Cell structure production device

Abstract

Provided is a cell structure production device that is, when producing cell structures, capable of appropriately controlling the dimensions of the cell structures.　This cell structure production device for producing cell structures comprises: a substrate supply part that supplies a substrate and in which at least one surface of the substrate has a cell attachment inhibiting ability; a base film forming part that is for forming a base film on the substrate and includes a base film forming application mechanism for applying a base film forming composition onto the substrate, and in which the base film has a cell adhesive property; and an inoculation part for inoculating cells onto the substrate including the base film.

Description

Cell structure manufacturing equipment

[0001] The present invention relates to a cell structure manufacturing apparatus for manufacturing cell structures such as spheroids.

[0002] Cell structures such as cell aggregates, cell clusters or spheroids are thought to be useful in drug discovery research, or cell therapy and regenerative therapy.

[0003] As a method for manufacturing a three-dimensional structure of cells such as a cell structure, for example, Patent Document 1 describes a three-dimensional structure manufacturing device that includes a storage plate formed with a large number of storage recesses each for storing a single cell mass, a support having a plurality of needle-shaped bodies that pierce and penetrate the cell mass, a suction nozzle connected to a negative pressure generating means for adsorbing and holding the cell mass, a moving means for moving and raising and lowering the suction nozzle between the storage plate and the support, and a control means for controlling the suction operation of the suction nozzle and the operation of the moving means.

[0004] Patent document 2 also describes a cell structure manufacturing device that includes multiple long, thin needles with pointed tips, a puncturing section that punctures and penetrates cell clusters with the tips of the needles and raises the needles after puncturing, a base section, and a control section.

[0005] Furthermore, Patent Documents 3 and 4 describe a cellular three-dimensional structure manufacturing device that includes a container for storing cell clumps, a support on which are arranged multiple needle-shaped bodies that pierce and penetrate the cell clumps, a suction nozzle that adsorbs and holds the cell clumps, a nozzle moving means for moving the suction nozzle, and a control means for controlling the suction operation of the suction nozzle and the operation of the nozzle moving means.

[0006] Furthermore, Patent Document 5 describes a cell cluster sheet manufacturing device for arranging multiple cell clusters in a plane on a mounting surface in a culture vessel, culturing the cell clusters, and producing a cell cluster sheet in which the cell clusters are fused together.

[0007] Furthermore, various materials have been proposed as base film forming agents for efficient cell culture. For example, Patent Document 6 discloses a method for producing a polymer used as a base film for cell culture and a cell culture vessel.

[0008] Patent Document 7 describes a coating film made of an ion complex material that has the ability to inhibit adhesion of biological substances.

[0009] Japanese Patent No. 5896104 Japanese Patent No. 6334837 Japanese Patent No. 6663555 Japanese Patent No. 6663556 Japanese Patent No. 6880384 International Publication No. 2020 / 040247 International Publication No. 2014 / 196650

[0010] As used herein, a cell structure refers to a collection of cells in which cells self-assemble and aggregate. Cell structures are also commonly referred to as cell aggregates, cell clusters, spheroids, spheres, or organoids. It has been reported that because a living body-like structure is constructed in a cell structure, the functions of the cells in the cell structure can be maintained for a long period of time, and physiological functions are improved. Therefore, there are growing expectations for the use of cell structures in drug discovery research, cell therapy, and regenerative therapy.

[0011] Furthermore, technologies for easily, quickly, uniformly, and mass-produced cell structures are important for the practical application of regenerative medicine and the efficiency of drug discovery testing. However, conventional production methods that utilize the random aggregation phenomenon of suspended cells using low-adhesion cell culture dishes (e.g., multi-well plates) only form one spheroid per well. Therefore, there are problems with ease of use and mass productivity.

[0012] Furthermore, when the cell structure is a spherical spheroid, if the diameter of the spheroid is too large, some of the cells in the spherical spheroid may die. Furthermore, if the diameter of the spheroid is too small, the therapeutic effect of the spheroid may be reduced. Therefore, appropriately controlling the dimensions of cell structures such as spheroids is an important technique for improving the yield of cell structure production. In conventional production methods, the error in the diameter of spheroids can be as high as 40%. To improve the yield of cell structure production, the error in the diameter of spheroids is preferably required to be within 20%.

[0013] Furthermore, in the production of cell structures, the base membrane used for cell culture generally requires the use of serum derived from a living organism to ensure uniform adhesion of cells to the base membrane. The use of serum derived from a living organism poses problems such as variations in quality, including proliferation ability due to individual differences, and safety risks such as allergies and viral contamination when serum derived from animals other than humans is used.

[0014] Therefore, an object of the present invention is to provide a cell structure manufacturing device that can appropriately control the dimensions of a cell structure when manufacturing the cell structure.

[0015] Another object of the present invention is to provide a cell structure manufacturing device that can produce uniform, high-quality cell structures without using serum derived from living organisms, that is easy to operate and suitable for mass production, and that can appropriately control the dimensions of the cell structures.

[0016] In order to solve the above problems, the present invention has the following configuration.

[0017] (Configuration 1) Configuration 1 of the present invention is a cell structure manufacturing apparatus for manufacturing a cell structure, comprising: a substrate supply unit that supplies a substrate, at least one surface of the substrate having the ability to inhibit cell adhesion; a base film forming unit that forms a base film on the substrate, the base film forming unit including a base film forming coating mechanism that applies a base film forming composition onto the substrate, the base film having cell adhesive properties; and a seeding unit that seeds cells onto the substrate including the base film.

[0018] (Configuration 2) Configuration 2 of the present invention is the cell structure manufacturing apparatus of Configuration 1, wherein the substrate supply unit further includes a coating film forming unit that forms a coating film having cell adhesion inhibitory properties on at least a portion of at least one surface of the raw material substrate, the coating film forming unit includes a coating film forming application mechanism that applies a coating film forming composition to the surface of the substrate, and the base film forming application mechanism applies the base film forming composition to at least a portion of the surface of the coating film of the substrate.

[0019] (Configuration 3) Configuration 3 of the present invention is a cell structure manufacturing device of configuration 2, in which the composition for forming a coating film contains a copolymer having a repeating unit (A) represented by the following formula (A) and a repeating unit (B) represented by the following formula (B):

[0020] (In the formula, R １ ~R ３ each independently represents a hydrogen atom or an alkyl group having 1 to 5 carbon atoms; X １ and X ２ each independently represents a single bond, an ester bond, an ether bond, an amide bond, or an alkylene group having 1 to 5 carbon atoms which may be interrupted by an oxygen atom.

[0021] (Configuration 4) Configuration 4 of the present invention is a cell structure manufacturing device of any of configurations 1 to 3, in which at least one selected from the application mechanism for forming a base film and the application mechanism for forming a coating film is a pointillist application mechanism.

[0022] (Configuration 5) Configuration 5 of the present invention is the cell structure manufacturing device according to any one of configurations 1 to 4, wherein the substrate has a substantially smooth surface.

[0023] (Configuration 6) Configuration 6 of the present invention is the cell structure manufacturing device according to any one of Configurations 1 to 4, wherein the surface of the substrate has irregularities.

[0024] (Configuration 7) Configuration 7 of the present invention is a cell structure manufacturing apparatus of any of configurations 1 to 6, in which the substrate or the raw substrate is flexible, the substrate supply section includes a winding-type substrate cassette or raw substrate cassette, and the substrate is supplied from the substrate cassette, or the raw substrate is supplied from the raw substrate cassette.

[0025] (Configuration 8) Configuration 8 of the present invention is a cell structure manufacturing apparatus of any of configurations 1 to 7, wherein the cell structure manufacturing apparatus further includes an aggregation culture section for culturing cells attached to the substrate including the base film.

[0026] (Configuration 9) Configuration 9 of the present invention is the cell structure manufacturing device of any one of configurations 1 to 8, in which the size error of the cell structure is within 20%.

[0027] (Configuration 10) Configuration 10 of the present invention is a cell structure manufacturing apparatus of any of configurations 1 to 9, wherein the cell structure manufacturing apparatus has an airtight mechanism that can make the inside of the cell structure manufacturing apparatus an airtight closed space, and the airtight mechanism can maintain the inside of the closed space in a sterile environment.

[0028] (Configuration 11) In Configuration 11 of the present invention, the composition for forming a base film is a compound represented by the following formula (I): [wherein Ua1 and Ua2 each independently represent a hydrogen atom or a linear or branched alkyl group having 1 to 5 carbon atoms; Ra1 represents a hydrogen atom or a linear or branched alkyl group having 1 to 5 carbon atoms; and Ra2 represents a linear or branched alkylene group having 1 to 5 carbon atoms], and a repeating unit derived from a monomer represented by the following formula (II): The cell structure manufacturing device of any one of structures 1 to 10 includes a copolymer containing a repeating unit derived from a monomer represented by the formula: [wherein Rb represents a hydrogen atom or a linear or branched alkyl group having 1 to 5 carbon atoms].

[0029] (Configuration 12) Configuration 12 of the present invention is the cell structure manufacturing device of configuration 11, wherein the base film-forming composition further contains a cell adhesive substance.

[0030] According to the present invention, it is possible to provide a cell structure manufacturing device that can appropriately control the dimensions of a cell structure when manufacturing the cell structure.

[0031] Furthermore, according to the present invention, it is possible to provide a cell structure manufacturing device that can produce homogeneous, high-quality cell structures without using serum derived from living organisms, that is easy to operate and suitable for mass production, and that can appropriately control the dimensions of the cell structures.

[0032] 1 is a schematic diagram showing an example of a cell structure manufacturing apparatus of this embodiment. FIG. 2 is a schematic cross-sectional view showing an example of an undercoat film pattern formed on the surface of a substrate. FIG. 3 is a schematic plan view showing another example of a cell structure manufacturing apparatus of this embodiment. FIG. 4 is a photograph of the results of microscopic observation of the cell adhesion test of Experimental Example A1. FIG. 5 is a photograph of the results of microscopic observation of the cell adhesion test of Comparative Experimental Example A3. FIG. 6 is a stereomicroscope photograph of the appearance of the cell aggregate manufacturing substrates produced in Experimental Examples B1 to B3 and Comparative Experimental Examples B1 and B2, which were subjected to the cell adhesion confirmation test of Test Example B1. FIG. 7 is a stereomicroscope photograph of the appearance of the cell aggregate manufacturing substrates produced in Experimental Examples B4 and B6, which were subjected to the cell adhesion / cell aggregate formation confirmation test of Test Example B2, taken 2 hours and 2 days later, respectively. FIG. 8 is a stereomicroscope photograph of the appearance of the cell aggregate manufacturing substrates produced in Experimental Example B5 and Comparative Experimental Example B3, which were subjected to the cell adhesion confirmation test of Test Example B3. 1 is a stereomicroscope photograph of the appearance of the cell aggregate production substrate prepared in Experimental Example B7, which was subjected to the cell adhesion confirmation test in Test Example B4. These are stereomicroscope photographs taken 2 hours and 3 days after cell aggregates (spheroids) were formed using the cell aggregate production substrates of Experimental Examples B8 to B12. This shows the results of size evaluation of spheroids formed using the cell aggregate production substrates of Experimental Examples B8 to B10. This is a table showing the relationship between the applied diameter, average spheroid diameter, and spheroid error for spheroids formed using the cell aggregate production substrates of Experimental Examples B8 to B12. This is a diagram showing the relationship between the applied area and spheroid diameter when spheroids are formed using the cell aggregate production substrates of Experimental Examples B8 to B12. This is a diagram showing the relationship between the applied area and spheroid volume when spheroids are formed using the cell aggregate production substrates of Experimental Examples B8 to B12. 10A and 10B are stereomicroscope photographs taken 2 hours and 2 days later of the appearance of the substrate for producing cell aggregates prepared in Comparative Experimental Example B4, which was subjected to the cell adhesion / cell aggregate formation confirmation test of Test Example B6. 10B are stereomicroscope photographs taken 2 hours and 2 days later of the appearance of the substrate for producing cell aggregates prepared in Comparative Experimental Example B5, which was subjected to the cell adhesion / cell aggregate formation confirmation test of Test Example B7.

[0033] Hereinafter, embodiments of the present invention will be described in detail with reference to the drawings. Note that the following embodiments are forms for embodying the present invention and are not intended to limit the scope of the present invention.

[0034] In this specification, "on" a substrate or a film includes not only cases where it is in direct contact with the upper surface of the substrate or film, but also cases where it is not in direct contact with the upper surface of the substrate or film. For example, "forming a film A on a substrate" not only means forming a film A directly on the surface of the substrate, but also includes forming a film A on the surface of another film formed on the surface of the substrate. In other words, "on" a substrate or a film includes cases where another film exists between the substrate or film and the object (film). Furthermore, "on" does not necessarily only mean the upper side in the vertical direction. "On" merely indicates the relative positional relationship between the substrate, the film, etc.

[0035] The cell structure manufacturing device 10 of this embodiment will be described with reference to FIG.

[0036] This embodiment is a cell structure manufacturing apparatus 10 for manufacturing a cell structure 1. The cell structure manufacturing apparatus 10 of this embodiment includes a substrate supply unit 20, a base film forming unit 40, and a seeding unit 50. The substrate supply unit 20 of the cell structure manufacturing apparatus 10 of this embodiment can further include a coating film forming unit 30.

[0037] <Substrate Supply Unit 20> The substrate supply unit 20 supplies a substrate 80. In this specification, the substrate 80 is a planar (plate-like or film-like) structure whose surface has the ability to inhibit adhesion of cells 56. In this specification, a planar (plate-like or film-like) structure whose surface does not have the ability to inhibit adhesion of cells 56 is referred to as a raw material substrate 82. The substrate 80 can be obtained by forming a coating film 84 having the ability to inhibit adhesion of cells 56 on the surface of the raw material substrate 82.

[0038] In this specification, a surface having the ability to inhibit adhesion of cells 56 means that no adhesion or spreading of cells 56 is observed on that surface when observed under a microscope, and cell structures 1 such as spheroids are formed in parts other than the surface.

[0039] Alternatively, having the ability to inhibit adhesion of cells 56 means that the luminescence intensity (%) (luminescence intensity of adherent cells on the coating film 84) / (luminescence intensity of adherent cells on a well without coating) compared to when no coating is performed with ATP assay is 50% or less, preferably 30% or less, and more preferably 10% or less.

[0040] In this embodiment, at least one surface of the substrate 80 has the ability to inhibit adhesion of the cells 56. As a result, it is possible to inhibit the cells 56 from adhering to the exposed portion of the substrate 80 (or the coating film 84 of the substrate 80).

[0041] The substrate 80 or raw material substrate 82 used in the cell structure manufacturing apparatus 10 of this embodiment is preferably flexible. Furthermore, in the cell structure manufacturing apparatus 10 of this embodiment, the substrate supply unit 20 preferably includes a winding-type substrate cassette or raw material substrate cassette 22.

[0042] In the example of the substrate supply unit 20 of the cell structure manufacturing apparatus 10 shown in Figure 1, the raw material substrate 82 is flexible and is supplied from a winding-type raw material substrate 82 cassette 22. Similarly, when using a substrate 80 that has the ability to inhibit adhesion of cells 56, the substrate supply unit 20 can include a winding-type substrate cassette. By using a winding-type substrate cassette or raw material substrate cassette 22, the cell structure 1 can be manufactured continuously.

[0043] The cell structure manufacturing apparatus 10 of this embodiment can employ a batch-type manufacturing method as shown in FIG. 3 . In this case, the shape of the raw material substrate 82 (or substrate 80) can be a rectangle of predetermined dimensions. In this case, the raw material substrate 82 of the predetermined shape is placed in the batch-type coating film forming unit 30, a coating film 84 is formed on the surface of the raw material substrate 82, and the raw material substrate 82 is removed from the coating film forming unit 30. In this manner, a rectangular substrate 80 can be obtained on which a coating film 84 capable of inhibiting adhesion of cells 56 has been formed. The substrate 80 of the predetermined dimensions can then be subjected to predetermined processing in the base film forming unit 40 and the seeding unit 50. Furthermore, when employing the manufacturing method shown in FIG. 3 , it is preferable that the raw material substrate 82 or substrate 80 is not flexible in order to facilitate transportation of the raw material substrate 82 or substrate 80.

[0044] The substrate 80 (or raw material substrate 82) used in the cell structure manufacturing apparatus 10 of this embodiment preferably has a substantially smooth surface. By using a substrate 80 (or raw material substrate 82) with a substantially smooth surface, the base film 90 described below can be formed at any location and with any dimensions. By controlling the dimensions of the base film 90, the dimensions of the resulting cell structure 1 can be controlled.

[0045] The surface of the substrate 80 (or raw material substrate 82) used in the cell structure manufacturing apparatus 10 of this embodiment can have irregularities. By having the surface of the substrate 80 (or raw material substrate 82) have appropriate irregularities, it is possible to identify the location where the base film 90 described below will be formed. This makes it easier to determine the location where the base film 90 will be formed. The base film 90 can be formed in the recesses among the irregularities on the surface of the substrate 80 (or raw material substrate 82). By controlling the dimensions of the recesses where the base film 90 is formed, the dimensions of the resulting cell structure 1 can be made appropriate. Therefore, by making the surface of the substrate 80 (or raw material substrate 82) have appropriate irregularities and dimensions and forming the base film 90 in the recesses, the dimensions of the resulting cell structure 1 can be made appropriate. When the surface of the substrate 80 (or raw material substrate 82) has appropriate irregularities, the raw material substrate 82 or the substrate 80 can be inflexible.

[0046] Examples of materials for the source substrate 82 include glass, metals, metal-containing compounds or semimetal-containing compounds, activated carbon, and resin. Examples of metals include typical metals (alkali metals: Li, Na, K, Rb, Cs; alkaline earth metals: Ca, Sr, Ba, Ra), magnesium group elements: Be, Mg, Zn, Cd, Hg, aluminum group elements: Al, Ga, In, rare earth elements: Y, La, Ce, Pr, Nd, Sm, Eu, tin group elements: Ti, Zr, Sn, Hf, Pb, Th, iron group elements: Fe, Co, Ni, earth elements: V, Nb, Ta, chromium group elements: Cr, Mo, W, U, manganese group elements: Mn, Re, noble metals: Cu, Ag, Au, platinum group elements: Ru, Rh, Pd, Os, Ir, Pt, etc. Examples of metal-containing compounds or metalloid-containing compounds include ceramics, which are sintered bodies whose basic component is a metal oxide and which are hardened by heat treatment at high temperatures; semiconductors such as silicon; inorganic solid materials such as molded bodies of inorganic compounds such as metal oxides or metalloid oxides (silicon oxide, alumina, etc.); metal carbides or metalloid carbides; metal nitrides or metalloid nitrides (silicon nitride, etc.); and metal borides or metalloid borides; aluminum, nickel titanium, and stainless steel (SUS304, SUS316, SUS316L, etc.).

[0047] Resins that can be used as the material for the raw substrate 82 may be natural resins or derivatives thereof, or synthetic resins. Examples of natural resins or derivatives thereof include cellulose, cellulose triacetate (CTA), nitrocellulose (NC), and cellulose with dextran sulfate fixed thereto. Examples of synthetic resins include polyacrylonitrile (PAN), polyimide (PI), polyester polymer alloy (PEPA), polystyrene (PS), polysulfone (PSF), polyethylene terephthalate (PET), polymethyl methacrylate (PMMA), and polyvinyl alcohol. Preferably used are polyvinyl alcohol (PVA), polyurethane (PU), ethylene vinyl alcohol (EVAL), polyethylene (PE), polyester, polypropylene (PP), polyvinylidene fluoride (PVDF), polyethersulfone (PES), polycarbonate (PC), cycloolefin polymer (COP), polyvinyl chloride (PVC), polytetrafluoroethylene (PTFE), ultra-high molecular weight polyethylene (UHPE), polydimethylsiloxane (PDMS), acrylonitrile-butadiene-styrene resin (ABS), or Teflon (registered trademark).

[0048] In the production of the cell structure 1 using the cell structure production apparatus 10 of this embodiment, no high-temperature treatment is required when forming the base film 90. Therefore, resins with low heat resistance can also be used as the raw material substrate 82.

[0049] The material of the raw material substrate 82 may be one type or a combination of two or more types. In this embodiment, for example, in order to mass-produce the cell structure 1, the material of the raw material substrate 82 may be a material that is flexible enough to be wound up like a belt conveyor (roll method). Materials for the substrate 80 used in the roll method include synthetic resins and natural polymers.

[0050] Furthermore, the raw material substrate 82 may be a substrate 80 used in a so-called cell culture vessel. Examples of such substrates include Petri dishes or dishes, such as a Petri dish, a tissue culture dish, or a multi-dish, flasks, such as a cell culture flask, a spinner flask, or a multi-tier flask, bags, such as a plastic bag, a Teflon (registered trademark) bag, or a culture bag, plates, such as a microplate, a microwell plate, a multi-plate, or a multi-well plate, chamber slides, tubes, trays, and bottles, such as roller bottles, which are generally used for culturing cells 56.

[0051] 1 , the substrate supply unit 20 can have a coating film forming unit 30. The coating film forming unit 30 is configured to form a coating film 84 having the ability to inhibit adhesion of cells 56 on at least a portion of at least one surface of a raw material substrate 82.

[0052] Raw material substrate 82 generally does not have the ability to inhibit adhesion of cells 56. Therefore, by forming coating film 84 having the ability to inhibit adhesion of cells 56 on at least one surface of raw material substrate 82 in coating film forming section 30, substrate 80 having the ability to inhibit adhesion can be obtained.

[0053] The coating film forming unit 30 includes a coating film forming application mechanism 32. The application method used by the coating film forming application mechanism 32 can be selected from, for example, spin coating, inkjet printing, screen printing, flexographic printing, gravure printing, offset printing, bar coating, slit coating, roll-to-roll printing, dip coating, solvent casting, pad printing, and spraying. The coating film forming application mechanism 32 can apply a coating film forming composition 38 to at least a portion of the surface of the substrate 80 in a predetermined pattern. The coating film forming application mechanism 32 can also apply the coating film forming composition 38 to the entire surface of the raw material substrate 82. The coating film forming composition 38 can also be applied in a predetermined pattern so that the application of the base film 90 described below and the formation of a cell structure 1 of predetermined dimensions can be properly performed. The dimensions of the base film 90 are important for forming a cell structure 1 of predetermined dimensions. In order to obtain flexibility in the dimensions of the base film 90, it is preferable that the coating film forming application mechanism 32 applies the coating film forming composition 38 to the entire surface of the raw material substrate 82. However, if the areas where the formation of the coating film 84 is not required are predetermined, the coating film can be applied in a predetermined pattern so as not to form the coating film 84 in the unnecessary areas. This allows the amount of the coating film forming composition 38 used to be reduced.

[0054] In the cell structure manufacturing apparatus 10 of this embodiment, the coating film forming application mechanism 32 is preferably a pointillism application mechanism, for example, an inkjet method using an inkjet printer. By using a pointillism application mechanism to apply the coating film forming composition 38, the coating film forming composition 38 can be applied in a predetermined pattern shape. Furthermore, by controlling the nozzle scanning speed of the pointillism application mechanism and / or the number of repeated pointillism applications, the thickness of the coating film 84 can be adjusted to a desired thickness.

[0055] In the example shown in Figure 1, in the coating film forming unit 30, a coating film forming composition 38 is supplied from a coating film forming composition tank 34 to a coating film forming application mechanism 32 (e.g., an inkjet printer). The coating film forming composition 38 is ejected from the coating film forming application mechanism 32 onto the entire surface of the substrate 80 (or in a predetermined pattern shape). Note that the coating film forming application mechanism 32 can have a nozzle driving function so that the nozzle of the coating film forming application mechanism 32 can perform a predetermined movement.

[0056] Specific examples of the coating film-forming composition 38 will be described later.

[0057] <Coating film drying mechanism 36> The cell structure manufacturing apparatus 10 of this embodiment may optionally include a coating film drying mechanism 36. When the coating film forming composition 38 contains a solvent, after application of the coating film forming composition 38, the solvent can be evaporated by the coating film drying mechanism 36 to form a coating film 84 of the coating film forming composition 38. The coating film drying mechanism 36 may be, for example, a heater and / or a blower.

[0058] Drying can be carried out, for example, in the atmosphere or under vacuum at a temperature within a range of −200° C. to 200° C. The coating film 84 can also be formed by drying at, for example, room temperature (10° C. to 35° C., e.g., 25° C.). To form the coating film 84 more quickly, drying may be carried out at, for example, 40° C. to 100° C. The drying temperature is not particularly limited. The drying temperature is preferably lower than the glass transition point of the raw material substrate 82, for example, 10° C. to 180° C., and more preferably, 20° C. to 100° C. The drying time is not particularly limited, but is, for example, 1 minute to 24 hours.

[0059] <Base film forming section 40> As shown in Figure 1, the cell structure manufacturing apparatus 10 of this embodiment has a base film forming section 40 for forming a base film 90. The base film forming section 40 includes a base film forming application mechanism 42 that applies a base film forming composition 48 onto the substrate 80. The base film 90 has cell adhesive properties, so that cells 56 can be attached to the surface of the base film 90.

[0060] At least a portion of the surface of the substrate 80 has the ability to inhibit adhesion of cells 56. A base film 90 can be formed on the surface having the ability to inhibit adhesion of cells 56 so that a portion of the surface having the ability to inhibit adhesion of cells 56 is exposed. This makes it possible to repel the cells 56 from the surface of the substrate 80 having the ability to inhibit adhesion of cells 56, and to make the cells 56 gather in the portion where the base film 90 is formed. As a result, the cell structure 1 can be manufactured.

[0061] The base film forming unit 40 includes a base film forming coating mechanism 42. The coating method used by the base film forming coating mechanism 42 can be selected from, for example, spin coating, ink jet coating, screen printing, slit coating, roll-to-roll coating, dip coating, solvent casting, pad printing, and spraying. The base film forming coating mechanism 42 can apply a base film forming composition 48 to at least a portion of the surface of the substrate 80 (or coating film 84) so ​​as to form a predetermined pattern. In this specification, the pattern of a predetermined shape formed by applying the base film forming composition 48 is referred to as an "base film pattern 90a."

[0062] In the cell structure manufacturing apparatus 10 of this embodiment, the base film forming coating mechanism is preferably a pointillism coating mechanism, for example, an inkjet method using an inkjet printer. By using a pointillism coating mechanism, or a line or area coating mechanism using a series of pointillism coatings to apply the base film forming composition 48, the base film forming composition 48 can be applied so as to form a predetermined base film pattern 90a. Furthermore, by controlling the nozzle scanning speed of the pointillism coating mechanism and / or the number of repeated pointillism applications, the film thickness of the base film pattern 90a can be adjusted to a desired thickness.

[0063] 1 , in the coating mechanism 42 for forming a base film, a base film forming composition 48 is supplied from a base film forming composition tank 44 to the coating mechanism 42 for forming a base film (e.g., an inkjet printer). The base film forming composition 48 is ejected from the coating mechanism 42 for forming a base film so as to form a base film pattern 90a having a predetermined shape. Note that the coating mechanism 42 for forming a base film may have a nozzle driving function so that the nozzle of the coating mechanism 42 for forming a base film can perform a predetermined movement.

[0064] Another application method involves, for example, immersing the substrate 80, optionally protecting the areas where the base film pattern 90a is not formed, in the base film-forming composition 48. Alternatively, the base film-forming composition 48 can be added to the substrate 80 (container), optionally protecting the areas where the base film pattern 90a is not formed, and then allowed to stand for a predetermined period of time. When the substrate 80 is a cell culture vessel, the base film-forming composition 48 can be added to the container, optionally protecting the areas where the base film pattern 90a is not formed, and then allowed to stand for a predetermined period of time. Addition can be performed, for example, by adding the base film-forming composition 48 in an amount 0.5 to 1 times the total volume of the container using a syringe or the like. The time and temperature for the standing can be appropriately selected depending on the material of the vessel or substrate 80 and the type of base film-forming agent for cell culture. For example, the standing time can be 1 minute to 24 hours, preferably 5 minutes to 3 hours, and the standing temperature can be 10 to 80°C. This allows the base film pattern 90 a to be properly formed on the substrate 80 .

[0065] The area to which the base film forming composition 48 is applied is important for forming a cell structure 1 of predetermined dimensions. The present inventors have found that when the cell structure 1 is a spheroid, there is a strong linear correlation between the area of ​​the base film pattern 90a and the diameter of the spheroid.

[0066] The shape of the pattern (undercoat film pattern 90a) of the undercoat film-forming composition 48 is arbitrary. The shape of the undercoat film pattern 90a may be a circle, a polygon such as a triangle or a rectangle, a star, a cross, or the like. In order to facilitate the cells 56 to assemble into the cell structure 1, the undercoat film pattern 90a is preferably a circle (dot pattern or spot).

[0067] When the base film pattern 90a of the base film-forming composition 48 is a circular dot pattern (spot), the ratio of the total area of ​​the dot patterns of the base film 90, the diameter of each dot pattern, and the spacing between dot patterns can be appropriately selected from a predetermined range depending on the type of cells 56 and substrate 80 used, the desired size of the cell aggregates, etc. The ratio of the total area of ​​the dot patterns of the base film 90 to the surface area of ​​the substrate 80 is preferably 30% or more, 40% or more, or 50% or more, and preferably 99% or less. The diameter of each dot pattern of the base film 90 may be, for example, 50 to 5,000 μm, or in some cases, 300 to 3,000 μm. The spacing between the centers of each dot pattern of the base film 90 may be, for example, 100 to 6,000 μm, or, as needed, 150 to 4,000 μm or 150 to 300 μm.

[0068] In this embodiment, by arranging independent micro-sized regions (dot patterns) to which cells 56 can adhere at high density, preferably in a regular pattern, on a substrate 80 having the ability to inhibit adhesion of cells 56, multiple spheroids of uniform size can be formed at once on a single substrate 80 (container).

[0069] The film thickness of the undercoat film 90 is, for example, 1 to 1000 nm, preferably 5 to 500 nm, preferably 5 to 300 nm, preferably 5 to 200 nm, preferably 5 to 150 nm, preferably 10 to 150 nm.

[0070] Furthermore, the base film pattern 90a on the surface of the substrate 80 obtained by the above-mentioned method can be used as a substrate 80 with a base film 90 for producing a cell structure 1, either as is without undergoing a drying process, or after washing with water or the medium of the sample to be subjected to cell culture (e.g., water, buffer solution, culture medium, etc.).

[0071] That is, after the formation of the base film pattern 90a on the surface of the substrate 80, the substrate 80 can be used as it is without a drying step within 48 hours, preferably within 24 hours, more preferably within 12 hours, even more preferably within 6 hours, even more preferably within 3 hours, and even more preferably within 1 hour, as a substrate 80 with a base film 90 for producing a cell structure 1, or after washing with water or a medium for the sample to be subjected to cell culture (e.g., water, a buffer solution, a culture medium, etc., particularly preferably a culture medium (e.g., DMEM medium (Dulbecco's modified Eagle's medium))).

[0072] Specific examples of the base film-forming composition 48 will be described later.

[0073] <Base film drying mechanism 46> The cell structure manufacturing apparatus 10 of this embodiment may, if necessary, include a base film drying mechanism 46. When the base film forming composition 48 contains a solvent, after the base film pattern 90a is formed, the solvent can be vaporized by the base film drying mechanism 46. The base film drying mechanism 46 may be, for example, a heater and / or a blower.

[0074] The base film 90 can be dried under the same conditions as those for drying the coating film 84 described above.

[0075] Specifically, the drying step of the substrate 80 having the undercoat film 90 can be carried out in the atmosphere or under vacuum, preferably at a temperature range of −200° C. to 200° C. The drying step removes the solvent in the undercoat film-forming composition 48, thereby allowing the composition to be completely fixed to the surface of the substrate 80 or the coating film 84.

[0076] The base film pattern 90a can also be formed by drying at room temperature (10°C to 35°C, preferably 20°C to 30°C, e.g., 25°C). To form spots more quickly, drying at, for example, 40°C to 80°C may be performed. Drying temperatures below -200°C require the use of an uncommon refrigerant, resulting in a lack of versatility, and drying takes a long time due to solvent sublimation, resulting in poor efficiency. Drying temperatures above 200°C result in thermal decomposition of the polymer. Furthermore, drying temperatures below the glass transition point of the source substrate 82 are preferred, with 10°C to 180°C being more preferred, and 20°C to 100°C being even more preferred. The substrate 80 with the base film pattern 90a for cell structure production of this embodiment is manufactured through the above-described simple process.

[0077] Furthermore, to remove impurities, unbonded polymers, and the like remaining on the base film pattern 90a, a washing step (washing step) may be performed in which the base film pattern 90a is washed with at least one solvent selected from water and an aqueous solution containing an electrolyte. Washing with running water or ultrasonic cleaning is preferred. The aqueous solution containing water and an electrolyte may be heated, for example, to a temperature ranging from 40°C to 95°C. Preferred aqueous solutions containing electrolytes include PBS, saline (containing only sodium chloride), Dulbecco's phosphate-buffered saline, Tris-buffered saline, HEPES-buffered saline, and Veronal-buffered saline, with PBS being particularly preferred. After bonding, the coating film 84 remains firmly bonded to the substrate without elution even when washed with water, PBS, alcohol, or the like.

[0078] The maximum and minimum thicknesses of the base film 90 (or base film pattern 90a) of this embodiment are in the range of 1 to 1000 nm, preferably 5 to 500 nm.

[0079] 1, the cell structure manufacturing apparatus 10 of this embodiment has a seeding unit 50 for seeding cells 56 on a substrate 80 including a base film 90. The seeding unit 50 includes a cell seeding mechanism 52 for seeding cells 56 on the base film 90.

[0080] As shown in FIG. 2 , a coating film 84 capable of inhibiting cell adhesion 56 is formed on the surface of the substrate 80 in the areas where the base film pattern 90a is not formed, or the surface of the substrate 80 having the ability to inhibit cell adhesion 56 is exposed. By seeding cells 56 on the base film pattern 90a having cell adhesive properties, the cells 56 gather on the base film pattern 90a, thereby obtaining a cell structure 1. The cells 56 can also be seeded on the portion of the coating film 84 (or substrate 80) adjacent to the base film pattern 90a. Because the surface of the coating film 84 (or substrate 80) has the ability to inhibit cell adhesion 56, the cells 56 seeded on the portion of the coating film 84 (or substrate 80) can gather on the adjacent base film pattern 90a and become part of the cell structure 1.

[0081] The seeding unit 50 includes a cell seeding mechanism 52. The cell seeding mechanism 52 can be selected from devices such as a device including a drop-type nozzle, a pointillist application mechanism such as printing by an inkjet printer, a screen printing device, a pad printing device, and a spray device. In order to prevent damage to the cells 56, the cell seeding mechanism 52 is preferably a device including a drop-type nozzle. The cell seeding mechanism 52 can seed the cells 56 onto the base film pattern 90a (and portions of the coating film 84 or substrate 80 adjacent to the base film pattern 90a).

[0082] In the example shown in FIG. 1 , cells 56 are supplied from a cell tank 54 to a cell seeding mechanism 52 (e.g., a device including a drip-type nozzle) in the seeding unit 50. The cells 56 are dripped from the cell seeding mechanism 52 onto the base film pattern 90a (and onto the coating film 84 or the portion of the substrate 80 that covers the base film pattern 90a). Note that the cell seeding mechanism 52 may have a nozzle driving function so that the nozzle of the cell seeding mechanism 52 can perform a predetermined movement.

[0083] The seeded cells 56 gather over a predetermined time and can form a cell structure 1 of a predetermined shape, such as a spheroid, on the base film pattern 90a.

[0084] The cell 56 in this embodiment is the most basic unit constituting an animal or plant, and has as its elements a cytoplasm and various organelles inside the cell membrane. In this case, the nucleus containing DNA may or may not be contained inside the cell 56. For example, the animal-derived cell 56 in this embodiment includes germ cells such as sperm and eggs, somatic cells that constitute an organism, stem cells (such as pluripotent stem cells), progenitor cells, cancer cells isolated from an organism, cells isolated from an organism that have acquired immortalization and are stably maintained outside the body (cell lines), cells isolated from an organism that have been artificially genetically modified, and cells isolated from an organism that have had their nuclei artificially replaced. Examples of somatic cells that make up a living organism include, but are not limited to, fibroblasts, bone marrow cells, B lymphocytes, T lymphocytes, neutrophils, erythrocytes, platelets, macrophages, monocytes, osteocytes, bone marrow cells, pericytes, dendritic cells, keratinocytes, adipocytes, mesenchymal cells, epithelial cells, epidermal cells, endothelial cells, vascular endothelial cells, hepatic parenchymal cells, chondrocytes, cumulus cells, nervous system cells, glial cells, neurons, oligodendrocytes, microglia, astrocytes, cardiac cells, esophageal cells, muscle cells (e.g., smooth muscle cells or skeletal muscle cells), pancreatic beta cells, melanocytes, hematopoietic progenitor cells (e.g., CD34-positive cells derived from umbilical cord blood), and mononuclear cells. The somatic cells include cells collected from any tissue, such as skin, kidney, spleen, adrenal gland, liver, lung, ovary, pancreas, uterus, stomach, colon, small intestine, large intestine, bladder, prostate, testis, thymus, muscle, connective tissue, bone, cartilage, vascular tissue, blood (including umbilical cord blood), bone marrow, heart, cardiac muscle, eye, brain, or neural tissue. Furthermore, the somatic cells include cells induced to differentiate from stem cells or progenitor cells.

[0085] Stem cells are cells that have the ability to replicate themselves and differentiate into cells of multiple 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, hepatic stem cells, pancreatic stem cells, muscle stem cells, germ stem cells, intestinal stem cells, cancer stem cells, and hair follicle stem cells. Among the stem cells, 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 germ cells. Cancer cells are cells that are derived from somatic cells and have acquired the ability to proliferate indefinitely. Cell lines are cells that have acquired the ability to proliferate indefinitely through artificial manipulation outside of the body. Among these, fibroblasts and stem cells are more preferred as the cells 56 in this embodiment. Furthermore, among stem cells, pluripotent stem cells are more preferred.

[0086] 1, the cell structure manufacturing apparatus 10 of this embodiment may further include, as necessary, an aggregation culture section 60 for aggregating and / or culturing cells 56 attached on a substrate 80 including a base film 90. The cells 56 aggregate to form a desired cell structure 1.

[0087] Although not shown in FIG. 1 , the aggregation culture section 60 preferably contains a predetermined culture medium. By containing a predetermined culture medium in the aggregation culture section 60, the cells 56 can be cultured in the culture medium. Any known culture medium can be used as the culture medium for the cells 56. By including the aggregation culture section 60 in the cell structure manufacturing apparatus 10 of this embodiment, the seeded cells 56 can form the cell structure 1 in a stable environment. The aggregation culture section 60 can be a culture medium tank containing a culture medium. In the cell structure manufacturing apparatus 10 of this embodiment, the direction of travel of the flexible substrate 80 seeded with the cells 56 can be changed to downward or obliquely downward, and the substrate 80 can be configured to be immersed in a culture medium tank containing a culture medium.

[0088] <Cell structure collection unit 70> As shown in Figure 1, the cell structure manufacturing device 10 of this embodiment can further include a cell structure collection unit 70 for collecting the obtained cell structure 1, if necessary. The cell structure 1 is detached from the substrate 80 including the base film 90 and collected by the cell structure collection mechanism 72 of the cell structure collection unit 70. Note that the detachment of the cell structure 1 from the substrate 80 is preferably carried out in a solution that is harmless to the cell structure 1, such as a culture medium.

[0089] Depending on the use of the cell structure 1, the cell structure manufacturing device 10 may not have a cell structure collection section 70, and the cell structure 1 may be placed on a substrate 80 including a base film 90 to produce the cell structure 1 as a product.

[0090] <Airtight Mechanism> The cell structure manufacturing apparatus 10 of this embodiment preferably has an airtight mechanism that can make the inside of the cell structure manufacturing apparatus 10 an airtight closed space. In this case, the airtight mechanism of the cell structure manufacturing apparatus 10 can maintain the inside of the closed space in a sterile environment.

[0091] In the cell structure manufacturing apparatus 10 of this embodiment, when the coating film 84 and / or the base film 90 are formed while the apparatus is open to the outside air, there is a possibility that unexpected germs may be introduced from the outside air. In order to prevent the proliferation of germs, when the cell structure manufacturing apparatus 10 is open to the outside air, it becomes necessary to add a sterilization step and / or a cleaning step as appropriate to the manufacturing process of the cell structure 1. On the other hand, the cell structure manufacturing apparatus 10 is configured to maintain a sterile environment inside the closed space using a predetermined airtight mechanism, thereby making the cleaning and sterilization steps unnecessary.

[0092] <Cleaning Process and Sterilization Process> If the cell structure manufacturing apparatus 10 does not have an airtight mechanism, contamination by various bacteria and the like may occur during each process. In such a case, it is preferable to perform a cleaning process and / or a sterilization process using a cleaning device after each process is completed, for example, after removing the raw material substrate 82 from the raw material substrate cassette 22, after forming the coating film 84, and / or after forming the base film 90. By performing the cleaning process after forming the coating film 84 and / or base film 90, excess film can be removed from the formed coating film 84 and / or base film 90. Furthermore, by performing the sterilization process, adhering various bacteria can be removed.

[0093] The cleaning step is not particularly limited as long as it is a step for cleaning the coating film 84 and / or the undercoat film 90. The cleaning step is carried out, for example, to remove impurities, unreacted monomers, and the like remaining in the coating film 84 and / or the undercoat film 90 from the coating film 84.

[0094] Washing can be performed by a known method. Examples of solvents used for washing include water and aqueous solutions containing electrolytes. The solvent is usually used at room temperature (e.g., 10 to 35°C). The solvent may be heated to a temperature in the range of 40 to 95°C, for example. Preferred aqueous solutions containing electrolytes are PBS, saline (containing only sodium chloride), Dulbecco's phosphate buffered saline, Tris-buffered saline, HEPES-buffered saline, and Veronal-buffered saline, with PBS being particularly preferred. The coating film 84 of this embodiment remains firmly attached to the substrate without elution even when washed with water, PBS, alcohol, etc.

[0095] The coating film 84 and / or the base film 90 may be washed with a medium (e.g., water, a buffer solution, a culture medium, etc.) of the sample used in cell culture. A preferred medium is a culture medium, and more preferred media are BME medium (Eagle's Basal Medium) and DMEM medium (Dulbecco's Modified Eagle's Medium).

[0096] The coating film 84 and / or the base film 90 preferably undergo a sterilization process by irradiation. The sterilization process is usually carried out at ambient temperature (e.g., about 0°C to about 40°C, preferably about 10°C to about 30°C, more preferably about 25°C). The radiation to be irradiated is not limited as long as it can perform sterilization, but gamma rays, X-rays, or electron beam irradiation are preferred. Gamma rays or electron beams are more preferred, and gamma rays are even more preferred. The dose of gamma rays may be, for example, a dose employed in a typical sterilization process; for example, irradiation of about 5 to 40 kGy is sufficient, and preferably 10 to 25 kGy.

[0097] <Dimensional control of cell structure 1> In the cell structure manufacturing apparatus 10 of this embodiment, the size error (dimensional error) of the cell structure 1 is preferably within 20%, more preferably within 15%, and even more preferably within 10%.

[0098] The size error of the cell structure 1 means the value obtained by dividing the standard deviation of the dimensions when 100 or more cell structures 1 are manufactured by the average dimension. If the cell structure manufacturing apparatus 10 of this embodiment is used, the base film pattern 90a can be formed with predetermined dimensions, making it easy to control the dimensions of the cell structure 1. Therefore, when the cell structure 1 is manufactured using the cell structure manufacturing apparatus 10 of this embodiment, the size error of the cell structure 1 can be reduced compared to conventional methods.

[0099] When the cell structure 1 is a spherical spheroid, if the diameter of the spheroid is too large, some of the cells 56 in the spherical spheroid may die. Furthermore, if the diameter of the spheroid is too small, the therapeutic effect of the spheroid may be reduced. Therefore, by appropriately controlling the dimensions of the cell structure 1, such as a spheroid, the yield of the cell structure 1 can be improved. Therefore, the mass productivity of the cell structure 1 can be improved.

[0100] <Batch-type cell structure manufacturing apparatus 10> Figure 3 shows a schematic plan view of another aspect of the cell structure manufacturing apparatus 10 of this embodiment. The cell structure manufacturing apparatus 10 shown in Figure 3 is a schematic plan view showing the layout of each part when a batch-type manufacturing method is adopted.

[0101] In the cell structure manufacturing apparatus 10 shown in FIG. 3 , the shape of the source substrate 82 (or substrate 80) is a rectangle of predetermined dimensions. The source substrate 82 (or substrate 80) is placed on the substrate supply unit 20. The source substrate 82 is removed from the substrate supply unit 20 by a moving mechanism 12, such as a robot. If the orientation of the source substrate 82 (or substrate 80) needs to be changed, it is rotated by the rotation mechanism 14. As described in the embodiment shown in FIG. 1 above, the source substrate 82 (or substrate 80) undergoes predetermined processing in the coating film forming unit 30 and the base film forming unit 40, and is then seeded in the seeding unit 50. After being cultured in the aggregation culture unit 60 as necessary, the cell structure 1 is collected in the cell structure collection unit 70. At this time, the cell structure 1 can be collected while still placed on the substrate 80.

[0102] <Method for manufacturing cell structure 1> The method for manufacturing cell structure 1 using cell structure manufacturing apparatus 10 of this embodiment includes at least a step of seeding cells 56 onto a substrate 80 on which a base film 90 (base film pattern 90a) is arranged in the seeding section 50. The method for manufacturing cell structure 1 may further include other steps as necessary.

[0103] For example, by seeding cells 56 on a coating film 84 on which a base film 90 of a predetermined size and shape has been arranged, the cell adhesion-inhibiting function of the coating film 84 inhibits the adhesion, spreading, and proliferation of the cells 56. As a result, a good cell structure 1 (for example, a spheroid such as a three-dimensional cell aggregate) can be obtained.

[0104] For example, the substrate 80 may have a cell-seeding surface that includes a coating film 84 capable of inhibiting cell adhesion and a dot-patterned base film 90 (base film pattern 90a) having cell adhesive properties and disposed on the coating film 84. When cells 56 are seeded on such a cell-seeding surface, the cells 56 are selectively cultured on the dot-patterned base film pattern 90a. As a result, a cell structure 1 (e.g., a spheroid such as a three-dimensional cell aggregate) of a predetermined size can be obtained.

[0105] <Coating film-forming composition 38> Next, a coating film-forming composition 38 that can be used in the cell structure manufacturing apparatus 10 of this embodiment will be described. In this embodiment, it is preferable to use the following coating film-forming composition 38 (sometimes referred to as the "coating film-forming composition of this embodiment"). By using the coating film-forming composition of this embodiment, it is possible to form a coating film 84 that is capable of inhibiting adhesion of biological materials and is difficult to dissolve in phosphate-buffered saline, and a coating film 84 that is capable of inhibiting adhesion of biological materials and is difficult to dissolve in phosphate-buffered saline.

[0106] The composition for forming a coating film of the present embodiment preferably contains a copolymer having a repeating unit (A) represented by the following formula (A) and a repeating unit (B) represented by the following formula (B).

[0107] (In the formula, R １ ~R ３ each independently represents a hydrogen atom or an alkyl group having 1 to 5 carbon atoms; X １ and X ２ each independently represents a single bond, an ester bond, an ether bond, an amide bond, or an alkylene group having 1 to 5 carbon atoms which may be interrupted by an oxygen atom.

[0108] More specifically, the coating film-forming composition of this embodiment is as follows.

[0109] [1] A coating film-forming composition containing a copolymer and used to inhibit adhesion of a biological material, wherein the copolymer is water-insoluble, the copolymer has a repeating unit (A) represented by the above formula (A) and a repeating unit (B) represented by the above formula (B), and the molar ratio (A:B) of the repeating unit (A) to the repeating unit (B) in the copolymer is 89:11 to 50:50.

[0110] [2] R １ and R ２ is a hydrogen atom, and R ３ is a methyl group, and X １ and X２ The composition for forming a coating film according to [1], wherein

[0111] [3] The composition for forming a coating film according to [1] or [2], wherein the viscosity-average degree of polymerization of the copolymer is 200 to 3,000.

[0112] [4] The composition for forming a coating film according to any one of [1] to [3], wherein the total mol % of the repeating unit (A) and the repeating unit (B) in all repeating units in the copolymer is 99.5 mol % or more.

[0113] [5] The composition for forming a coating film according to any one of [1] to [4], wherein the copolymer does not have an azide group.

[0114] [6] The composition for forming a coating film according to any one of [1] to [5], which contains a solvent.

[0115] [7] The composition for forming a coating film according to [6], wherein the solvent contains an alcohol.

[0116] According to the present embodiment, it is possible to provide a coating film that can inhibit adhesion of biological materials and is difficult to dissolve in phosphate-buffered saline, and a composition for forming a coating film that can form a coating film that can inhibit adhesion of biological materials and is difficult to dissolve in phosphate-buffered saline.

[0117] The coating film-forming composition of this embodiment is used to inhibit adhesion of biological materials. The coating film-forming composition contains at least a copolymer, and further contains other components such as a solvent, as necessary. The coating film-forming composition of this embodiment is capable of forming a coating film that is difficult to dissolve in phosphate-buffered saline. The use of the coating film-forming composition of this embodiment is not particularly limited as long as it is used to inhibit adhesion of biological materials, and is not limited to the formation of a coating film that comes into contact with phosphate-buffered saline.

[0118] <<Copolymer>> The copolymer is water-insoluble. Here, "water-soluble" means that 1.0 g or more of the copolymer can be dissolved in 100 g of water at 25° C. "Water-insoluble" means that the copolymer does not fall under the category of "water-soluble," i.e., the copolymer has a solubility of less than 1.0 g in 100 g of water at 25° C.

[0119] The copolymer 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 the repeating unit (A) to the repeating unit (B) in the copolymer is 89:11 to 50:50. (In the formula, R １ ~R ３ each independently represents a hydrogen atom or an alkyl group having 1 to 5 carbon atoms; X １ and X ２ each independently represents a single bond, an ester bond, an ether bond, an amide bond, or an alkylene group having 1 to 5 carbon atoms which may be interrupted by an oxygen atom.

[0120] The copolymer may have two or more types of repeating units (A). The copolymer may have two or more types of repeating units (B). The copolymer preferably has one type of repeating unit (A) and one type of repeating unit (B).

[0121] Examples of alkyl groups having 1 to 5 carbon atoms include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, s-butyl, t-butyl, n-pentyl, 1-methylbutyl, 2-methylbutyl, 3-methylbutyl, 1,1-dimethylpropyl, 1,2-dimethylpropyl, 2,2-dimethylpropyl, and 1-ethylpropyl groups. １ ~R ３ are each independently preferably a hydrogen atom, a methyl group, or an ethyl group.

[0122] The above "ester bond" means -C(=O)-O- or -O-C(=O)-, an "ether bond" means -O-, and an "amide bond" means -NHC(=O)- or -C(=O)NH-.

[0123] The alkylene group having 1 to 5 carbon atoms may be interrupted by an oxygen atom. Examples of the alkylene group having 1 to 5 carbon atoms include a methylene group, an ethylene group, a propylene group, a trimethylene group, a tetramethylene group, a 1-methylpropylene group, a 2-methylpropylene group, a dimethylethylene group, an ethylethylene group, a pentamethylene group, a 1-methyl-tetramethylene group, a 2-methyl-tetramethylene group, a 1,1-dimethyl-trimethylene group, a 1,2-dimethyl-trimethylene group, a 2,2-dimethyl-trimethylene group, and a 1-ethyl-trimethylene group. １ and X ２ is preferably a methylene group, an ethylene group, or a propylene group. The phrase "optionally interrupted by an oxygen atom" means that one or more carbon-carbon bonds in the alkylene group having 1 to 5 carbon atoms are bonded via an ether bond.

[0124] The copolymer may be, for example, R １ and R ２ is a hydrogen atom, and R ３ is a methyl group, and X １ and X ２ A copolymer in which is a single bond is preferred.

[0125] The molar ratio (A:B) of the repeating unit (A) to the repeating unit (B) is 89:11 to 50:50. When the total number of moles of the repeating unit (A) and the repeating unit (B) in the copolymer is 100, the molar ratio (A:B) of the repeating unit (A) to the repeating unit (B) can be expressed as (100-m):m. In this case, the range of m is 11 to 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 to 49, 12 to 48, 15 to 48, 20 to 49, 20 to 45, 22 to 49, or 22 to 45.

[0126] The total mol% of the repeating units (A) and (B) in all repeating units in the copolymer is not particularly limited, but is preferably 90 mol% or more, more preferably 95 mol% or more, even more preferably 99.5 mol% or more, and particularly preferably 100%.

[0127] In this embodiment, in order to obtain a coating film that is difficult to dissolve in phosphate buffered saline, the molar ratio of the repeating unit (A) to the repeating unit (B) in the copolymer is set to a specific range. Therefore, in this embodiment, a coating film that is difficult to dissolve in phosphate buffered saline is obtained without crosslinking the copolymer. Therefore, the copolymer does not need to have a photosensitive group for crosslinking the copolymer. In other words, it is preferable that the copolymer does not have a photosensitive group. Examples of photosensitive groups include azide groups.

[0128] In this embodiment, the copolymer does not need to have a photosensitive group for crosslinking the copolymer. Therefore, when forming a coating film, it is not necessary to perform light irradiation for crosslinking the copolymer. Therefore, the process for forming the coating film can be simplified.

[0129] The viscosity-average degree of polymerization of the copolymer (hereinafter sometimes referred to as "degree of polymerization") is not particularly limited, but from the viewpoint of suitably obtaining the effects of the present embodiment, it is preferably 200 to 3,000, more preferably 200 to 2,500, and particularly preferably 200 to 2,000.

[0130] The viscosity average degree of polymerization is measured when the copolymer is in a fully saponified state. The "viscosity average degree of polymerization" of the polyvinyl alcohol obtained by fully saponifying is a value calculated from the intrinsic viscosity [η] (g / dL) measured at 30°C using an Ostwald viscometer with ion-exchanged water as a solvent, using the following formula: log(P) = 1.613 × log([η] × 10 ４ / 8.29)

[0131] In the above formula, P represents the viscosity-average degree of polymerization, which can be determined in accordance with JIS K 6726.

[0132] The method for producing the copolymer is not particularly limited, and examples thereof include a method in which a compound represented by the following formula (C) is polymerized to produce a homopolymer, and the obtained homopolymer is partially hydrolyzed by a known saponification reaction to obtain the copolymer. (In the formula, R １ , R ３ , and X １ has the same meaning as above.)

[0133] Furthermore, examples of a method for producing the copolymer include a method in which a compound represented by the following formula (C) and a compound represented by the following formula (D) are copolymerized to obtain the copolymer. (In the formula, R １ ~R ３ , X １ , and X ２ has the same meaning as above.)

[0134] The copolymer may be a random copolymer or a block copolymer. Commercially available copolymers may be used. Specific examples of commercially available copolymers include polyvinyl acetate (manufactured by Nippon Vinyl Acetate & Poval, trade name JMR-10L (registered trademark)).

[0135] The content of the copolymer in the film-forming components in the coating film-forming composition is not particularly limited, but is preferably 80% by mass or more, more preferably 90% by mass or more, and particularly preferably 95% by mass or more. The film-forming components refer to all components of the composition excluding the solvent component.

[0136] The content of the copolymer in the composition for forming a coating film is not particularly limited, but from the viewpoint of easily forming a coating film having a desired thickness, it is preferably 0.1 to 10 mass %, more preferably 0.1 to 8 mass %, and particularly preferably 0.1 to 5 mass %.

[0137] <<Solvent>> Examples of the solvent include water, phosphate buffered saline (PBS), alcohol, and water-soluble organic solvents (excluding alcohol).

[0138] 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-heptanol, 2-methyl-2-butanol (t-amyl alcohol), 2-methyl-3-pentanol, ... Examples of the alcohols include cyclohexanol, 2-hexanol, 3-hexanol, 2,3-dimethyl-2-butanol, 3,3-dimethyl-1-butanol, 3,3-dimethyl-2-butanol, 2-ethyl-1-butanol, 2-methyl-1-pentanol, 2-methyl-2-pentanol, 2-methyl-3-pentanol, 3-methyl-1-pentanol, 3-methyl-2-pentanol, 3-methyl-3-pentanol, 4-methyl-1-pentanol, 4-methyl-2-pentanol, 4-methyl-3-pentanol, and cyclohexanol. These may be used alone or in combination of two or more.

[0139] The water-soluble organic solvent refers to an organic solvent that can be mixed with water and alcohol in any ratio 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, 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.

[0140] In the composition for forming a coating film, water, phosphate buffered saline (PBS), alcohol, or a water-soluble organic solvent may be used alone as the solvent. In the composition for forming a coating film, two or more of water, phosphate buffered saline (PBS), alcohol, and a water-soluble organic solvent may be used in combination as the solvent. From the viewpoint of the solubility of the copolymer, the solvent is preferably selected from water, alcohol, a water-soluble organic solvent, and a combination of two or more thereof, and more preferably selected from water, ethanol, a water-soluble organic solvent, and a combination of two or more thereof.

[0141] The following combinations of solvents are preferred: Water and alcohol; Water, alcohol and a water-soluble organic solvent; Alcohol and a water-soluble organic solvent.

[0142] The following combinations of solvents are more preferred: Water and ethanol Water, ethanol and propylene glycol monomethyl ether Ethanol and propylene glycol monomethyl ether

[0143] The mixing ratio (mass ratio) of water:alcohol in the composition for forming a coating film is, for example, 1:99 to 70:30, or 1:99 to 50:50.

[0144] In the composition for forming a coating film, the mixing ratio (mass ratio (A:B:C)) of water:alcohol:water-soluble organic solvent is, for example, 5 to 30:65 to 92:1 to 30 (provided that A+B+C is 100).

[0145] In the composition for forming a coating film, the mixing ratio (mass ratio) of alcohol to water-soluble organic solvent is, for example, 1:99 to 97:3.

[0146] The content of the solvent in the composition for forming a coating film 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, more preferably 92% by mass or more, and particularly preferably 95% by mass or more.

[0147] <<Other Components>> The coating film-forming composition may contain other components as needed. Examples of the other components include a pH adjuster, a preservative, a surfactant, an anti-mold agent, and sugars.

[0148] Having the ability to inhibit adhesion of cells 56 means that adhesion and spreading of cells 56 are not observed under a microscope, and cell aggregates (spheroids) are formed.

[0149] Alternatively, having the ability to inhibit adhesion of cells 56 means that the luminescence intensity (%) (luminescence intensity of adherent cells on the coated film) / (luminescence intensity of adherent cells on a well without coating) compared to when no ATPassay coating is used is 50% or less, preferably 30% or less, and more preferably 10% or less.

[0150] <<Coating film 84>> The coating film 84 of the coating film-forming composition of the present embodiment (sometimes referred to as the "coating film of the present embodiment") is obtained by applying the above-mentioned coating film-forming composition of the present embodiment. In other words, the coating film of the present embodiment is an applied film of the coating film-forming composition of the present embodiment. The coating film of the present embodiment is used to inhibit adhesion of biological materials.

[0151] The coating film of this embodiment is characterized by being hardly soluble in phosphate buffered saline. The use of the coating film-forming composition of this embodiment is not particularly limited as long as it is used to inhibit adhesion of biological materials, and is not limited to uses that involve contact with phosphate buffered saline.

[0152] In the coating film of this embodiment, the molar ratio of the repeating unit (A) to the repeating unit (B) in the copolymer is set to a specific range in order to obtain a coating film that is difficult to dissolve in phosphate buffered saline. Therefore, in the coating film of this embodiment, a coating film that is difficult to dissolve in phosphate buffered saline is obtained without crosslinking the copolymer. Therefore, in the coating film of this embodiment, the copolymer does not need to be crosslinked. This also simplifies the process for forming the coating film of this embodiment.

[0153] The content of the copolymer in the coating film of the present embodiment is not particularly limited, but is preferably 80% by mass or more, more preferably 90% by mass or more, and particularly preferably 95% by mass or more.

[0154] <<Film Thickness of Coating Film>> The film thickness of the coating film of this embodiment is not particularly limited, but is, for example, 1 to 10,000 nm, preferably 5 to 1,000 nm, more preferably 10 to 500 nm, still more preferably 20 to 300 nm, even more preferably 50 to 250 nm, and particularly preferably 100 to 250 nm. Note that this film thickness can also be applied to other types of coating films.

[0155] <<Another embodiment of the coating film-forming composition 38>> As another embodiment of the coating film 84 of this embodiment, the following can be used.

[0156] As another embodiment of the coating film 84 of this embodiment, for example, a coating film-forming composition described in International Publication No. 2014 / 196650 can be used. The coating film-forming composition is a copolymer (P) containing 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): [In the formula, U ａ１１ , U ａ１２ , U ｂ１１ , U ｂ１２ and U ｂ１３ each independently represents a hydrogen atom or a linear or branched alkyl group having 1 to 5 carbon atoms; An － represents an anion selected from the group consisting of halide ions, inorganic acid ions, hydroxide ions, and isothiocyanate ions] and a solvent, and then drying the composition for forming a coating film. The coating film may be contained on at least a portion of the surface of the substrate, but is preferably applied over the entire surface on which cell aggregates are produced (i.e., the surface on which the spots of this embodiment are present) or over the entire surface of the substrate.

[0157] The entire disclosures of WO 2014 / 196650 and WO 2016 / 093293 are incorporated herein by reference.

[0158] Having the ability to inhibit adhesion of cells 56 means that adhesion and spreading of cells 56 are not observed under a microscope, and cell aggregates (spheroids) are formed. Alternatively, it means that the luminescence intensity (%) (luminescence intensity of adherent cells on the coated film) / (luminescence intensity of adherent cells on a well without coating) is 50% or less, preferably 30% or less, and more preferably 10% or less, when compared with the case without ATPassay coating.

[0159] Another coating film of this embodiment may be a copolymer of an ethylenically unsaturated monomer, or a polysaccharide or its derivative. Examples of the ethylenically unsaturated monomer include one or more ethylenically unsaturated monomers selected from the group consisting of (meth)acrylic acid and its esters, vinyl acetate, vinylpyrrolidone, ethylene, vinyl alcohol, and hydrophilic functional derivatives thereof. Examples of polysaccharides or their derivatives include cellulose-based polymers such as hydroxyalkyl cellulose (e.g., hydroxyethyl cellulose or hydroxypropyl cellulose), starch, dextran, and curdlan.

[0160] The hydrophilic functional derivative refers to an ethylenically unsaturated monomer having a hydrophilic functional group or structure. Examples of the hydrophilic functional group or structure include a betaine structure, an amide structure, an alkylene glycol residue, an amino group, and a sulfinyl group.

[0161] The betaine structure means a monovalent or divalent group of a compound having an amphoteric center of a quaternary ammonium type cation structure and an acidic anion structure, and is, for example, a phosphorylcholine group: Examples of ethylenically unsaturated monomers having such a structure include 2-methacryloyloxyethyl phosphorylcholine (MPC).

[0162] The amide structure has the following formula: [where R１６ , R １７ and R １８ are each independently a hydrogen atom or an organic group (for example, a methyl group, a hydroxymethyl group, or a hydroxyethyl group). Examples of ethylenically unsaturated monomers having such a structure include (meth)acrylamide, N-(hydroxymethyl)(meth)acrylamide, and N-isopropyl(meth)acrylamide. Furthermore, monomers or polymers having such a structure are disclosed, for example, in JP-A-2010-169604.

[0163] The alkylene glycol residue refers to an alkyleneoxy group (-Alk-O-) remaining after one or both terminal hydroxyl groups of alkylene glycol (HO-Alk-OH; where Alk is an alkylene group having 1 to 10 carbon atoms) undergo a condensation reaction with another compound, and also encompasses poly(alkyleneoxy) groups in which alkyleneoxy units are repeated. Examples of ethylenically unsaturated monomers having such a structure include 2-hydroxyethyl (meth)acrylate and methoxypolyethylene glycol (meth)acrylate. Furthermore, monomers or polymers having such a structure are disclosed, for example, in JP 2008-533489 A.

[0164] An amino group has the formula: -NH ２ , -NHR １９ or -NR ２０ R ２１ [where R １９ , R ２０ and R ２１ are each independently an organic group (for example, a linear or branched alkyl group having 1 to 5 carbon atoms). The amino group in the present invention includes quaternized or salified amino groups. Examples of ethylenically unsaturated monomers having such a structure include dimethylaminoethyl (meth)acrylate, 2-(t-butylamino)ethyl (meth)acrylate, and methacryloylcholine chloride.

[0165] The sulfinyl group has the following formula: [where R２２ is an organic group (for example, an organic group having 1 to 10 carbon atoms, preferably an alkyl group having 1 to 10 carbon atoms and one or more hydroxy groups). Examples of polymers having such a structure include copolymers disclosed in JP-A-2014-48278 and the like.

[0166] According to the coating film of this embodiment, it is possible to form a coating film that is capable of suppressing adhesion of biological materials and is difficult to dissolve in phosphate buffered saline, and a coating film that is capable of suppressing adhesion of biological materials and is difficult to dissolve in phosphate buffered saline. The coating film of this embodiment can be preferably used as the coating film 84 used in the cell structure manufacturing apparatus 10 of this embodiment.

[0167] <Base film forming composition 48> Next, a base film forming composition 48 that can be used in the cell structure manufacturing device 10 of this embodiment will be described. In this embodiment, it is preferable to use the base film forming composition 48 described below (sometimes referred to as the "base film forming composition of this embodiment"). By using the base film forming composition of this embodiment, uniform adhesion of cells 56 to the base film 90 can be achieved under serum-free culture conditions derived from animals, making it possible to manufacture high-quality cell structures 1. Thus, by using the base film forming composition of this embodiment, it is possible to achieve mass production of homogeneous, high-quality cell structures 1 for use in the field of regenerative medicine.

[0168] The composition for forming a base film of this embodiment contains a compound represented by the following formula (I): [wherein Ua1 and Ua2 each independently represent a hydrogen atom or a linear or branched alkyl group having 1 to 5 carbon atoms; Ra1 represents a hydrogen atom or a linear or branched alkyl group having 1 to 5 carbon atoms; and Ra2 represents a linear or branched alkylene group having 1 to 5 carbon atoms], and a repeating unit derived from a monomer represented by the following formula (II): It is preferable to include a copolymer containing a repeating unit derived from a monomer represented by the formula: [wherein Rb represents a hydrogen atom or a linear or branched alkyl group having 1 to 5 carbon atoms].

[0169] More specifically, the composition for forming an undercoat film according to this embodiment is as follows.

[0170] [1] The compound of the formula (I) [wherein U ａ１ and U ａ２ each independently represents a hydrogen atom or a linear or branched alkyl group having 1 to 5 carbon atoms; R a１ represents a hydrogen atom or a linear or branched alkyl group having 1 to 5 carbon atoms; R a２ represents a linear or branched alkylene group having 1 to 5 carbon atoms], a cell adhesive substance, and a solvent.

[0171] [2] The polymer further comprises a compound represented by the formula (II) [wherein R ｂ represents a hydrogen atom or a linear or branched alkyl group having 1 to 5 carbon atoms].

[0172] [3] The composition for forming an undercoat film according to [1] or [2], wherein the weight ratio of the polymer to the cell adhesive substance is 100:0.1 to 100:100.

[0173] [4] The composition for forming a base film according to any one of [1] to [3], wherein the cell adhesive substance contains a glycoprotein.

[0174] <<Base Film Forming Composition 48>> The base film forming composition of this embodiment is a composition represented by the following formula (I): [In the formula, U ａ１ and U ａ２ each independently represents a hydrogen atom or a linear or branched alkyl group having 1 to 5 carbon atoms; R a１ represents a hydrogen atom or a linear or branched alkyl group having 1 to 5 carbon atoms; R a２ represents a linear or branched alkylene group having 1 to 5 carbon atoms], a polymer containing a repeating unit derived from a monomer represented by the formula:

[0175] (Polymer) The polymer contained in the composition for forming a base film of this embodiment is a polymer containing a repeating unit derived from the monomer represented by the above formula (I). The polymer contains a repeating unit derived from the monomer represented by the above formula (I) and a repeating unit derived from the following formula (II): [In the formula, R ｂ represents a hydrogen atom or a linear or branched alkyl group having 1 to 5 carbon atoms].

[0176] In this specification, unless otherwise defined, examples of a "linear or branched alkyl group having 1 to 5 carbon atoms" include a methyl group, an ethyl group, an n-propyl group, an isopropyl group, an n-butyl group, an isobutyl group, an s-butyl group, a t-butyl group, an n-pentyl group, a 1-methylbutyl group, a 2-methylbutyl group, a 3-methylbutyl group, a 1,1-dimethylpropyl group, a 1,2-dimethylpropyl group, a 2,2-dimethylpropyl group, and a 1-ethylpropyl group.

[0177] R a１ and R ｂ are preferably each independently selected from a hydrogen atom and a methyl group. ａ１ and U ａ２ are each independently preferably selected from a hydrogen atom, a methyl group, an ethyl group, an n-propyl group, an isopropyl group, and an n-butyl group, more preferably a methyl group or an ethyl group, and most preferably a methyl group.

[0178] In this specification, unless otherwise defined, examples of a "linear or branched alkylene group having 1 to 5 carbon atoms" include a methylene group, an ethylene group, a propylene group, a trimethylene group, a tetramethylene group, a 1-methylpropylene group, a 2-methylpropylene group, a dimethylethylene group, an ethylethylene group, a pentamethylene group, a 1-methyl-tetramethylene group, a 2-methyl-tetramethylene group, a 1,1-dimethyl-trimethylene group, a 1,2-dimethyl-trimethylene group, a 2,2-dimethyl-trimethylene group, and a 1-ethyl-trimethylene group. a２ is preferably selected from an ethylene group and a propylene group.

[0179] Therefore, examples of the cationic monomer represented by formula (I) include 2-N,N-dimethylaminoethyl methacrylate and N,N-dimethylaminomethyl methacrylate, with 2-N,N-dimethylaminoethyl methacrylate being preferred. Examples of the anionic monomer represented by formula (II) include acrylic acid and methacrylic acid, with methacrylic acid being preferred.

[0180] The molar ratio of units derived from the monomer represented by formula (I) to units derived from the monomer represented by formula (II) in the polymer is 100 / 0 to 50 / 50, preferably 98 / 2 to 50 / 50, more preferably 98 / 2 to 60 / 40, and particularly preferably 98 / 2 to 70 / 30. When the molar ratio of formula (II) is 50 or less, a decrease in the adhesive strength of cells 56 due to the anionic nature of the polymer can be suppressed.

[0181] (Monomer Having Two or More Carbon-Carbon Unsaturated Bonds) The polymer may be a polymer obtained by polymerizing a monomer represented by formula (I) / formula (II) together with a monomer having two or more carbon-carbon unsaturated bonds. Specific examples of the monomer having two or more carbon-carbon unsaturated bonds include a monomer having two or more carbon-carbon double bonds, such as a polyfunctional acrylate compound, a polyfunctional acrylamide compound, a polyfunctional polyester, or an isoprene compound.

[0182] Preferred specific examples include monomers represented by the following formulas (III) to (V).

[0183] In the formula, R ｃ and R ｄ each independently represents a hydrogen atom or a linear or branched alkyl group having 1 to 5 carbon atoms; R e represents a linear or branched alkylene group having 1 to 5 carbon atoms, and n represents a number from 1 to 50. Among these, the monomer represented by formula (III) is preferred.

[0184] The molar ratio of the monomers represented by formulas (III) to (V) to the entire polymer is preferably 0 to 50%, more preferably 2 to 25%. When the molar ratio of formulas (III) to (V) is 50% or less, gelation of the solid content during production due to high molecular weight caused by excessive crosslinking can be suppressed, and production can be facilitated.

[0185] R ｃ and R ｄ are preferably each independently selected from a hydrogen atom and a methyl group.

[0186] R ｅ is preferably selected from methylene, ethylene and propylene groups, with ethylene being most preferred.

[0187] n is a number from 1 to 50. n is preferably a number from 1 to 30, and n is preferably a number from 1 to 10.

[0188] The difference between the mol % of the monomer represented by formula (II) relative to the entire polymer and the mol % of the monomer represented by formula (II) relative to the total amount of monomers charged in the preparation step is 0 to 10 mol %. The polymer of this embodiment is produced by the production method described below, and the difference between the monomer charge ratio and the actual measured value of the produced polymer is small, being 0 to 10 mol %, more preferably 0 to 8 mol %.

[0189] The number average molecular weight (Mn) of the polymer is 20,000 to 1,000,000, and more preferably 50,000 to 800,000. The ratio (Mw / Mn) of the weight average molecular weight (Mw) to the number average molecular weight (Mn) of the polymer is 1.01 to 10.00, preferably 1.2 to 8.0, preferably 1.4 to 6.0, preferably 1.5 to 5.0, and preferably 1.6 to 4.5. The number average molecular weight (Mn) and number average molecular weight (Mn) can be determined, for example, by gel filtration chromatography as described in the examples.

[0190] By using the polymer of this embodiment as a base film 90 for cell culture, it is possible to form a cell structure 1 (a spheroid, such as a cell aggregate) by adhering cells 56 and then detaching them. The cell structure 1 refers to a structure formed as a result of aggregation of cells 56, and is not limited to a spherical or ring shape. Compared to cell aggregates produced by non-adhesive culture on conventional low-adhesion plates, this method has advantages such as the ability to adjust the size of the cell structure 1 by specifying the adhesion area (cell aggregates of any size can be produced).

[0191] The entire disclosures of International Publication No. 2020 / 040247 and Japanese Patent Application No. 2020-028120 are incorporated herein by reference.

[0192] (Cell adhesive substance) The base film-forming composition of this embodiment preferably further contains a cell adhesive substance. When the base film-forming composition 48 contains a cell adhesive substance, it can promote the adhesion, spreading, proliferation, and differentiation of the cells 56. As a result, it is possible to easily form the desired cell structure 1.

[0193] Examples of cell adhesive substances that can be used include known substances derived from living organisms, such as extracellular matrix (ECM) proteins, glycoproteins, and peptides, as well as synthetic compounds (low molecular weight, high molecular weight). The cell adhesive substance is preferably a compound not derived from living organisms, such as a synthetic compound (low molecular weight, high molecular weight). A low molecular weight is, for example, a compound with a weight-average molecular weight of 2,000 or less, and a high molecular weight is, for example, a compound with a weight-average molecular weight of 2,000 or more, with an upper limit of, for example, 1,000,000.

[0194] Examples of extracellular matrix (ECM) proteins include collagen (e.g., Merck's type I collagen (product numbers C9791, C7661, C1809, C2249, C2124), type II collagen (product number C9301), type IV collagen (product numbers C0543, C5533), elastin (e.g., Merck's product numbers E1625, E6527), fibronectin (e.g., Merck's product numbers F1141, F0635, F2518, F0895, F4759, F2006), laminin (e.g., Merck's product numbers L6724, L2020, L4544), laminin fragments (e.g., Matricsome's product number 892011), and vitronectin (e.g., VTN-N (Gibco), Vitronectin, Human Recombinant, Animal Free (PeproTech), Merck product numbers: V0132, V9881, V8379, 08-126, SRP3186).

[0195] The cell adhesive substance is preferably a glycoprotein, specifically selected from vitronectin, integrin, cadherin, fibronectin, laminin, tenascin, osteopontin, and bone sialoprotein, and preferably a protein having an RGD amino acid sequence.

[0196] Examples of peptides include ECM peptide (MAPTrix ​​(registered trademark) from Kollodis Bio Sciences) and RGD peptide (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.: 180-01531).

[0197] Examples of synthetic compounds (polymers) include polylysine (e.g., Merck products: P4707, P4832, P7280, P9155, P6407, P6282, P7405, P5899) and polyornithine (e.g., Merck product number P4975). Examples of synthetic compounds (low molecular weights) include adhesamine (e.g., Nagase & Co.: AD-00000-0201) and synthetic cyclic RGD peptide (e.g., IRIS BIOTECH: LS-3920.0010).

[0198] The ratio (by mass) of the polymer to the cell adhesive substance in the base film-forming composition of this embodiment is not limited as long as a base film-forming composition 48 capable of cell culture can be formed. However, the ratio (by mass) of the polymer to the cell adhesive substance is preferably 100:0.1 to 100:100. When the ratio of the cell adhesive substance is 0.1 or more, cell adhesiveness is sufficiently exhibited, and when the ratio of the cell adhesive substance is 100 or less, aggregation of cells 56 after cell adhesion (formation of cell structure 1) can be facilitated.

[0199] The composition for forming a base film according to this embodiment contains a solvent. The solvent is not limited as long as it can dissolve the polymer, but is preferably an aqueous solution containing water.

[0200] The aqueous solution may be water, a salt-containing aqueous solution such as physiological saline or a phosphate buffer solution, or a mixed solvent of water or a salt-containing aqueous solution with an alcohol. 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, and 1-hexanol. Examples of suitable solvents include 2-methyl-1-pentanol, 2-methyl-2-pentanol, 2-methyl-3-pentanol, 3-methyl-1-pentanol, 3-methyl-2-pentanol, 3-methyl-3-pentanol, 4-methyl-1-pentanol, 4-methyl-2-pentanol, 4-methyl-3-pentanol, and cyclohexanol. These may be used alone or in combination. The water content in the aqueous solution is, for example, 50% to 100% by mass, 80% to 100% by mass, or 90% to 100% by mass.

[0201] Furthermore, in addition to the above-mentioned polymer, cell adhesive substance, and solvent, other substances may be added to the base film-forming composition 48 as needed, provided that they do not impair the performance of the resulting base film 90. Examples of other substances include pH adjusters, crosslinking agents, preservatives, surfactants, primers that improve adhesion to the container or substrate 80, antifungal agents, and sugars.

[0202] Below, as examples, we will specifically explain the coating film forming composition 38 and the base film forming composition 48 that can be used in the cell structure manufacturing apparatus 10 of this embodiment, but the present invention is not limited to these.

[0203] <Coating Film-Forming Composition 38> First, Experimental Example A1 of the coating film-forming composition 38 will be described.

[0204] Experimental Example A1 Preparation of Coating Film-Forming Composition 38 Polyvinyl acetate (JMR-10L (registered trademark) manufactured by Nippon Vinyl Acetate & Poval (degree of polymerization: 250, degree of saponification: 35.8%)) was dissolved in water / ethanol (3 / 7 mass ratio) to a concentration of 10 mg / g to prepare coating film-forming composition 38. The obtained coating film-forming composition 38 was transparent and uniform.

[0205] (Formation of Coating Film 84 on HMDS-Treated Silicon Wafer) The coating film-forming composition 38 obtained above was spin-coated at 1500 rpm for 60 seconds onto an HMDS (1,1,1,3,3,3-hexamethyldisilazane)-treated silicon wafer, and then dried in an oven at 70°C for 24 hours as a drying step, to obtain a coating film 84 on the HMDS-treated silicon wafer. The film thickness of the coating film 84 on the HMDS-treated silicon wafer was measured using a spectroscopic ellipsometer. The wafer was then thoroughly washed with PBS (phosphate buffered saline) and dried in an oven at 50°C for 1 hour, and the film thickness of the coating film 84 on the HMDS-treated silicon wafer was measured using a spectroscopic ellipsometer. The remaining film ratio was calculated from the film thickness after washing with PBS relative to the film thickness after application.

[0206] (Preparation of Coated Plate for Cell Culture) Using the composition 38 for forming a coating film obtained above, a coated plate for cell culture was prepared by the following method (i) or (ii).

[0207] (i) (Experimental Examples A1, 2, Comparative Experimental Examples A1, 2, 5, 6) The coating film-forming composition 38 obtained above was added to separate wells of a 96-well cell culture plate (Corning, #351172, volume 0.37 mL, made of polystyrene) at 200 μL / well. After standing at room temperature for 1 hour, excess composition was removed. The plate was then dried in an oven at 70°C for 24 hours. Each coated well was then washed three times with 250 μL of pure water, dried in an oven at 50°C for 1 hour, and then used for testing.

[0208] (ii) (Experimental Examples A3 to A20, Comparative Experimental Examples A3 and A4) The coating film-forming composition 38 obtained above was added to separate wells of a 24-well cell culture plate (manufactured by Corning, #351147, volume 1 mL, made of polystyrene) at 800 μL / well, and after standing at room temperature for 1 hour, excess composition was removed. The plate was then dried in an oven at 70°C for 24 hours. Each coated well was then washed three times with 1.5 mL of pure water, dried in an oven at 50°C for 1 hour, and then used for testing.

[0209] (Cell Preparation) Mouse embryonic fibroblast cells (manufactured by DS Pharma Biomedical) were used. The medium used for cell culture was BME medium (manufactured by Thermo Fisher Scientific) containing 10% FBS (manufactured by Sigma-Aldrich) and L-glutamine-penicillin-streptomycin stabilizing solution (manufactured by Thermo Fisher Scientific). The cells were incubated at 37°C / CO ２ The cells were cultured in a 10 cm diameter Petri dish (10 mL of medium) for at least two days in an incubator maintained at 5% carbon dioxide. Subsequently, the cells were washed with 5 mL of PBS, and then detached by adding 1 mL of 0.25 w / v% trypsin-1 mmol / L EDTA solution (Fujifilm Wako Pure Chemical Industries, Ltd.). The cells were then suspended in 10 mL of the above medium. The suspension was centrifuged (Tomy Seiko Co., Ltd., Model No. LC-200, 1000 rpm / 3 min, room temperature), the supernatant was removed, and the above medium was added to prepare a cell suspension.

[0210] (Cell Adhesion Experiment) Each cell suspension was added to each well of the plate prepared above at a concentration of 1 × 10 for a 96-well cell culture plate. ４ 150 μL each for cells / well, 5 × 10 for 24-well cell culture plates ４ Then, the cells were incubated at 37°C for 3 days in a 5% carbon dioxide atmosphere with CO ２The plates were then placed in an incubator. After three days of culture, cell adhesion to each well of the plate prepared above was compared based on observation (magnification: 4x) using an inverted microscope (CKX31, manufactured by Olympus Corporation). The observation results were evaluated using the following evaluation criteria to confirm the cell adhesion inhibitory effect. [Evaluation criteria] ◯: Indicates a state in which cells did not adhere to or spread on the bottom surface of the substrate, and cell aggregates (spheroids) formed in the wells. ×: Indicates a state in which cells adhered to and spread on the bottom surface of the substrate. Figure 4 shows a photograph of the microscopic observation results of the cell adhesion test of Experimental Example A1. In the coating film 84 obtained from the coating film-forming composition 38 of Experimental Example A1, due to the hydrophilization of the substrate surface, no adhesion or spreading of cells 56 was observed, and cell aggregates (spheroids) were observed in the wells.

[0211] (Preparation of Coated Plate for Protein Adhesion Inhibition Test) The coating film-forming composition 38 obtained above was added to separate wells of a 96-well cell culture plate (Corning, #9017, volume 0.36 mL, made of polystyrene) at 150 μL / well, 5 wells at a time. After immersion at room temperature for 1 hour, the liquid was drained and dried in an oven at 50°C for 24 hours. Each coated well was then washed three times with 200 μL of pure water, dried in an oven at 70°C for 1 hour, and then used in the test. As a negative control, wells of an uncoated 96-well cell culture plate (Corning, #9017, volume 0.36 mL, made of polystyrene) were used.

[0212] (Preparation of IgG-HRP Dilution) Goat anti-mouse IgG antibody-HRP conjugate (manufactured by Southern Biotechnology Associates) was diluted with PBS to a concentration of 1 mg / g to prepare an IgG-HRP dilution.

[0213] (Protein Adhesion Experiment) 100 μL / well of IgG-HRP diluted solution was added to each well of the plate prepared above, including the negative control, and the plate was left to stand at room temperature for 30 minutes. After 30 minutes, the IgG-HRP diluted solution was drained, and each well was washed three times with 200 μL of PBS. 100 μL / well of TMB solution (Sera Care, SureBlue) was added, and one minute later, 100 μL / well of TMB STOP solution (Sera Care) was added. Absorbance at 450 nm and 650 nm was measured using a microplate reader (TECAN, Infinite M200PRO). The absorbance at 650 nm was subtracted from the absorbance at 450 nm to obtain the average absorbance for the five wells. The average absorbance in the negative control wells was taken as 100% protein adsorption rate, and the protein adsorption rate of the wells coated with the coating film-forming composition 38 obtained above was calculated.

[0214] <Experimental Example A2, Comparative Experimental Examples A1-2> A coating film-forming composition 38 was prepared in the same manner as in Experimental Example A1, except that the degree of polymerization and degree of saponification of polyvinyl acetate and the composition of each solvent were changed as described in Table 1. The obtained coating film-forming composition 38 was transparent and uniform. The obtained coating film-forming composition 38 was treated in the same manner as in Experimental Example A1 to form a coating film 84 on an HMDS-treated silicon wafer, and a coating plate for cell culture and a coating plate for a protein adhesion inhibition test were prepared. The remaining film rate was determined in the same manner as in Experimental Example A1. A cell adhesion experiment was conducted in the same manner as in Experimental Example A1. A protein adhesion experiment was conducted in the same manner as in Experimental Example A1.

[0215] <Experimental Examples A3-11, Comparative Experimental Examples A3-4> A coating film-forming composition 38 was prepared in the same manner as in Experimental Example A1, except that the degree of polymerization and degree of saponification of polyvinyl acetate and the composition of each solvent were changed as described in Table 1. The resulting coating film-forming composition 38 was transparent and uniform. A coating film 84 was formed on an HMDS-treated silicon wafer using the resulting coating film-forming composition 38 in the same manner as in Experimental Example A1. A cell culture coating plate was also prepared using the method described in (ii) above. A protein adhesion inhibition test was also prepared in the same manner as in Experimental Example A1. The remaining film rate was determined in the same manner as in Experimental Example A1. A cell adhesion experiment was conducted in the same manner as in Experimental Example A1. Figure 5 shows a photograph of the microscopic observation results of the cell adhesion test of Comparative Experimental Example A3. In the coating film 84 obtained from the coating film-forming composition 38 of Comparative Experimental Example A3, the substrate surface was not sufficiently hydrophilized, and the cells adhered to and spread on the bottom surface of the plate.

[0216] <Experimental Examples A12 to A20> A coating film 84 was formed on an HMDS-treated silicon wafer, a cell culture plate, and a coating plate for a protein adhesion inhibition test in the same manner as in Experimental Example A3, except that the degree of polymerization and degree of saponification of polyvinyl acetate and the composition of each solvent were changed as described in Table 1. The plates were then irradiated with gamma rays (25 kGy). The remaining film rate was determined in the same manner as in Experimental Example A1. A cell adhesion experiment was conducted in the same manner as in Experimental Example A1. A protein adhesion experiment was conducted in the same manner as in Experimental Example A1.

[0217] <Experimental Example A21> (Preparation of a composition for forming a coating film) A composition for forming a coating film was prepared in the same manner as in Experimental Example A20, except that the concentration was dissolved to 3 mg / g. The obtained composition for forming a coating film was transparent and uniform.

[0218] (Preparation of a coated plate for cell culture by inkjet printing) Using an inkjet device (Seiko Epson Corporation, R&D inkjet device) and an inkjet head (Seiko Epson Corporation, Precision Core Head S800-A1), an appropriate amount of the coating film-forming composition prepared above was applied to a polystyrene substrate measuring 79 mm x 121 mm in a perfect circle with a diameter of 18 mm. This was dried in an oven at 70°C for 24 hours. This was attached to a bottomless 24-well plate (CS Tech Co., Ltd.) to prepare a coated plate for cell culture.

[0219] (Cell Preparation) Human adipose tissue-derived mesenchymal stem cells (ADSCs) (Cellsource, Inc.) were used. The medium used for cell culture was Mesenchymal Stem Cell Growth Medium 2 (PromoCell), a low-serum medium containing an L-glutamine-penicillin-streptomycin stabilizing solution (Thermo Fisher Scientific). The cells were statically cultured for at least two days in a 10 cm diameter Petri dish (10 mL of medium) at 37°C in a CO2 incubator with a 5% carbon dioxide concentration. The cells were subsequently washed with 5 mL of PBS, and then detached by adding 1 mL of TrypLE Select Enzyme (Thermo Fisher Scientific). The cells were then suspended in 10 mL of the above medium. This suspension was centrifuged (Tomy Seiko Co., Ltd., Model No. LC-200, 1000 rpm / 3 minutes, room temperature), the supernatant was removed, and the above medium was added to prepare a cell suspension.

[0220] (Cell Adhesion Experiment) A cell adhesion experiment was carried out in the same manner as in Experimental Example 1, except that the cell suspension prepared by the above-mentioned method was used. In the coating film 84 obtained from the coating film-forming composition 38 of Experimental Example A21, by hydrophilizing the substrate surface, no adhesion or spreading of cells 56 was observed, and the formation of cell aggregates (spheroids) was observed in the wells.

[0221] Comparative Experimental Example A5 Photosensitive polyvinyl alcohol was obtained according to Synthesis Example 5 and Experimental Example A3 of JP 2003-292477 A. The obtained photosensitive polyvinyl alcohol was dissolved in ethanol to a concentration of 5 mg / g to prepare a coating film-forming composition 38. Note that Gohsenol EG-30P (manufactured by Mitsubishi Chemical Corporation, saponification degree 86.3-69.0) was used as the polyvinyl alcohol. The obtained coating film-forming composition 38 was treated in the same manner as Experimental Example A1 to form a coating film 84 on an HMDS-treated silicon wafer, and a cell culture coating plate was produced. The remaining film rate was determined in the same manner as Experimental Example A1. A cell adhesion experiment was conducted in the same manner as Experimental Example A1.

[0222] Comparative Experimental Example A6 In Comparative Experimental Example A5, when the coating film 84 was formed on the HMDS-treated silicon wafer and a coated plate for cell culture was produced, after the drying step, an ultra-high pressure mercury lamp (ultraviolet irradiance 20 mW / cm ２ A coating film 84 was formed on an HMDS-treated silicon wafer and a coated plate for cell culture was prepared in the same manner as in Comparative Experimental Example A5, except that the exposure was performed for 5 seconds using a UT-150 (illuminance meter manufactured by USHIO). The remaining film rate was determined in the same manner as in Experimental Example A1. A cell adhesion experiment was conducted in the same manner as in Experimental Example A1.

[0223]

[0224]

[0225] In Table 1, EtOH represents ethanol, and PGME represents propylene glycol monomethyl ether.

[0226] The degree of saponification of polyvinyl acetate in Experimental Examples A1 to A20 and Comparative Experimental Examples A1 to A4 corresponds to the molar ratio (A:B) of repeating units (A) to repeating units (B). For example, the molar ratio (A:B) of polyvinyl acetate with a saponification degree of 35.8 mol% is 64.2:35.8. For example, the molar ratio (A:B) of polyvinyl acetate with a saponification degree of 65.4 mol% is 34.6:65.4.

[0227] The polyvinyl acetates used in Experimental Examples A1 to A20 and Comparative Experimental Examples A3 and A4 were all water-insoluble. The polyvinyl acetates used in Comparative Experimental Examples A1 and A2 were all water-soluble. The photosensitive polyvinyl alcohol used in Comparative Experimental Example A5 was water-soluble.

[0228] The coating films 84 obtained from the coating film-forming compositions 38 of Experimental Examples A1 to A20 had a higher film retention rate even after washing with PBS than the coating films 84 obtained from the coating film-forming compositions 38 of Comparative Experimental Examples A1, A2, and A5. The coating films 84 obtained from the coating film-forming compositions 38 of Experimental Examples A1 to A20 showed no cell adhesion or spreading, and the formation of cell aggregates (spheroids) was observed in the wells. On the other hand, the coating films 84 obtained from the coating film-forming compositions 38 of Comparative Experimental Examples A3, A4, and A5 showed cells adhering to and spreading on the bottom surface of the plate. Furthermore, UV irradiation was required for the coating film 84 obtained from the coating film-forming composition 38 of Comparative Experimental Example A5 to achieve a film retention rate and cell adhesion inhibitory effect comparable to those of Experimental Example A. The coating films 84 obtained from the coating film-forming compositions 38 of Experimental Examples A1 to A20 showed a lower protein adhesion rate than Comparative Experimental Example A3 and the substrate 80 without the coating film 84.

[0229] From the above results, it is clear that the coating film 84 obtained from the coating film-forming compositions 38 of Experimental Examples A1 to A20 has the ability to inhibit adhesion of cells 56. Therefore, this coating film-forming composition 38 can be preferably used as the coating film-forming composition 38 for forming the coating film 84 in the coating film forming section 30 of the cell structure manufacturing apparatus 10 of this embodiment.

[0230] <Base film-forming composition 48> The base film-forming composition will be described in more detail below using Experimental Example B and Comparative Experimental Example B, but the base film-forming composition is not limited to the following Experimental Example B. In the following Experimental Example B and Comparative Experimental Example B, a base film 90 formed on a substrate 80 using base film-forming composition 48 is referred to as a substrate for producing cell aggregates. Furthermore, the base film-forming composition 48 used in Experimental Example B and Comparative Experimental Example B is referred to as a base film-forming agent.

[0231] <Method for measuring weight-average molecular weight> The weight-average molecular weights shown in the following synthesis examples are the results of gel filtration chromatography (hereinafter abbreviated as GFC). (Measurement conditions) Apparatus: HLC-8320GPC (manufactured by Tosoh Corporation) GFC column: TSKgel G 6000 + 3000 PWXL-CP Flow rate: 1.0 mL / min Eluent: salt-containing water / organic mixed solvent Column temperature: 40°C Detector: RI Injection concentration: polymer solids content 0.05 mass% Injection volume: 100 μL Calibration curve: cubic approximation curve Standard sample: polyethylene oxide (manufactured by Agilent) × 10 types

[0232] Synthesis Example 1 24.00 g of 2-(dimethylamino)ethyl methacrylate (Tokyo Chemical Industry Co., Ltd.), 1.46 g of methacrylic acid (Tokyo Chemical Industry Co., Ltd.), 5.09 g of ethylene glycol dimethacrylate (Tokyo Chemical Industry Co., Ltd.), 0.31 g of dimethyl 1,1'-azobis(1-cyclohexanecarboxylate) (VE-073, Fujifilm Wako Pure Chemical Industries, Ltd.), and 111.09 g of 2-propanol were mixed and polymerized dropwise into 166.62 g of 2-propanol at reflux temperature to synthesize a polymer. The reaction product was reprecipitated in hexane, a poor solvent, and the precipitate was collected by filtration and dried under reduced pressure. The weight-average molecular weight of this polymer measured by GFC was 228,000 (hereinafter referred to as "Synthesis Example Polymer 1").

[0233] Synthesis Example 2: 8.00 g of 2-(dimethylamino)ethyl methacrylate (Tokyo Chemical Industry Co., Ltd.), 1.88 g of methacrylic acid (Tokyo Chemical Industry Co., Ltd.), 1.98 g of ethylene glycol dimethacrylate (Tokyo Chemical Industry Co., Ltd.), 0.12 g of dimethyl 1,1'-azobis(1-cyclohexanecarboxylate) (VE-073, Fujifilm Wako Pure Chemical Industries, Ltd.), and 43.10 g of 2-propanol were mixed and polymerized dropwise into 64.65 g of 2-propanol at reflux temperature to synthesize a polymer. The reaction product was reprecipitated in hexane, a poor solvent, and the precipitate was collected by filtration and dried under reduced pressure. The weight-average molecular weight of this polymer measured by GFC was 438,000 (hereinafter referred to as "Synthesis Example Polymer 2").

[0234] Experimental Example B1: 0.0025 g of the polymer obtained in Synthesis Example 1 above was mixed with 99.5 g of purified water and 0.5 mL of 0.5 mg / mL vitronectin VTN-N (Gibco) and thoroughly stirred to prepare a primer film-forming agent. Using an inkjet device (Microjet Co., Ltd., model number: LaboJet-600) and an inkjet head (model number: 500-SC), an appropriate amount of the primer film-forming agent was applied to the culture surface of a culture dish (diameter: 35 mm) (Sumitomo Bakelite Co., Ltd., MS9035X) capable of inhibiting cell adhesion 56. The mixture was dried in a thermostatic oven at 70°C for one day to prepare a substrate for cell aggregate production.

[0235] Experimental Example B2 A primer film-forming agent was prepared by adding 99 g of purified water and 1 mL of 0.5 mg / mL vitronectin VTN-N (Gibco) to 0.0025 g of the polymer obtained in Synthesis Example 1 and thoroughly stirring the mixture. The primer film-forming agent was applied using an inkjet device and dried in the same manner as in Experimental Example B1, to prepare a substrate for producing cell aggregates.

[0236] Experimental Example B3 A primer film-forming agent was prepared by adding 99 g of purified water and 1.0 mL of 0.5 mg / mL vitronectin VTN-N (Gibco) to 0.005 g of the polymer obtained in Synthesis Example 2 and thoroughly stirring the mixture. The primer film-forming agent was applied using an inkjet device and dried in the same manner as in Experimental Example B1, to prepare a substrate for producing cell aggregates.

[0237] Experimental Example B4 A primer film-forming agent was prepared by adding 99 g of purified water and 1.0 mL of 0.5 mg / mL vitronectin VTN-N (Gibco) to 0.005 g of the polymer obtained in Synthesis Example 1 and thoroughly stirring the mixture. The primer film-forming agent was applied using an inkjet device and dried in the same manner as in Experimental Example B1, to prepare a substrate for producing cell aggregates.

[0238] Experimental Example B5: 0.015 g of the polymer obtained in Synthesis Example 1 was mixed with 94 g of purified water and 6.0 mL of 0.5 mg / mL vitronectin VTN-N (Gibco) and thoroughly stirred to prepare a primer film-forming agent. Using an inkjet device (Microjet Co., Ltd., model number: LaboJet-600) and an inkjet head (model number: IJHBS-1000), an appropriate amount of primer film-forming agent was applied to the culture surface of a culture plate (Sumitomo Bakelite Co., Ltd., PrimeSurface® Plate 24F, model number: MS-90240) capable of inhibiting cell adhesion. The mixture was dried in a thermostatic oven at 70°C for one day to prepare a substrate for cell aggregate production. Sterilization was performed by gamma ray irradiation at 25 kGy.

[0239] Experimental Example B6: 0.005 g of the polymer obtained in Synthesis Example 1 above was mixed with 98 g of purified water and 2.0 mL of 0.5 mg / mL vitronectin VTN-N (Gibco) and thoroughly stirred to prepare a primer film-forming agent. Using an inkjet device (Microjet Co., Ltd., model number: LaboJet-600) and an inkjet head (model number: IJHBS-1000), an appropriate amount of primer film-forming agent was applied to the culture surface of a culture plate (Sumitomo Bakelite Co., Ltd., PrimeSurface® Plate 24F, model number: MS-90240) capable of inhibiting cell adhesion. The mixture was dried in a thermostatic oven at 70°C for one day to prepare a substrate for cell aggregate production. Sterilization was performed by gamma ray irradiation at 25 kGy.

[0240] Experimental Example B7: 0.005 g of the polymer obtained in Synthesis Example 1 above was mixed with 31.3 g of purified water and 2.0 mL of 0.5 mg / mL vitronectin VTN-N (Gibco) and thoroughly stirred to prepare a primer film-forming agent. Using an inkjet device (Microjet Co., Ltd., model number: LaboJet-600) and an inkjet head (model number: 200-SC), an appropriate amount of primer film-forming agent was applied to the culture surface of a culture plate (Sumitomo Bakelite Co., Ltd., PrimeSurface® Plate 24F, model number: MS-90240) capable of inhibiting cell adhesion. The mixture was dried in a thermostatic oven at 70°C for one day to prepare a substrate for cell aggregate production. Sterilization was performed by gamma ray irradiation at 25 kGy.

[0241] Experimental Example B8: 0.005 g of the polymer obtained in Synthesis Example 1 was added to 62.7 g of sterile water and 4.00 g of Recombinant Human Vitronectin (Peprotech) diluted to 0.5 mg / mL with sterile water, and the mixture was thoroughly stirred to prepare a base film-forming agent. The base film-forming agent prepared in Experimental Example B8 was applied to the culture surface of a sheet capable of inhibiting cell adhesion (the sheet capable of inhibiting cell adhesion prepared in Experimental Example A21) using an inkjet device (Seiko Epson Corporation, R&D inkjet device) and an inkjet head (Seiko Epson Corporation, Precision Core Head S800-A1) to achieve a spot diameter of 170 μm and a center-to-center spacing of 250 μm. The coating was then dried in a constant-temperature dryer at 70°C for one day. The coated wells were then attached to a bottomless 24-well plate (CS Tech Co., Ltd.) to prepare a substrate for cell aggregate production. Sterilization was carried out by gamma irradiation at 25 kGy.

[0242] <Experimental Example B9> A substrate for forming cell aggregates was prepared in the same manner as in Experimental Example B8, except that the spot diameter of the base film forming agent was 250 μm and the distance between the centers of the spots was 350 μm.

[0243] <Experimental Example B10> A substrate for forming cell aggregates was prepared in the same manner as in Experimental Example B8, except that the spot diameter of the base film forming agent was 400 µm and the distance between the centers of the spots was 500 µm.

[0244] <Experimental Example B11> A substrate for forming cell aggregates was prepared in the same manner as in Experimental Example B8, except that the spot diameter of the base film forming agent was changed to 700 µm and the distance between the centers of the spots was changed to 800 µm.

[0245] <Experimental Example B12> A substrate for forming cell aggregates was prepared in the same manner as in Experimental Example B8, except that the spot diameter of the base film forming agent was 900 µm and the distance between the centers of the spots was 1000 µm.

[0246] Comparative Experimental Example B1 A primer film-forming agent was prepared by adding 100.0 g of pure water to 0.0025 g of the polymer obtained in Synthesis Example 1 and stirring thoroughly. The primer film-forming agent was applied using an inkjet device and dried in the same manner as in Experimental Example B1, to prepare a substrate for producing cell aggregates.

[0247] Comparative Experimental Example B2 A primer film-forming agent was prepared by adding 100.0 g of pure water to 0.005 g of the polymer obtained in Synthesis Example 2 and thoroughly stirring the mixture. The primer film-forming agent was applied using an inkjet device and dried in the same manner as in Experimental Example B1, to prepare a substrate for producing cell aggregates.

[0248] Comparative Experimental Example B3: 0.005 g of the polymer obtained in Synthesis Example 1 was added with 33.3 g of pure water and thoroughly stirred to prepare a base film-forming agent 10. Using an inkjet device (Microjet Corporation, model number: LaboJet-600) and an inkjet head (model number: 200-SC), an appropriate amount of the base film-forming agent was applied to the culture surface of a culture dish (diameter: 35 mm) (Sumitomo Bakelite Co., Ltd., MS9035X) capable of inhibiting cell adhesion 56. The mixture was dried in a thermostatic oven at 70°C for one day to prepare a substrate for producing cell aggregates. Sterilization was performed by irradiating with gamma rays at 25 kGy.

[0249] Comparative Experimental Example B4 4.8 g of pure water and 0.2 mL of 0.5 mg / mL vitronectin VTN-N (Gibco) were added and thoroughly stirred to prepare a base film forming agent 11. In the same manner as in Experimental Example B1, the base film forming agent was applied using an inkjet device and dried to prepare a substrate for producing cell aggregates.

[0250] Comparative Experimental Example B5 2.1 g of pure water and 1.2 mL of 0.5 mg / mL vitronectin VTN-N (Gibco) were added and thoroughly stirred to prepare a base film forming agent 12. In the same manner as in Experimental Example B1, the base film forming agent 12 was applied using an inkjet device and dried to prepare a substrate for producing cell aggregates.

[0251] <Test Example B1: Cell Adhesion Confirmation Test with Mouse Fibroblasts in FBS-Free Medium of Experimental Examples B1-3 and Comparative Experimental Examples B1-2> (Preparation of Cells 56) Mouse embryonic fibroblasts (C3H10T1 / T2 cells: manufactured by DS Pharma Biomedical Co., Ltd.) were used as cells 56. For cell culture, a medium was used in which FBS (manufactured by Sigma-Aldrich) was added to the basal medium (manufactured by Gibco) to make 10% FBS (manufactured by Sigma-Aldrich) and 1% Glutamine / Penicillin / Streptmycin (manufactured by Gibco). The cells were incubated at 37°C / CO ２ The cells were cultured in a 10 cm diameter dish (10 mL of medium) for at least two days in an incubator maintained at 5% carbon dioxide. Subsequently, the cells were washed with 3 mL of PBS solution (Fujifilm Wako Pure Chemical Industries, Ltd.), after which 3 mL of trypsin-EDTA solution (PromoCell) was added and the mixture was left at room temperature for 3 minutes to detach the cells. Cell 56 was recovered by adding 7 mL of BME medium without FBS (bovine bovine serum) or glutamine / penicillin / streptmycin. The suspension was centrifuged (Tomy Seiko Co., Ltd., Model No. LC-230, 200 × g / 3 minutes, room temperature), the supernatant was removed, and the above medium was added to prepare a cell suspension.

[0252] (Cell Adhesion Confirmation Test) 2.0 mL of cell suspension was added to the substrates for producing cell aggregates prepared in Experimental Examples B1 to B3 and Comparative Experimental Examples B1 and B2. The cell density was 1.5 × 10 for Experimental Example B1, Experimental Example B2, and Comparative Experimental Example B1. ５ cells / cm 2 , Experimental Example B3, and Comparative Experimental Example B2: 3.0 × 10 ５ cells / cm 2 After that, the medium was incubated at 37°C / CO2 with a 5% carbon dioxide concentration. ２The wells were then left to stand for 2 hours in an incubator. After standing, the non-adherent cells and medium were removed, and the wells were washed with PBS, leaving only the adherent cells on the wells. After washing, 2.0 mL of fresh medium was added, and the adherent cells were observed and photographed using a stereomicroscope SZX16 (Olympus Corporation). As a result, as shown in Figure 6, selective cell adhesion to the base film 90 on the substrate 80 prepared in Experimental Examples B1-3 and Comparative Experimental Examples B1 and B2 was confirmed. In Experimental Examples B1-3, cell adhesion was uniform, with no gaps. In contrast, in Comparative Experimental Examples B1-2, cell adhesion was found to be uneven, with gaps present. From the above, it was found that the inclusion of an additive in the base film-forming agent that promotes cell 56 adhesion and spreading can achieve uniform cell adhesion on the base film 90 in serum (FBS)-free medium.

[0253] <Test Example B2: Cell adhesion and cell aggregate formation confirmation test with mouse fibroblasts in FBS-free medium of Experimental Examples B4 and B6> (Preparation of cells 56) Cells 56 were prepared in the same manner as in Test Example B1. (Cell adhesion confirmation test) A cell suspension of 3.0 × 10 ５ cells / cm 2 After that, the mixture was incubated at 37°C / CO2 while maintaining a 5% carbon dioxide concentration. ２ The wells were then left to stand for 2 hours in an incubator. After standing, the non-adherent cells and medium were removed, and the wells were washed with PBS, leaving only the adherent cells on the wells. After washing, 2.0 mL of fresh medium was added, and the adherent cells were observed and photographed using a stereomicroscope (Olympus Corporation) SZX16. The next day, the presence or absence of cell aggregate formation was observed and photographed. As a result, as shown in Figure 7, selective adhesion of cells 56 to the base film 90 on the fabricated substrate 80 was confirmed. Furthermore, uniform adhesion occurred without gaps. After two days, the adhered cells 56 were confirmed to have detached from the Petri dish and aggregated, forming cell aggregates (spheroids). From the above, it was found that cells 56 could detach and form cell aggregates after uniform cell adhesion on a base film 90 containing an additive that promotes cell 56 adhesion and spreading.

[0254] <Test Example B3: Cell adhesion confirmation test in serum-free medium using human adipose tissue-derived mesenchymal stem cells of Experimental Example B5 and Comparative Experimental Example B3> (Preparation of cells 56) Human adipose tissue-derived mesenchymal stem cells (ADSC: manufactured by Cellsource Co., Ltd.) were used as cells 56. A low-serum medium, Mesenchymal Stem Cell Growth Medium 2 (manufactured by Takara Bio Inc.: serum concentration 2%) was used for cell culture. The cells were incubated at 37°C / CO ２ The cells were cultured in a 10 cm diameter dish (10 mL of medium) for at least two days in an incubator maintained at 5% carbon dioxide. Subsequently, the cells were washed with 3 mL of PBS solution (Fujifilm Wako Pure Chemical Industries, Ltd.), after which 3 mL of trypsin-EDTA solution (PromoCell) was added and the mixture was left to stand at room temperature for 3 minutes to detach the cells. Cells 56 were recovered by adding 7 mL of serum-free Mesenchymal Stem Cell Growth Medium DXF medium. The suspension was centrifuged (Tomy Seiko Co., Ltd., Model No. LC-230, 200 × g / 3 minutes, room temperature), the supernatant was removed, and the above medium was added to prepare a cell suspension.

[0255] (Cell Adhesion Confirmation Test) A cell suspension was applied to the substrates for producing cell aggregates prepared in Experimental Example B5 and Comparative Experimental Example B3 at a concentration of 3.0 × 10 ５ cells / cm 2 After that, the mixture was incubated at 37°C / CO2 while maintaining a 5% carbon dioxide concentration. ２The wells were then left to stand for 2 hours in an incubator. After standing, the non-adherent cells and medium were removed, and the wells were washed with PBS, leaving only the adherent cells in the wells. After washing, 2.0 mL of fresh medium was added, and the adherent cells 56 were observed and photographed using a stereomicroscope (Olympus Corporation) SZX16. As shown in Figure 8, in Experimental Example B5, selective adhesion of cells 56 to the base film 90 on the substrate 80 was confirmed. Furthermore, uniform adhesion was observed without gaps. In contrast, gaps were present in the cell adhesion in Comparative Experimental Example B3, indicating uneven cell adhesion. From the above, it was found that the inclusion of additives in the base film forming agent that promote cell 56 adhesion and spreading can achieve uniform cell adhesion on the base film 90 in serum-free medium, even when using ADSCs.

[0256] Test Example B4: Cell Adhesion Confirmation Test in Low-Serum Medium Using Human Adipose Tissue-Derived Mesenchymal Stem Cells of Test Example B7 (Preparation of Cells 56) Cells 56 were prepared in the same manner as Test Example B3, except that the culture medium after cell detachment was changed to Mesenchymal Stem Cell Growth Medium 2, a low-serum medium. (Cell Adhesion Confirmation Test) A cell adhesion confirmation test was performed on the cell aggregate production substrate prepared in Test Example B7 using the same method as Test Example B3. As a result, as shown in Figure 9, selective adhesion of cells 56 to the base film 90 portion on the prepared substrate 80 was confirmed. Furthermore, there were no gaps in the cell adhesion, and uniform adhesion occurred. From the above, it was found that by including an additive that promotes the adhesion and spreading of cells 56 in the base film forming agent, uniform cell adhesion can be achieved on the base film 90 even in low-serum medium when using ADSCs.

[0257] <Test Example B5: Spheroid diameter confirmation test in serum-free medium using human adipose tissue-derived mesenchymal stem cells of Experimental Examples B8 to B12> (Preparation of Cells 56) A cell suspension was prepared by the method described in Experimental Example B3. (Test to confirm cell adhesion and spheroid formation) 2.9 x 10 cell suspensions were added to the substrates for cell aggregate production prepared in Experimental Examples B8 to B12. ５ ~6.0 x 10 ５Then, the cells were incubated at 37°C / CO2 with the carbon dioxide concentration maintained at 5%. ２ The dish was left to stand in an incubator for 2 hours. After standing, the state of the adhered cells was observed and photographed using a stereomicroscope SZX16 (Olympus Corporation). After a further 3 days, the presence or absence of cell aggregate formation was observed and photographed using a Cell3imager duos (Screen Holdings Co., Ltd.). As a result, as shown in Figure 10, selective cell adhesion to the base film portion on the prepared substrate was confirmed. Furthermore, there were no gaps in the cell adhesion, and uniform adhesion occurred. Furthermore, the cell adhesion diameter was equivalent to the coated diameter. After a further 3 days, it was confirmed that the adhered cells had detached from the Petri dish and aggregated, forming cell aggregates (spheroids).

[0258] (Evaluation of spheroid size distribution) Wells in which spheroids were formed on the cell aggregate production substrates of Experimental Examples B8 to B10 in the above test were photographed using a Cell3imager duos and size evaluation was performed. As a result, as shown in Figure 11, there were peak tops at spheroid diameters of 100 μm, 120 μm, and 150 μm, respectively. Furthermore, the narrow distribution of spheroid diameters indicated that uniform spheroids were formed.

[0259] (Confirmation of spheroid size error, coated diameter, spheroid diameter, and volume) Wells in which spheroids were formed on the cell aggregate production substrates of Experimental Examples B8 to B12 in the above test were photographed using a Cell3imager duos. The diameters of 50 or more randomly selected spheroids were measured using the image processing software ImageJ, and the average spheroid diameter and size error were calculated. The results are shown in Figure 12. As shown in Figure 12, the average spheroid diameter increased as the coated diameter of the base film increased. Furthermore, the size error was small, ranging from 3 to 13%, indicating that uniform spheroids were formed.

[0260] The relationship between the applied area of ​​the base film for forming cell aggregates and the spheroid diameter in Experimental Examples B8 to B12 was graphed. The results are shown in Figure 13. As shown in Figure 13, it was shown that the distribution follows a power approximation relational equation. This is a result that is consistent with the theoretical formula. Furthermore, the relationship between the applied area of ​​the base film and the spheroid volume was calculated and graphed, and the results are shown in Figure 14. The results showed that there is a linear relationship between the applied area of ​​the base film and the spheroid volume. The correlation coefficient R 2 = 0.9989, indicating a strong correlation. These results demonstrate that it is possible to control the spheroid diameter and volume by controlling the coating area of ​​the base film.

[0261] Test Example B6: Cell Adhesion and Cell Aggregate Formation Confirmation Test with Mouse Fibroblasts in FBS-Free Medium of Comparative Experimental Example B4 (Additives Only, No Polymer) (Preparation of Cells 56) Cells 56 were prepared using the same method as in Test Example B1. (Cell Adhesion Confirmation Test) A cell adhesion and cell aggregate formation confirmation test was performed using the same method as in Test Example B2 on the cell aggregate production substrate prepared in Comparative Experimental Example B4. As a result, as shown in Figure 15, no cell adhesion was observed to the base film 90 portion on the prepared substrate 80. From the above, it was found that cell adhesion could not be achieved with a base film 90 that does not contain a polymer and contains only additives that promote the adhesion and spreading of cells 56. This demonstrates that the cell adhesion effect is only achieved by combining a polymer and an additive.

[0262] Test Example B7: Cell Adhesion and Cell Aggregate Formation Confirmation Test for Mouse Fibroblasts in FBS-Containing Medium of Comparative Test Example B5 (Additives Only, No Polymer) (Preparation of Cells 56) Cells 56 were prepared in the same manner as Test Example B1, except that the culture medium after cell detachment was changed to BME medium containing 10% FBS (bovine serum) and 1% glutamine / penicillin / streptmycin. (Cell Adhesion Confirmation Test) A cell adhesion and cell aggregate formation confirmation test was performed on the cell aggregate production substrate prepared in Comparative Test Example B5 in the same manner as Test Example B2. As a result, as shown in Figure 16, selective adhesion of cells 56 to the base film 90 portion on the prepared substrate 80 was confirmed. Furthermore, uniform adhesion occurred without any gaps in the cell adhesion. Furthermore, it was confirmed that the adhered cells 56 remained adhered to the Petri dish after two days. From the above, it was found that in the base film 90 that does not contain a polymer and contains only additives that promote the adhesion and spreading of cells 56, uniform cell adhesion is achieved in serum medium, but the cells do not subsequently detach, and therefore cell aggregates cannot be formed.

[0263] From the above results, it is clear that if a cell aggregate manufacturing substrate having a base film 90 obtained from the base film forming composition 48 obtained in Experimental Examples B1 to B12 is used, it is possible to selectively adhere cells 56 to the base film 90 portion. Therefore, this base film forming composition 48 can be preferably used as the base film forming composition 48 for forming the base film 90 in the base film forming section 40 of the cell structure manufacturing apparatus 10 of this embodiment.

[0264] REFERENCE SIGNS LIST 1 Cell structure 10 Cell structure manufacturing device 12 Moving mechanism 14 Rotating mechanism 20 Substrate supply section 22 Raw substrate cassette 30 Coating film forming section 32 Coating film forming application mechanism 34 Coating film forming composition tank 36 Coating film drying mechanism 38 Coating film forming composition 40 Base film forming section 42 Base film forming application mechanism 44 Base film forming composition tank 46 Base film drying mechanism 48 Base film forming composition 50 Seeding section 52 Cell seeding mechanism 54 Cell tank 56 Cells 60 Aggregation culture section 70 Cell structure collection section 72 Cell structure collection mechanism 80 Substrate 82 Raw substrate 84 Coating film 90 Base film 90a Base film pattern

Claims

1. A cell structure manufacturing apparatus for manufacturing a cell structure, comprising: a substrate supply unit that supplies a substrate, at least one surface of the substrate having cell adhesion inhibitory ability; a base film forming unit that forms a base film on the substrate, the base film forming unit including a base film forming coating mechanism that coats the substrate with a base film forming composition, and the base film has cell adhesiveness; a seeding unit that seeds cells onto the substrate including the base film; A cell structure manufacturing device comprising:

2. the substrate supply unit further includes a coating film forming unit that forms a coating film having cell adhesion inhibitory ability on at least a portion of at least one surface of the raw material substrate; the coating film forming unit includes a coating film forming application mechanism that applies a coating film forming composition to the surface of the substrate, The cell structure manufacturing device according to claim 1 , wherein the base film forming application mechanism applies the base film forming composition to at least a portion of the surface of the coating film on the substrate.

3. The cell structure manufacturing apparatus of claim 2, wherein the composition for forming a coating film comprises a copolymer having a repeating unit (A) represented by the following formula (A) and a repeating unit (B) represented by the following formula (B): 【Chemical 1】 (In the formula, R １ ~R ３ each independently represents a hydrogen atom or an alkyl group having 1 to 5 carbon atoms; X １ and X ２ each independently represents a single bond, an ester bond, an ether bond, an amide bond, or an alkylene group having 1 to 5 carbon atoms which may be interrupted by an oxygen atom.

4. 4. The cell structure manufacturing device according to claim 1, wherein at least one selected from the base film forming application mechanism and the coating film forming application mechanism is a pointillism type application mechanism.

5. The cell structure manufacturing device according to any one of claims 1 to 3, wherein the substrate has a substantially smooth surface.

6. The cell structure manufacturing device according to any one of claims 1 to 3, wherein the surface of the substrate has irregularities.

7. A cell structure manufacturing apparatus described in any one of claims 1 to 3, wherein the substrate or the raw material substrate is flexible, the substrate supply unit includes a winding-type substrate cassette or raw material substrate cassette, and the substrate is supplied from the substrate cassette or the raw material substrate is supplied from the raw material substrate cassette.

8. The cell structure manufacturing apparatus according to any one of claims 1 to 3, further comprising an aggregation culture section for aggregating or culturing cells attached to the substrate including the base film.

9. The cell structure manufacturing device according to any one of claims 1 to 3, wherein the size error of the cell structure is within 20%.

10. A cell structure manufacturing apparatus described in any one of claims 1 to 3, wherein the cell structure manufacturing apparatus has an airtight mechanism that can make the inside of the cell structure manufacturing apparatus an airtight closed space, and the airtight mechanism can maintain the inside of the closed space in a sterile environment.

11. The base film forming composition has the following formula (I): 【Chemistry 2】 [In the formula, U ａ１ and U ａ２ each independently represents a hydrogen atom or a linear or branched alkyl group having 1 to 5 carbon atoms; R a１ represents a hydrogen atom or a linear or branched alkyl group having 1 to 5 carbon atoms; R a２ represents a linear or branched alkylene group having 1 to 5 carbon atoms], and a repeating unit derived from a monomer represented by the following formula (II): 【Chemistry 3】 [In the formula, R ｂ A cell structure manufacturing apparatus described in any one of claims 1 to 3, comprising a copolymer containing a repeating unit derived from a monomer represented by the formula: [wherein represents a hydrogen atom or a linear or branched alkyl group having 1 to 5 carbon atoms].

12. The cell structure manufacturing apparatus according to claim 11 , wherein the base film forming composition further contains a cell adhesive substance.

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