethanol (manufactured by Kanto Kagaku Co., Ltd.) were added to a three-necked flask equipped with a cooling tube, and the temperature was raised to the reflux temperature while stirring. While maintaining this state, the above mixture was dripped into a boiling liquid of ion-exchanged water and ethanol in a three-necked flask over a period of 1 hour using a dripping pump via a Teflon (registered trademark) tube. After the dropwise addition, the mixture was heated and stirred while maintaining the above environment for 23 hours. After the reaction was completed, the mixture was cooled to obtain a copolymer-containing varnish with a solid content of about 10.63% by mass. The weight average molecular weight of the obtained liquid according to GFC was about 300,000.

<Preparation example 1>
To 5.00 g of the copolymer-containing varnish obtained in Synthesis Example 1 above, 56.39 g of pure water, 28.50 g of ethanol, and 1 mol/L aqueous sodium hydroxide solution (1N) (manufactured by Kanto Kagaku Co., Ltd.) A coating agent was prepared by adding .85 g and stirring thoroughly. pH was 7.5. The resulting coating agent was spin-coated onto a silicon wafer at 1500 rpm for 60 seconds, and dried in an oven at 50° C. for 24 hours. Thereafter, it was thoroughly washed with PBS (phosphate buffered saline) and pure water to obtain a silicon wafer on which a coating film was formed. When the thickness of the coating film on the silicon wafer was confirmed using an optical interference film thickness meter, it was 55 Å.

<Preparation example 2>
To 1.00 g of the copolymer-containing varnish obtained in Synthesis Example 2 above, 1.28 g of pure water, 9.12 g of ethanol, and 0.43 g of 1N hydrochloric acid (manufactured by Kanto Kagaku Co., Ltd.) were added and thoroughly stirred. A coating agent was prepared. pH was 3.5. The obtained coating agent was spin-coated on an HMDS-treated silicon wafer at 1500 rpm/60 sec, and dried in an oven at 50° C. for 24 hours as a drying step. Thereafter, it was thoroughly washed with PBS and then dried in an oven at 50° C. for 1 hour to obtain a coating film on the HMDS-treated silicon wafer.

<Preparation example 3>
Polyvinyl acetate (JMR-150L (registered trademark) manufactured by Japan Vinyl & Poval Co., Ltd. (degree of polymerization 1480, degree of saponification 22.7%)) was mixed with ethanol/1-methoxy-2-propanol (7/3 mass ratio). A coating agent was prepared by dissolving the solution at a concentration of 10 mg/g. The resulting composition was transparent and uniform.

<Example 1>
SYLGARD 184 silicone elastomer (manufactured by Dow Corning) was mixed and stirred at a ratio of 10.00 g of main ingredient and 1.00 g of curing agent, and poured into a mold.
After removing bubbles using a vacuum pump, it was dried in an oven at 100°C for 1 hour. After cooling to room temperature, the cured product of polydimethylsiloxane (PDMS) was taken out from the mold. The cured product was immersed in pure water and sterilized in an autoclave at 120° C. for 15 minutes.
The mold used is a mold that allows the containers for cell structure production shown in FIGS. 1, 2, 3A, 3B, and 5 to be produced by integral molding, and the produced container for cell structure production The characteristics are as follows. The symbols correspond to those in the above figure.
- As shown in FIG. 1, the outer shape of the cell structure manufacturing container is cylindrical with circular upper and lower surfaces, the diameter of the circle is 15 mm, and the height is 11.5 mm.
- The thickness of the outer peripheral wall 2 forming the culture space 4 is 4 mm near the top surface.
- As shown in FIG. 2, a plurality of recesses 10 and a partition wall 11 that partitions the plurality of recesses 10 are formed in the culture space 4 surrounded by the outer peripheral wall 2. The number of recesses 10 in the cell structure manufacturing container is 19.
- Also, as shown in FIG. 2, the plurality of recesses 10 are arranged in a honeycomb arrangement.
- From the cross-sectional view of FIG. 3A, the five recesses 10, the partition wall 11 that partitions them, and the outer peripheral wall 2 higher than the partition wall 11 at the outermost side can be confirmed. The outer peripheral wall 2 is higher than the partition wall 11 by about 8 mm.
- In FIG. 3A, the shape of the top 11a of the partition wall 11 (in other words, the top 11a of the partition wall 11 in a cross section including the center line of each of the two adjacent recesses 10) is arcuate.
- The depth (d) of the recess 10 is 2.0 mm.
- The equivalent diameter (r) of the recess 10 is 1.0 mm.
- The width (w) of the partition wall 11 that partitions two adjacent recesses 10 is 0.3 mm.
- As shown in FIG. 3B, the shape of the central portion 10e of the recessed portion 10 excluding the bottom portion 10b and the opening portion 10d is cylindrical (non-tapered shape). The length (l) of the recess in the central portion 10e in the depth direction is 1.5 mm.
・As shown in FIGS. 2 and 5, at the center of an equilateral triangle in the partition wall 11 surrounded by three mutually adjacent recesses 10 arranged so as to form an equilateral triangle when the center points of the openings are connected. The corresponding vertex point 11b is the highest on the partition wall 11.
Note that the depth of the recess in the produced cell structure manufacturing container was 2.0 times the equivalent diameter of the recess at the center in the depth direction of the recess.

250 μL of the coating agent prepared in Preparation Example 1 was dispensed into the culture space of the prepared cell structure manufacturing container. After being allowed to stand for 1 hour, it was removed and dried in an oven at 50°C for 24 hours. Thereafter, the coated culture space was washed three times each with 300 μL of pure water. As a result, a container for producing a cell structure with a base film was obtained.

<Example 2>
The mold was changed from the mold used in Example 1 so that the shape of the bottom of the recess became a pen shape (inverted conical shape) as shown in FIG. A container for producing a cell structure was obtained in the same manner as in Example 1, except that the mold was changed to one in which (l) was 1.0 mm. Furthermore, in the same manner as in Example 1, a container for producing a cell structure with a base film was obtained.
Note that the depth (d') of the bottom portion 10b shown in FIG. 4 is 1.0 mm.
Further, the depth of the recess in the produced cell structure manufacturing container was 2.0 times the equivalent diameter of the recess at the center in the depth direction of the recess.

<Example 3>
A cell structure manufacturing container with a base film was obtained in the same manner as in Example 1, except that the coating agent prepared in Preparation Example 2 was used in place of the coating agent prepared in Preparation Example 1. .
That is, 250 μL of the coating agent prepared in Preparation Example 2 was dispensed into the culture space of the cell structure production container prepared in Example 1. After being allowed to stand for 1 hour, it was removed and dried in an oven at 50°C for 24 hours. Thereafter, the coated culture space was washed three times each with 300 μL of pure water. As a result, a container for producing a cell structure with a base film was obtained.

<Example 4>
A container for producing a cell structure with a base film was obtained in the same manner as in Example 1, except that the coating agent prepared in Preparation Example 3 was used instead of the coating agent prepared in Preparation Example 1. .
That is, 150 μL of the coating agent produced in Preparation Example 3 was dispensed into the culture space of the cell structure production container produced in Example 1. After being allowed to stand for 1 hour, it was removed and dried in an oven at 50°C for 24 hours. Thereafter, the coated culture space was washed three times each with 300 μL of pure water. As a result, a container for producing a cell structure with a base film was obtained.

<Comparative example 1>
A container for cell structure production was obtained in the same manner as in Example 1, except that the mold used in Example 1 was changed to a mold in which the entire top of the compartment wall was flat. . It should be noted that the obtained container for producing cell structures has no difference from the container for producing cell structures in Example 1, except that the entire top of the partition wall is flat.
Furthermore, in the same manner as in Example 1, a container for producing a cell structure with a base film was obtained.
Note that the depth of the recess in the produced cell structure manufacturing container was 2.0 times the equivalent diameter of the recess at the center in the depth direction of the recess.

<Comparative example 2>
The mold was changed from the mold used in Example 1 so that the depth of the recess was 1.0 mm, and the length (l) in the depth direction of the recess in the non-tapered central part was 0.5 mm. A container for cell structure production was obtained in the same manner as in Example 1, except that the mold was changed to the following. In addition, in the obtained cell structure manufacturing container, the depth of the recessed part is 1.0 mm, and the length (l) in the depth direction of the recessed part in the central part, which is non-tapered, is 0.5 mm. Other than that, there is no difference from the cell structure manufacturing container of Example 1.
Furthermore, in the same manner as in Example 1, a container for producing a cell structure with a base film was obtained.
Note that the depth of the recess in the produced cell structure manufacturing container is 1.0 times the equivalent diameter of the recess at the center in the depth direction of the recess.

(Preparation of cell suspension of human adipose-derived mesenchymal stem cells)
Human adipose tissue-derived mesenchymal stem cells (ADSC: manufactured by Cellsource Co., Ltd.) were used as cells.
A low serum medium Mesenchymal Stem Cell Growth Medium 2 (manufactured by Takara Bio Inc.: serum concentration 2%) was used for culturing the cells.
The cells were statically cultured in a 10 cm diameter petri dish (medium 10 mL) for 2 days or more while maintaining a 5% carbon dioxide concentration in a 37° C./CO ₂ incubator. Subsequently, the cells were washed with 3 mL of PBS solution (manufactured by Fuji Film Wako Pure Chemical Industries, Ltd.), and then 3 mL of trypsin-EDTA solution (manufactured by PromoCell) was added and left at room temperature for 3 minutes to detach the cells. 7 mL of the above low serum medium was added and the cells were collected. After centrifuging this suspension (manufactured by Tomy Seiko Co., Ltd., model number LC-230, 200 x g/3 minutes, room temperature), the supernatant was removed, and the above medium was added to obtain human adipose-derived mesenchymal stem cells. A cell suspension was prepared.

(Preparation of cell suspension of mouse fetal fibroblasts)
Mouse fetal fibroblast cells (C3H10T1/T2 cells: manufactured by DS Pharma Biomedical Co., Ltd.) were used as cells.
For cell culture, 10% FBS (manufactured by Sigma-Aldrich) and 1% Glutamine/Penicillin/Streptmycin (manufactured by Gibco) were added to BME medium (manufactured by Gibco) as a basal medium. A medium was used. The cells were statically cultured in a 10 cm diameter petri dish (medium 10 mL) for 2 days or more while maintaining a 5% carbon dioxide concentration in a 37° C./CO ₂ incubator. Subsequently, the cells were washed with 3 mL of PBS solution (manufactured by Fuji Film Wako Pure Chemical Industries, Ltd.), and then 3 mL of trypsin-EDTA solution (manufactured by PromoCell) was added and left at room temperature for 3 minutes to detach the cells. 7 mL of the above medium was added and the cells were collected. After centrifuging this suspension (manufactured by Tomy Seiko Co., Ltd., model number LC-230, 200 x g/3 minutes, room temperature), the supernatant was removed, and the above medium was added to form mouse fetal fibroblast cells. A suspension was prepared.

(Spheroid formation test; Example 1 and Comparative Example 1)
A cell suspension of human adipose-derived mesenchymal stem cells was added to each container for producing cell structures with base membranes obtained in Example 1 and Comparative Example 1 at a concentration of 19×10 ⁴ cells/container. 200 μL was added. Thereafter, it was left standing in a CO ₂ incubator at 37° C. for 1 day while maintaining a 5% carbon dioxide concentration.

(Observation of spheroid formation; Example 1 and Comparative Example 1)
After one day of culturing in the above spheroid formation test, the formation of cell structures in each container for producing cell structures with base membranes of Example 1 and Comparative Example 1 was observed using an inverted microscope (Olympus Corporation, CKX31) (magnification: 2 times). The observation results of Example 1 (after 1 day of culture) are shown in FIG. The observation results of Comparative Example 1 are shown in FIGS. 7A and 7B. The results are shown in Table 1 regarding the number of spheroids formed and spheroid size data.
Note that FIG. 7A is a photograph focused on the bottom of the recess, and FIG. 7B is a photograph focused on the top surface of the partition wall. In FIG. 7B, the arrows indicate spheroids present on the upper surface of the compartment wall.

The average diameter was measured using image analysis software ImageJ. The diameter of the formed cell structure was calculated on ImageJ, and the sum of the diameters of all cell structures was divided by the number of cell structures.
The size distribution was calculated by considering the diameters calculated above as independent samples, calculating the standard deviation, and then calculating the ratio of the standard deviation to the average diameter. The smaller the value, the higher the uniformity of size.

Spheroids were formed in both Example 1 and Comparative Example 1. In Example 1, spheroids existed only within the recesses (dimples), and the average diameter was about 400 μm, with a size error of 12%.
On the other hand, in Comparative Example 1, many spheroids were present in addition to the recesses (dimples) on the upper surface of the partition walls that partitioned the recesses, and the total number was 50 or more. The average diameter was approximately 200 μm, with a size error of 61%. Compared to Example 1, the size was smaller and the error was larger. This is because when the upper surface of the partition wall is flat as in Comparative Example 1, cells remain there and form small spheroids. By making the upper surface (top) of the partition wall convex as in Example 1, almost all the cells can be collected in the recesses (dimples) and spheroids of uniform size can be formed.

(Spheroid formation test; Example 2, influence of recess bottom structure)
A cell suspension of mouse fibroblasts (200 μL) was added to each container for producing a cell structure with a base membrane obtained in Example 2 at a concentration of 23×10 ⁴ cells/container. Thereafter, it was left standing in a CO ₂ incubator at 37° C. for 1 day while maintaining a 5% carbon dioxide concentration.

(Observation of spheroid formation; Example 2)
After one day of culturing in the above spheroid formation test, the formation of cell structures in the container for producing cell structures with a base membrane in Example 2 was observed using an inverted microscope (CKX31, manufactured by Olympus) (magnification: 4x, 10x). Comparisons were made based on. The observation results of Example 2 (after 1 day of culture) are shown in FIG. 8A (4x magnification) and FIG. 8B (10x magnification).
In Example 2, spheroids were formed. In Example 2, spheroids were present only within the recesses (dimples). Even when the bottom shape of the recess is changed as in Example 2, almost all the cells can be collected in the recess (dimple) and spheroids of uniform size can be formed as in Example 1.

(Effect of medium exchange)
The effects of the depth of the recesses were confirmed using the containers for producing cell structures with base membranes of Example 1 and Comparative Example 2.
200 μL of a cell suspension of human adipose-derived mesenchymal stem cells was added to each container for producing cell structures with base membranes in Example 1 and Comparative Example 2 so that 19×10 ⁴ cells/container were obtained. . Thereafter, it was left standing in a CO ₂ incubator at 37° C. for 1 day while maintaining a 5% carbon dioxide concentration. Thereafter, 150 μL of the medium was aspirated using a micropipettor. Thereafter, 150 μL of new medium was added using a micropipettor. The formation of cell structures after medium exchange was compared based on observation using an inverted microscope (CKX31, manufactured by Olympus Corporation) (magnification: 2x).
The observation results of Example 1 are shown in FIG. The observation results of Comparative Example 2 are shown in FIG. In Example 1, the arrangement of the cell structures was maintained even after medium exchange. On the other hand, in Comparative Example 2, the cell structures escaped due to medium exchange and were present in different recesses and on the upper surfaces of compartment walls. Note that in FIG. 10, the white arrow indicates the recess from which the cell structure escaped due to medium exchange. This is because the depth of the recesses was shallower than in Example 1, and the formed cell structures were lifted up due to the influence of the flow during medium exchange.

(Preparation of mouse skin-derived epithelial cells and mesenchymal cells)
An 18-day-old fetus (C57BL/6 mouse) was collected from a pregnant mouse (manufactured by CLEA Japan). Skin tissue was collected from the dorsal skin of the collected fetus. The collected skin was treated with dispase (manufactured by Roche) at 4° C. for 1 hour at 20 rpm, and epithelial tissue and mesenchymal tissue were separated using tweezers. Each of the epithelial tissue and mesenchymal tissue was treated with 100 U/mL collagenase (manufactured by Fuji Film Wako Pure Chemical Industries, Ltd.) at 37° C. for 80 minutes. Furthermore, the epithelial tissue was treated with 0.25% trypsin (manufactured by Thermo Fisher) containing 100 U/mL collagenase at 37°C for 10 minutes, and then the epithelial cells and mesenchymal cells were each treated with a cell strainer (manufactured by Corning). Unification processing was performed using . 5 μL of Vybrant™ Cell-labeling Solutions (manufactured by Thermo Fisher) was added to 1 mL of a suspension of mesenchymal cells, and the cells were stained by incubating for 20 minutes.

(Hair follicle primordium formation test)
Subsequently, cells (mouse skin-derived epithelial cells and mesenchymal cells) were collected from each solution by centrifugation and mixed in a medium at a ratio of 1:1 to prepare a cell suspension. 200 μL of the prepared cell suspension was added to the base membrane-equipped cell structure manufacturing container obtained in Example 1 so that the amount was 19×10 ⁴ cells/vessel. The medium is a 1:1 mixture of mesenchymal cell culture medium (DMEM (manufactured by Sigma-Aldrich) + 10% FBS (manufactured by Sigma-Aldrich) + 1% Penicillin/Streptomycin (manufactured by Sigma-Aldrich)) and HuMedia-KG2 (manufactured by Kurabo Industries). A mixed medium was used. Thereafter, it was left standing in a CO ² incubator at 37° C. for 3 days while maintaining a 5% carbon dioxide concentration.

(Observation of hair follicle primordium formation)
After 3 days of culture in the hair follicle primordium formation test, the formation of cell structures in the container for producing cell structures with a base membrane of Example 1 was observed using a phase contrast fluorescence microscope BZ-X700 (manufactured by Keyence Corporation). The observation results are shown in FIGS. 11A to 11C.
FIG. 11A is a photograph of the bright field observation results. FIG. 11B is a photograph of the fluorescence observation results. FIG. 11C is a photograph in which bright field observation results and fluorescence observation results are superimposed.
As a result, it was confirmed by bright field observation that two separated dumbbell-shaped structures were formed. Further, as a result of fluorescence observation, mesenchymal cells stained with VybrantTM Cell-labeling Solutions and unstained epithelial cells were clearly separated, indicating the formation of hair follicle primordia. From the above, it is thought that this culture vessel can be applied to the preparation of organ primordia and organoids formed from multiple cells, including hair follicle primordia.

(Hair regeneration test using patch method)
When preparing the epithelial cells and mesenchymal cells, the cells were prepared without staining mesenchymal cells with Vybrant™ Cell-labeling Solutions. Furthermore, seeds were seeded under the same conditions as above in the container for manufacturing the cell structure with the base membrane obtained in Example 1, and the seeds were placed in a CO ² incubator at 37° C. for 3 days while maintaining a carbon dioxide concentration of 5%. The cells were cultured to form hair follicle primordia. A transplant hole was made subcutaneously in an ICR nude mouse (manufactured by Oriental Yeast Co., Ltd.) under exhalation anesthesia with isoflurane (manufactured by Bioresearch Center Co., Ltd.) using a 20G ophthalmic V lance (manufactured by Nippon Alcon Co., Ltd.) (patch method). Nineteen hair follicle primordia prepared in each container were transplanted into one transplant hole using a micropipette (manufactured by Co., Ltd.). The mice to which the hair follicle primordia were transplanted were observed using a microscope (manufactured by Keyence Corporation) on the 28th day after the transplantation.
As shown in FIG. 12, hair was formed as a black mass at the hair follicle primordium transplantation site, and a large amount of hair was observed within the black mass. The quantitative result of the number of regenerated hairs was 39±8 (n=3).

(Study of low adhesion coating materials; Examples 3 and 4)
(Spheroid formation test)
100 μL of a cell suspension of mouse fetal fibroblasts was added to each container for producing a cell structure with a base membrane prepared in Example 3 at a concentration of 1×10 ⁴ cells/container. Thereafter, it was left standing in a CO ₂ incubator at 37° C. for 1 day while maintaining a 5% carbon dioxide concentration.

(Observation of spheroid formation)
After one day of culturing in the above spheroid formation test, the formation of cell structures in the container for producing cell structures with a base membrane in Example 3 was observed using an inverted microscope (CKX31, manufactured by Olympus Corporation) (magnification: 4x, 10x) Comparisons were made based on. The observation results (after 1 day of culture) are shown in FIGS. 13A and 13B. Almost all cells were collected within the dimples, and uniform spheroids were formed only within the dimples.

(Spheroid formation test)
100 μL of a cell suspension of human adipose-derived mesenchymal stem cells was added to each container for producing a cell structure with a base membrane prepared in Example 4 so that the amount was 5×10 ⁴ cells/container. Thereafter, it was left standing in a CO ₂ incubator at 37° C. for 1 day while maintaining a 5% carbon dioxide concentration.

(Observation of spheroid formation)
After one day of culturing in the above spheroid formation test, the formation of cell structures in the container for producing cell structures with a base film in Example 4 was compared based on observation using an inverted microscope (CKX31, manufactured by Olympus) (magnification: 4x). did. The observation results (after 1 day of culture) are shown in FIG. Almost all cells were collected within the dimples, and spheroids were formed only within the dimples.

According to the cell structure production container of the present invention, a plurality of uniformly sized cell structures can be produced in large quantities.

1 Bottom surface 2 Peripheral wall 3 Opening 4 Culture space 10 Recess 10a Opening surface 10b Bottom 10c Center in depth direction 10d Opening 10e Center 11 Compartment wall 11a Top 11b Vertex location 11c Vertex 100 Container for manufacturing cell structures

Claims

comprising a plurality of recesses and a partition wall that partitions the plurality of recesses,
The top of the partition wall has a convex shape,
The depth of the recess is more than 1.0 times the equivalent diameter of the recess at the center in the depth direction of the recess.
Container for producing cell structures.
The cell structure production according to claim 1, wherein the width of the partition wall that partitions two adjacent recesses is 0.1 mm to 0.5 mm when measured at the center of the depth direction of the recess. container.
The container for producing cell structures according to claim 1, which is made of a gas-permeable elastic material.
The container for producing cell structures according to claim 3, wherein the gas-permeable elastic material is silicone.
The cell structure manufacturing container according to claim 1, wherein the depth of the recess is 1.5 times or more the equivalent diameter of the recess at the center in the depth direction of the recess.
The container for producing a cell structure according to claim 1, wherein the central portion of the recess excluding the bottom and the opening has a non-tapered shape.
The cell structure manufacturing container according to claim 6, wherein the length in the depth direction of the concave portion of the non-tapered central portion is 0.5 times or more the depth of the concave portion.
The cell structure according to claim 1, wherein the shape of the top of the partition wall in a cross section including the center line of each of the two adjacent recesses is a convex arc or a shape having a corner at the top. Manufacturing containers.
The width of the partition wall that partitions two adjacent recesses is at least 0.1 times and at most 0.5 times the equivalent diameter of the recess when measured at the center of the depth direction of the recess; The container for producing a cell structure according to claim 1.
The plurality of recesses are arranged such that the distances between the center point of the opening of the specific recess and the center points of each of the openings of two or more recesses arranged adjacent to the specific recess are equal to each other. The container for producing a cell structure according to claim 1, wherein the container is arranged in a container for producing a cell structure according to claim 1.
A point corresponding to the center of the equilateral triangle in the partition wall surrounded by three mutually adjacent recesses arranged to form an equilateral triangle when the center points of the openings are connected is the highest in the partition wall, The container for producing a cell structure according to claim 10.
The cell structure manufacturing container according to claim 1, wherein the bottoms of the plurality of recesses have an arc shape or an inverted triangular shape in cross section.
The container for producing a cell structure according to claim 1, wherein the equivalent diameter of the recess is 0.3 mm to 1.5 mm.
The container for producing a cell structure according to claim 1, wherein the depth of the recess is 0.5 mm to 3.0 mm.
The container for producing a cell structure according to claim 1, having 5 to 2000 recesses.
The container for producing cell structures according to claim 1, which has a cylindrical outer shape.
The cell structure manufacturing container according to claim 1, which can be stored in a recessed part of a multiwell plate.
The container for producing a cell structure according to claim 1, wherein at least a part of the surface of the container for producing a cell structure is provided with a coating film having the ability to suppress adhesion of cells.
A method for manufacturing a cell structure manufacturing container according to any one of claims 1 to 18, comprising:
A method for manufacturing a cell structure manufacturing container, the method comprising molding the cell structure manufacturing container using a mold.
A cell structure produced using the cell structure production container according to any one of claims 1 to 18.
A method for producing a cell structure, comprising the step of adding a medium in which cells are dispersed to a culture space of a container for producing a cell structure according to any one of claims 1 to 18.