JP2017131144A - Materials for preparing three-dimensional cell aggregates, compositions for preparing three-dimensional cell aggregates, three-dimensional cell aggregate preparation sets, composition-receiving containers, and production methods of three-dimensional cell aggregates - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide materials for preparing three-dimensional cell aggregates that suppress particle size change due to temperature changes and have less cytotoxicity.SOLUTION: The present invention discloses a material for preparing three-dimensional cell aggregates comprising at least two kinds of polymers whose particle size change rates are different. The at least two kinds of polymers comprise a polymer constituting a core and a polymer constituting a shell covering the surface of the core, and preferably, for example, an aspect in which the polymer constituting the core and the polymer constituting the shell form a polymer particle having a core-shell structure, an aspect in which the particle size change rate of the polymer constituting the shell is larger than the particle size change rate of the polymer constituting the core, and an aspect in which the polymer constituting the core is at least one selected from collagen, gelatin, proteoglycan, hyaluronic acid, fibronectin, laminin, tenascin, entactin, elastin and fibrillin.SELECTED DRAWING: None

Description

  The present invention relates to a material for preparing a three-dimensional cell assembly, a composition for preparing a three-dimensional cell assembly, a set for preparing a three-dimensional cell assembly, a composition container, and a method for preparing a three-dimensional cell assembly.

  In recent years, in the field of cell culture technology, a method has been developed in which cells are arranged on a substrate and the regeneration of damaged living tissue is induced using a cell sheet prepared in vitro.

  The cell sheet comprises a polymer or oligomer having an upper critical solution temperature in the range of 0 ° C. to 80 ° C. or a polymer or oligomer having a lower critical solution temperature in the range of 0 ° C. to 80 ° C. of the substrate. Covering the surface, culturing the cells on the polymer or oligomer, and bringing the cells to a temperature not lower than the upper critical solution temperature or lower than the lower critical solution temperature, thereby causing the cells to undergo phase change between the sol state and the gel state. It can peel from the said base | substrate.

  However, there is a problem that separation is difficult due to strong adhesion of cells to the substrate, and cells cannot be cultured efficiently and in large quantities. Furthermore, in handling cells, it is desired that the toxicity to cells is low, but none of the prior art has been sufficiently studied from the viewpoint of toxicity to cells.

Thus, a thermoresponsive cell culture bead using a polymer whose hydration power changes within a temperature range of 0 ° C. or more and 80 ° C. or less on the bead surface has been proposed (for example, see Patent Document 1).
In addition, gelatin hydrogel particles used for producing particle-containing cell aggregates have been proposed (see, for example, Patent Document 2).

  An object of the present invention is to provide a material for preparing a three-dimensional cell aggregate that suppresses a change in particle diameter due to temperature and has little cytotoxicity.

  The material for preparing a three-dimensional cell assembly of the present invention as a means for solving the above-described problems includes at least two kinds of polymers having different particle size change rates.

  According to the present invention, it is possible to provide a material for producing a three-dimensional cell assembly that suppresses a change in particle diameter due to temperature and has little cytotoxicity.

FIG. 1 is a schematic view showing a phase change from a sol state to a gel state of a composition for preparing a three-dimensional cell assembly containing at least two kinds of polymers having different particle size change rates. FIG. 2 is a schematic diagram illustrating an example of an inkjet discharge apparatus. FIG. 3 is a schematic diagram illustrating another example of the inkjet discharge apparatus. 4 is a graph showing the particle size distribution of polymer particles A having a core-shell structure in Example 1. FIG. FIG. 5A is a photograph showing a state at 25 ° C. of a composition for preparing a three-dimensional cell aggregate containing the polymer particles A containing a polymer constituting a shell having a lower critical solution temperature of 32 ° C. FIG. 5B is a photograph showing a state at 37 ° C. of a composition for preparing a three-dimensional cell aggregate containing the polymer particles A containing a polymer constituting a shell having a lower critical solution temperature of 32 ° C. FIG. 6 is a graph showing the relationship between the shear viscosity and the temperature of the composition A for preparing a three-dimensional cell assembly. FIG. 7A is a schematic diagram in which a composition for preparing a three-dimensional cell assembly in a sol state is applied on cells applied on a substrate. FIG. 7B is a schematic diagram in which cells are applied onto the layer after the composition for preparing a three-dimensional cell assembly in a sol state applied on cells arranged on a substrate is gelled to form a layer. FIG. 7C shows that the composition for preparing a three-dimensional cell assembly in a gel state is removed from a cell, a composition for preparing a three-dimensional cell assembly, and a cell stack in which cells are stacked in this order by removing the gel. It is a schematic diagram which obtains a cell aggregate. FIG. 8 is a photograph of the three-dimensional cell assembly produced in Example 2 observed with a fluorescent confocal microscope. FIG. 9 is a cross-sectional observation image obtained by observing the three-dimensional cell assembly produced in Example 2 with a stereomicroscope.

(Three-dimensional cell assembly material)
The material for preparing a three-dimensional cell assembly of the present invention contains at least two kinds of polymers having different particle size change rates, and contains other components as necessary.
The material for producing a three-dimensional cell assembly of the present invention is difficult to remove from the produced three-dimensional cell assembly because the conventional temperature-responsive cell culture beads have a large particle size change rate due to temperature. And based on the problem of cytotoxicity. Further, the conventional gelatin hydrogel particles are based on the problem that a precise and complex three-dimensional cell aggregate of cells cannot be produced because it does not have temperature responsiveness.

<Polymer>
The polymer includes at least two types of polymers having different particle size change rates, and may include different types of polymers having the same particle size change rate.
As the at least two kinds of polymers, it is preferable that at least one kind of polymer is a polymer whose particle size changes with temperature, so-called temperature-responsive polymer.
The temperature-responsive polymer may change from a sol state to a gel state or from a gel state to a sol state due to a change in particle size due to temperature when it is contained in a composition for preparing a three-dimensional cell assembly. preferable. The polymer can be identified using, for example, a GC-MS apparatus (apparatus name: QP2010, manufactured by Shimadzu Corporation).

The at least two kinds of polymers include a polymer constituting a core and a polymer constituting a shell covering the surface of the core, and the polymer constituting the core and the polymer constituting the shell are a core shell. It is preferable to form polymer particles having a structure. The core-shell structure means a form in which at least two polymers having different compositions are present in phase separation in the particles. The core-shell structure may be not only a form in which the shell completely covers the core but also a part of the core. Further, a part of the polymer of the shell may form a domain or the like in the core. It may have a multilayer structure of three or more layers including one or more layers having different compositions between the core and the shell.
Moreover, there is no restriction | limiting in particular that it has the said core shell structure, According to the objective, it can select suitably, For example, Unexamined-Japanese-Patent No. 2004-051657, the homepage of the Japan Foundation for Materials Science and Technology ( (http://www.mst.or.jp/case/eachcase/c0142.html) and the like.

<< Polymer particles >>
Examples of the shape of the polymer particles include a spherical shape such as a true spherical shape or a semi-true spherical shape; and an indefinite shape from the viewpoint that the polymer can be prevented from being excessively adsorbed by the cell and can be less toxic to the cell. Can be mentioned. These may be used alone or in combination of two or more.
In the polymer particles having the core-shell structure, it is preferable that the polymer constituting the core does not have temperature responsiveness, and the polymer constituting the shell has temperature responsiveness.

The volume average particle size of the polymer particles in the case where the temperature is equal to or lower than the lower critical solution temperature of the polymer constituting the shell is preferably smaller than the volume average particle size of cells in a free state, more preferably 1.0 μm or less, 0 .50 μm or less is particularly preferable. When the volume average particle size is 1.0 μm or less, the polymer particles do not settle in the liquid composition and can be stably discharged from the inkjet nozzle.
The volume average particle size of the polymer particles having the core-shell structure when exceeding the lower critical solution temperature of the polymer constituting the shell is preferably 0.8 μm or less, and more preferably 0.4 μm or less. When the volume average particle diameter is 0.8 μm or less, when a laminate composed of the cells and the composition for preparing a three-dimensional cell aggregate is prepared and the cells are cultured, the distance between the cells is reduced. And does not inhibit the adhesion between cells.

As the volume average particle size, using a concentrated system particle size analyzer (trade name: FPAR-1000, manufactured by Otsuka Electronics Co., Ltd.), using the sample solution obtained by the following sample solution preparation example, It can be measured according to measurement conditions.
-Sample solution preparation-
The synthesized powdery polymer is dispersed at a concentration of 0.5% by mass in pure water obtained using a pure water production apparatus (trade name: GSH-2000, manufactured by ADVANTEC). The sample liquid can be prepared by stirring the liquid volume for measurement at 5 mL and dispersing the 20 mm rotor with a stirrer at 200 rpm for about one day.
-Measurement conditions-
Solvent: water (refractive index: 1.3314, viscosity at 25 ° C .: 0.884 cP, optimal light amount adjustment is appropriately set by ND filter)
Measurement probe: Concentration probe Measurement routine: Measurement: 180 ° C. at 25 ° C. → Measurement: 600 ° C. at 25 ° C. (The liquid temperature gradually changes from 25 ° C. to 35 ° C. when the body side is changed to 35 ° C. Diameter change is monitored) → Measurement: 35 ° C for 180 seconds

The at least two polymers are accompanied by a change in particle size at a temperature around the critical solution temperature of the polymer.
Examples of the at least two kinds of polymers include a polymer having an upper critical solution temperature and a polymer having a lower critical solution temperature. Among these, a polymer having a lower critical solution temperature is preferable from the viewpoint that cells are not loaded.
The lower critical solution temperature can be calculated by measuring the cloud point of the polymer according to a total light transmittance measurement method based on the JIS K 7105 method.
The upper critical solution temperature means a temperature at which the composition enters a sol state at a temperature equal to or higher than the critical solution temperature and enters a gel state at a temperature lower than the critical solution temperature.
The lower critical solution temperature means a temperature at which the composition becomes a sol at a temperature lower than the critical solution temperature and becomes a gel at a temperature exceeding the critical solution temperature.
The critical solution temperature means a temperature at which the composition undergoes a phase change between a sol state and a gel state due to a change in temperature.

  Examples of the particle size change include swelling, which is a phenomenon in which the polymer absorbs a liquid (dispersion medium) to increase the particle size. The degree of the particle size change can be expressed by the particle size change rate, and the particle size change rate is the volume average particle size of the polymer at the lower critical solution temperature or lower than the lower critical solution temperature. The volume average particle diameter is preferably 200% or less, and more preferably 150% or less. When the particle size change rate is 200% or less, it is easy to create a three-dimensional cell aggregate preparation without damaging the structure of the three-dimensional cell aggregate by making a composition for preparing the three-dimensional cell aggregate. In addition, the polymer can be removed from the three-dimensional cell aggregate.

When the temperature is lowered from a temperature A higher than the lower critical solution temperature of the polymer to a temperature B lower than the lower critical solution temperature, the particle size change rate can be expressed by the following equation.
Change rate of particle size (%) = (Volume average particle size of polymer at temperature B below the lower critical solution temperature / Volume average particle size of polymer at temperature A exceeding the lower critical solution temperature) × 100

  The at least two polymers are preferably less toxic to cells.

-Polymer constituting the core-
The polymer constituting the core in the core-shell structure is not particularly limited as long as the rate of change in particle size due to temperature is small and the cytotoxicity is small, and can be appropriately selected according to the purpose. For example, collagen, gelatin, proteoglycan , Hyaluronic acid, fibronectin, laminin, tenascin, entactin, elastin, fibrillin and the like. Among these, gelatin is preferable.

The particle size change rate of the polymer constituting the core is preferably 10% or less, and more preferably 5% or less.
In addition, the said particle size change rate can be calculated | required similarly to the said polymer.

The volume average particle size of the polymer constituting the core at a temperature below the lower critical solution temperature of the polymer constituting the shell is preferably 0.10 μm or more and 0.5 μm or less, more preferably 0.20 μm or more and 0.30 μm or less. preferable.
The volume average particle size of the polymer constituting the core at a temperature exceeding the lower critical solution temperature of the polymer constituting the shell is preferably 0.10 μm or more and 0.5 μm or less, more preferably 0.20 μm or more and 0.30 μm or less. preferable.
The volume average particle diameter can be measured in the same manner as the volume average particle diameter of the polymer particles having the core-shell structure.

-Polymer constituting the shell-
As the polymer constituting the shell in the core-shell structure, as long as the composition for preparing a three-dimensional cell assembly is contained in the composition for preparing a three-dimensional cell assembly, the composition for preparing the three-dimensional cell assembly is a phase change due to a temperature change. The size, shape, structure, material, etc. are not particularly limited and can be appropriately selected according to the purpose.
As the polymer constituting the shell, a temperature-responsive polymer is preferable.

The lower limit critical dissolution temperature of the polymer constituting the shell is preferably 0 ° C. or higher and 50 ° C. or lower. When cells are cultured at 37 ° C., 0 ° C. or higher and 35 ° C. or lower is more preferable. When the lower critical lysis temperature is 0 ° C. or higher, the cells are not excessively cooled and the cell is not loaded, and when it is 50 ° C. or lower, the cells are not excessively heated. Is not loaded.
The lower critical solution temperature can be calculated by measuring the cloud point of the polymer constituting the shell according to the total light transmittance measurement method based on the JIS K 7105 method.

The particle size change rate of the polymer constituting the shell is preferably larger than the particle size change rate of the polymer constituting the core, more preferably greater than the particle size change rate of the polymer constituting the core and 450% or less. More preferably, it is larger than the particle size change rate of the polymer constituting the core and not more than 400%.
In addition, the said particle size change rate can be calculated | required similarly to the said polymer using the polymer which comprises the said shell before comprising a shell.

The average thickness of the polymer constituting the shell at a temperature not higher than the lower critical solution temperature of the polymer constituting the shell is preferably 0.05 μm or more and 0.5 μm or less, and more preferably 0.10 μm or more and 0.25 μm or less.
The average thickness of the polymer constituting the shell at a temperature exceeding the lower critical solution temperature of the polymer constituting the shell is preferably 0.01 μm or more and 0.10 μm or less, and more preferably 0.03 μm or more and 0.07 μm or less.
The average thickness can be calculated by the following formula.
Average thickness (μm) = (Volume average particle diameter of polymer having core-shell structure (μm) −Volume average particle diameter of polymer constituting core (μm)) / 2

  Examples of the polymer constituting the shell include alkyl acrylamide polymers such as polyisopropylacrylamide (PNIPAM) and polydiethylacrylamide; polyvinyl methyl ether, polyvinyl ether having oxyethylene in the side chain; methyl cellulose, carboxymethyl cellulose, and the like. . These may be used alone or in combination of two or more.

  The polymer constituting the core and the polymer constituting the shell may contain a hydrophilic polymer other than the temperature-responsive polymer. When the hydrophilic polymer is included in the polymer constituting the shell, the surface of the core-shell structure is preferably formed of a temperature-responsive polymer.

There is no restriction | limiting in particular as a weight average molecular weight of hydrophilic polymers other than the said temperature-responsive polymer, According to the objective, it can select suitably, 1,000-50,000 are preferable, 2,000-50, Is more preferably 5,000 or less, and particularly preferably 5,000 or more and 50,000 or less. When the weight average molecular weight is 1,000 or more, hydrophilicity can be improved. When the weight average molecular weight is 50,000 or less, the polymer itself can be prevented from becoming bulky, and the phase can be changed by temperature change. It becomes easy.
The said weight average molecular weight can be calculated | required by measuring a tetrahydrofuran soluble part, for example using a gel permeation chromatography (GPC).

  Examples of the hydrophilic polymer other than the temperature-responsive polymer include polyvinyl alcohol; polyvinyl pyrrolidone; polyacrylate; polyacrylamide; cellulose derivatives such as ethyl cellulose and hydroxyethyl cellulose; methyl starch, ethyl starch, hydroxyethyl starch, carboxymethyl, and the like. Starch derivatives such as starch; alginic acid derivatives such as propylene glycol alginate; derivatives of animal polymers such as gelatin, casein, albumin and collagen; derivatives of plant polymers such as guar gum, locust bean gum, quince seed gum and carrageenan; xanthan gum; Examples thereof include derivatives of microbial polymers such as dextran, hyaluronic acid, pullulan, and curdlan. These may be used alone or in combination of two or more.

[Method for producing polymer particles having core-shell structure]
As a method for producing the polymer particles, the hydrophilic polymer core is coated with a temperature-responsive polymer shell by polymerizing a temperature-responsive monomer in a particle dispersion after making the hydrophilic polymer into particles. You can get things.
Examples of the method for making gelatin into particles as the hydrophilic polymer include a coacervation method in which an aqueous gelatin solution is finely precipitated by adding an organic solvent such as acetone or ethanol. The micronized gelatin can be made fine particles with a conventional crosslinking agent such as an aldehyde-based crosslinking agent to obtain core particles. Not only the coacervation method but also hydrophilic core particles can be obtained by polymerization of a hydrophilic monomer and a crosslinking agent by emulsion polymerization including suspension polymerization in a general W / O emulsion. In the liquid in which the hydrophilic core particles are dispersed, when a monomer that is insolubilized at a temperature exceeding the lower critical solution temperature, such as N-isopropylacrylamide, is polymerized, the temperature-responsive polymer is insolubilized or precipitated on the core particles. Core-shell particles are obtained.

(Composition for producing three-dimensional cell aggregates)
The composition for preparing a three-dimensional cell assembly includes the material for preparing a three-dimensional cell assembly of the present invention, and further includes other components as necessary.
As the composition for preparing a three-dimensional cell assembly, the three-dimensional cell assembly preparation material containing the at least two kinds of polymers is converted into a three-dimensional cell assembly in which one component is in a state of particles such as a colloid by temperature change. Phase change from a sol state to a gel state or from a gel state to a sol state between a sol state dispersed in the composition for preparation and a fluid state and a gel state having high shear viscosity and no fluidity Can do.
Cells can be fixed by changing the state of the composition for preparing a three-dimensional cell aggregate containing the polymer from the sol state to the gel state. Moreover, it becomes easy to remove the polymer from the three-dimensional cell aggregate by changing the state of the composition for preparing a three-dimensional cell aggregate containing the polymer from a gel state to a sol state. The phase change from the sol state to the gel state is preferably performed by raising the temperature to a temperature exceeding the lower critical solution temperature of the polymer constituting the shell.

  As a change point of the phase change between the sol state and the gel state, for example, when the phase change is made from the sol state to the gel state, the polymer contracts or forms an aggregate network between the polymers. A point where the shear viscosity suddenly increases and the fluidity of the composition for preparing a three-dimensional cell assembly disappears; when the phase is changed from the gel state to the sol state, the polymer swells, For example, the shear viscosity is drastically lowered by dispersion, and the composition for preparing a three-dimensional cell aggregate is fluid.

  FIG. 1 is a schematic view showing a phase change from a sol state to a gel state of a composition for preparing a three-dimensional cell assembly containing at least two kinds of polymers having different particle size change rates. As shown in FIG. 1, for example, the composition D for preparing a three-dimensional cell assembly applied to the substrate 1 is a temperature not higher than the lower critical solution temperature of the polymer 2 contained in the composition D for preparing a three-dimensional cell assembly. Is in a sol state, has a low viscosity and does not agglomerate, and is fluidly present in the solution. When the substrate 1 is heated to a temperature exceeding the lower limit critical dissolution temperature of the polymer 2 with respect to the composition D for preparing a three-dimensional cell assembly provided on the substrate 1, the composition for preparing a three-dimensional cell assembly in a sol state D is gelled, loses its fluidity, becomes a gel-state composition for producing a three-dimensional cell assembly D ′, forms a pseudo-aggregate network, and contains the gel-state three-dimensional cell assembly preparation composition D ′ Layer is formed. On the layer, the composition D for preparing a three-dimensional cell assembly in the sol state is further applied, and a layer that is gelled and changed in phase to the composition D ′ for three-dimensional cell assembly preparation is laminated. Is done. By repeating this multiple times, it is possible to form a three-dimensional laminate in which a plurality of layers containing the gel-like three-dimensional cell assembly preparation composition D ′ are laminated. As in the case of the composition D for producing a three-dimensional cell aggregate, a three-dimensional cell aggregate can be formed by applying cells and forming a three-dimensional laminate in the same manner as described above.

The shear viscosity of the composition for preparing a three-dimensional cell assembly in the sol state is not particularly limited and may be appropriately selected depending on the intended purpose, but is preferably 0.1 Pa · s or less, preferably 0.01 Pa · s. The following is more preferable. When the shear viscosity is 0.1 Pa · s or less, the viscosity of the composition for preparing a three-dimensional cell assembly can be lowered, and therefore, it can be suitably used for an ink jet method.
There is no restriction | limiting in particular as shear viscosity of the composition for three-dimensional cell aggregate preparation of the said gel state, Although it can select suitably according to the objective, 0.2 Pa.s or more is preferable and 1 Pa.s or more is preferable. More preferably, 10 Pa · s or more is particularly preferable. When the shear viscosity is 0.2 Pa · s or more, the viscosity of the composition for preparing a three-dimensional cell assembly can be increased, and thus the cells can be immobilized.

The shear viscosity can be determined by the following measurement method.
The polymer in the composition for preparing a three-dimensional cell assembly is dispersed in pure water at a concentration of 10% by mass, and autoclaving (trade name: MLS-3030, manufactured by Panasonic Corporation) is performed at 121 ° C. for 20 minutes. . Thereafter, Dulbecco's modified Eagle containing 10% by volume fetal bovine serum (FBS, manufactured by Gibco) and 1% by weight antibiotic (trade name: Antibiotic-Antilytic Coated Solution Solution (100 ×), manufactured by Wako Pure Chemical Industries, Ltd.) The sterilized polymer is diluted to 1% by mass in a medium (Wako Pure Chemical Industries, Ltd.) and dispersed using a stirrer (trade name: ReximRS-4A, manufactured by AsOne). After confirming the dispersion, the liquid viscosity is measured with a rheometer (trade name: MCR301, manufactured by Anton Paar). Measurement with a rheometer is performed at a shear rate of 10 (1 / s) at 25 ° C. for 100 seconds (10 points every 10 seconds), and then raised to 37 ° C., with a shear rate of 0.1 (1 / s) Can be obtained by measuring for 100 seconds (10 points every 10 seconds).

  When the particle size changes, the three-dimensional cell aggregate preparation composition may be anisotropically deformed or may be isotropically changed. Among these, it is preferable that the three-dimensional cell aggregate preparation composition is deformed only in a specific direction when the particle size is changed in order to prevent the cells from being easily damaged at the time of peeling.

  The polymer particles are preferably dispersed uniformly in the composition for preparing a three-dimensional cell assembly in a sol state. When the polymer particles are uniformly dispersed, the polymer particles can be suitably used for an ink jet method.

  A known temperature raising device can be used as a device for phase-changing the sol-shaped composition for producing a three-dimensional cell aggregate containing the polymer into a gel state. The temperature raising device is not particularly limited as long as the temperature can be raised to a temperature exceeding the lower critical solution temperature of the polymer, and examples thereof include known culture plates and incubators.

  The content of the polymer is preferably 0.1% by mass or more and 10% by mass or less, and preferably 0.5% by mass or more and 3% by mass with respect to the total amount of the composition for preparing a three-dimensional cell aggregate before being arranged on the substrate. % Or less is more preferable. When the content is 0.1% by mass or more, gelation can be suitably performed, and when the content is 10% by mass or less, toxicity to cells can be reduced.

<Other ingredients>
There is no restriction | limiting in particular as said other component, According to the objective, it can select suitably, For example, a crosslinking agent, surfactant, pH adjuster, antiseptic | preservative, antioxidant, etc. are mentioned.

(3D cell assembly production set)
The set for preparing a three-dimensional cell assembly of the present invention has a composition for preparing a three-dimensional cell assembly and cells, and further includes other components as necessary.

As the composition for preparing a three-dimensional cell assembly, the composition for preparing a three-dimensional cell assembly of the present invention can be suitably used.
The volume average particle size of the polymer particles in the sol-state three-dimensional cell aggregate preparation composition is preferably smaller than the volume average particle size of the cells in the free state.

<Cell>
As long as the cells can be applied on the substrate, the type thereof is not particularly limited and can be appropriately selected according to the purpose. Taxonomically, for example, eukaryotic cells, prokaryotic cells, It can be used for all cells, regardless of whether they are multicellular organism cells or unicellular organism cells.
Examples of the eukaryotic cells include animal cells, insect cells, plant cells, and fungi. These may be used alone or in combination of two or more. Among these, animal cells are preferable, and from the point that the cells form a three-dimensional cell aggregate, the cells adhere to each other and have cell adhesiveness that cannot be isolated without physicochemical treatment. Adhesive cells having are more preferred.

The adherent cell is not particularly limited and may be appropriately selected depending on the intended purpose. Examples thereof include differentiated cells and pre-differentiation cells.
Examples of the differentiated cells include liver cells that are liver parenchymal cells; stellate cells; Kupffer cells; vascular endothelial cells; endothelial cells such as canalic endothelial cells and corneal endothelial cells; fibroblasts; osteoblasts; Bone cells; periodontal ligament-derived cells; epidermal cells such as epidermal keratinocytes; tracheal epithelial cells; gastrointestinal epithelial cells; cervical epithelial cells; epithelial cells such as corneal epithelial cells; mammary cells; Examples include muscle cells such as cardiomyocytes; kidney cells; pancreatic islets of Langerhans; nerve cells such as peripheral nerve cells and optic nerve cells; chondrocytes; These may be used alone or in combination of two or more. The adherent cells may be primary cells collected directly from tissues or organs, or may be obtained by passage of them for several generations.
The cells before differentiation are not particularly limited and can be appropriately selected according to the purpose. For example, pluripotent stem cells such as embryonic stem cells which are undifferentiated cells, mesenchymal stem cells having multipotency Unipotent stem cells such as vascular endothelial progenitor cells having unipotency; cells that have completed differentiation; iPS cells and the like. These may be used alone or in combination of two or more.

  Examples of the prokaryotic cells include eubacteria and archaea. These may be used alone or in combination of two or more.

  The volume average particle size of the cells in the free state is preferably 100 μm or less, more preferably 50 μm or less, and particularly preferably 20 μm or less. If the volume average particle diameter is 100 μm or less, it can be suitably used in an ink jet method.

The volume average particle diameter of the cells can be measured by the following measurement method.
In an incubator (trade name: KM-CC17RU2, manufactured by Panasonic Corporation, 37 ° C., 5% CO ₂ environment), 1% by mass antibiotic (Antibiotic-Antilytic Coated Solution Solution (100 ×), manufactured by Wako Pure Chemical Industries, Ltd.) ) Cell culture in Dulbecco's modified Eagle medium (manufactured by Wako Pure Chemical Industries, Ltd., hereinafter also referred to as “D-MEM”), and 100 mm dish with an aspirator (trade name: VACUSIP, manufactured by INTEGRA) 10% by volume fetal bovine serum (hereinafter also referred to as “FBS”) and the medium are removed. 3 mL of phosphate buffered saline (made by Wako Pure Chemical Industries, Ltd., hereinafter sometimes referred to as “PBS (−)”) is added to the dish, and PBS (−) is removed by suction with an aspirator, and the surface is washed. . After repeating the washing operation with PBS (−) three times, 3 mL of 0.1 mass% trypsin solution (Trypsin, from Porcine Pancreas, manufactured by Wako Pure Chemical Industries, Ltd.) was added, and the mixture was heated in an incubator for 5 minutes. Detach cells from the dish. After confirming cell detachment with a phase contrast microscope, 4 mL of D-MEM containing 10% by mass FBS and 1% by mass antibiotic is added to inactivate trypsin. Transfer to a centrifuge tube, centrifuge (trade name: H-19FM, manufactured by KOKUSAN, 1.2 × 10 ³ rpm (234G), 5 min, 5 ° C.), and remove the supernatant with an aspirator. After removal, 1 mL of D-MEM containing 10% by mass FBS and 1% by mass antibiotic is added to the centrifuge tube, and gently pipetting to disperse the cells. 10 μL of the cell suspension is taken out into an Eppendorf tube, 10 μL of 0.4 mass% trypan blue staining solution is added, and the cells are stained by pipetting. 10 μL is taken out from the stained cell suspension, placed on a plastic slide made of PMMA, and can be measured using an automatic cell counter (trade name: Countess Automated Cell Counter, manufactured by Invitrogen). The number of cells and cell viability can also be determined by the same measurement method. When the cells are in a free state, the cells have a substantially spherical shape, and thus the volume average particle diameter can be measured.

(Composition container)
The composition storage container of the present invention contains the composition for preparing a three-dimensional cell assembly of the present invention in a container, and further has other members as necessary.

  The container is not particularly limited, and its shape, structure, size, material and the like can be appropriately selected according to the purpose. For example, a composition bag formed of an aluminum laminate film, a resin film, or the like The thing etc. which have are mentioned.

(Method for producing three-dimensional cell aggregate)
The method for producing a three-dimensional cell aggregate of the present invention includes a composition application step, preferably a cell application step, and further includes other steps as necessary.
By sequentially repeating the composition application step and the cell application step, a laminate (three-dimensional cell assembly) composed of a composition for preparing a three-dimensional cell assembly and cells can be prepared.

<Composition application process>
The said composition provision process is a process of providing the said composition for three-dimensional cell aggregate preparation on a base | substrate. Here, the term “on the substrate” means that a composition for preparing a three-dimensional cell assembly is applied on the substrate, then cells are applied on the composition for preparing a three-dimensional cell assembly, and then a tertiary is applied on the cells. This also includes the case where a composition for preparing original cell aggregates is further applied.

  The application temperature in the composition application step is preferably equal to or lower than the minimum temperature of the lower critical solution temperature of the polymer having the lower critical solution temperature.

<Cell application process>
The cell application step is a step of applying cells on the substrate.
The term “on the substrate” includes the case where cells are provided on the composition for preparing a three-dimensional cell assembly after the composition for preparing a three-dimensional cell assembly is applied on the substrate.
The cells are preferably applied as a dispersion liquid dispersed in a liquid such as a culture medium.

There is no restriction | limiting in particular as the number of the cells in the said dispersion liquid, According to the objective, it can select suitably, 5 * 10 < ⁵ > / mL or more and 5 * 10 < ⁸ > / mL or less are preferable, and 5 * 10 < ⁵ >. More preferably, the number per piece / mL is 5 × 10 ⁷ pieces / mL or less. When the number of cells is 5 × 10 ⁵ cells / mL or more and 5 × 10 ⁸ cells / mL or less, the discharged droplets can surely contain cells, which is suitable for precise cell arrangement. The number of cells can be measured using an automatic cell counter (trade name: Countess Automated Cell Counter, manufactured by Invitrogen) in the same manner as in the method for measuring the volume average particle diameter.

-Method for applying composition for preparing three-dimensional cell aggregate and method for applying cells-
Examples of the method for applying the composition for preparing a three-dimensional cell assembly and the method for applying the cells include an inkjet method, a dispenser method, a pipette method, and an aspirator method. These may be used alone or in combination of two or more. Among these, the inkjet method is preferable from the viewpoint that precise and complicated cell arrangement can be performed at the level of one cell.

  The ink jet method is not particularly limited, and a known method can be used. In order to carry out the ink jet method, a known ink jet discharge device can be suitably used as the cell and composition applying means.

<< Substrate >>
The substrate is not particularly limited in size, shape, structure, material and the like as long as it does not inhibit cell activity or proliferation, and can be appropriately selected according to the purpose.
Examples of the shape include three-dimensional shapes such as dishes, multi-plates, flasks, and cell inserts; flat membrane shapes.
Examples of the structure include a porous structure.
Examples of the material of the base include organic materials and inorganic materials. These may be used alone or in combination of two or more.
Examples of the organic material include polyethylene terephthalate (PET), polystyrene (PS), polycarbonate (PC), TAC (triacetyl cellulose), polyimide (PI), nylon (Ny), low density polyethylene (LDPE), and medium density. Acrylic materials such as polyethylene (MDPE), vinyl chloride, vinylidene chloride, polyphenylene sulfide, polyether sulfone, polyethylene naphthalate, polypropylene, urethane acrylate, cellulose, and the like. These may be used alone or in combination of two or more.
Examples of the inorganic material include glass and ceramic. These may be used alone or in combination of two or more.

Before applying the cell and the composition for preparing a three-dimensional cell aggregate on the substrate, it is preferable to apply an organic substance that promotes cell adhesion to the surface of the substrate according to a known method. By applying the organic matter, adhesion of the cells can be promoted.
There is no restriction | limiting in particular as said organic substance, It is a substance in connection with adhesion | attachment of cells or an extracellular matrix, for example, peptides derived from organisms, such as laminin, collagen, fibronectin, gelatin; And proteins having the same structure as the above; peptides such as arginine-glycine-aspartic acid (RGD) and the like. These may be used alone or in combination of two or more.

The cell viability after applying the cells on the substrate is preferably 90% by number or more, more preferably 95% by number or more, with respect to the survival rate in the dispersion before the cells are applied on the substrate. A number% or more is particularly preferred. When the cell viability is 90% by number or more, a load due to the application of cells onto the substrate is reduced, and a three-dimensional cell aggregate can be formed.
Examples of the cell viability measuring apparatus include an automatic cell counter (trade name: Countess Automated Cell Counter, manufactured by Invitrogen) and the like in the same manner as the volume average particle diameter measuring method.

<< Inkjet dispenser >>
The inkjet discharge device is not particularly limited as long as it can apply a composition for preparing cells and a three-dimensional cell aggregate on a substrate. And a composition containing part for preparing original cell aggregates, and further having other means as required.
Examples of the means for applying the composition for preparing cells and three-dimensional cell aggregates include means for applying the composition for preparing cells and three-dimensional cell aggregates on a substrate, and for preparing cells and three-dimensional cell aggregates. Means for simultaneously applying the composition on a substrate; means for applying a composition for preparing a three-dimensional cell aggregate on the cells after the cells have been applied on the substrate, and the like.

  The means for applying the composition for preparing cells and three-dimensional cell aggregates preferably has a nozzle capable of applying the composition for preparing cells and three-dimensional cell aggregates onto the substrate by an ink jet method. In addition, as the nozzle, a nozzle (ejection head) in a known inkjet printer can be suitably used, and the inkjet printer is suitably used as a composition applying means for producing the cells and three-dimensional cell aggregates. can do. In addition, as said inkjet printer, SG7100 by Ricoh Co., Ltd. etc. are mentioned suitably, for example. The ink jet printer is preferable in that the application amount can be increased because the amount of the composition for preparing cells and three-dimensional cell aggregates that can be dripped from the head portion is large and the application area is large.

  The cell and 3D cell assembly preparation composition applying means applies a stimulus to the cell and the 3D cell assembly preparation composition to provide the cell and 3D cell assembly preparation composition. If it is a means to make it form a laminated body, there will be no restriction | limiting in particular, According to the objective, it can select suitably, A continuous injection type, an on-demand type, etc. are mentioned. Examples of the on-demand type include a piezo method, a thermal method, and an electrostatic method. Among these, the piezo method is particularly preferable.

Specifically, the composition applying means for preparing the cells and the three-dimensional cell aggregate preferably has a liquid chamber part, a fluid resistance part, a diaphragm, a nozzle member, and the like. The liquid chamber part, the fluid resistance part It is preferable that at least a part of the diaphragm and the nozzle member is formed of a material containing at least one of silicone and nickel.
Moreover, there is no restriction | limiting in particular as a nozzle diameter of the said nozzle, In order to avoid a cell clogging a nozzle part, 2 times or more with respect to the diameter of a cell is preferable. Since the size of animal cells, particularly human cells, is generally 5 μm or more and 30 μm or less, the nozzle diameter is preferably 10 μm or more and 200 μm or less in accordance with the cells to be used. On the other hand, 60 μm or less is more preferable because it becomes difficult to achieve the original purpose of forming microdroplets when the droplets are too large. Among these, when it is 10 μm or more, a dispersion containing cells can be suitably discharged, and therefore, it is particularly preferably 10 μm or more and 60 μm or less from the viewpoint that it can be suitably used for an ink jet method.
The diameter of the cell can be measured using a stereomicroscope.

The cell and the composition for preparing a three-dimensional cell aggregate is a member in which the cell and the composition for preparing a three-dimensional cell aggregate are accommodated, and the size, shape, material, and the like are not particularly limited. However, it can be appropriately selected according to the purpose, and examples thereof include a storage tank, a bag, a cartridge, and a tank.
When using means for simultaneously arranging the cells and the composition for producing a three-dimensional cell aggregate on a substrate, a mixed dispersion liquid in which the cells and the composition for producing a three-dimensional cell aggregate are mixed is contained in the member. It is preferable to do.
When using means for applying a composition for preparing a three-dimensional cell aggregate on the cells after the cells have been applied on the substrate, a dispersion containing the cells and the preparation for the three-dimensional cell aggregates are used. It is preferable that the member which accommodates the composition is separate.

  Here, FIG. 2 shows an example of an inkjet discharge apparatus. This ink jet discharge apparatus has an ink jet head 40.

  The inkjet discharge apparatus applies a mixed dispersion liquid 20 in a sol state in which cells and a composition for producing a three-dimensional cell aggregate are mixed onto a substrate 10 using an inkjet head 40. At this time, the temperature of the substrate 10 is raised to a temperature exceeding the lower critical solution temperature of the polymer by using the temperature adjusting device 50. Before applying the mixed dispersion 20 on the substrate 10 by the temperature adjusting device 50, it is preferable to warm the substrate 10 and keep it at a temperature exceeding the lower critical solution temperature of the polymer. As a result, the composition for preparing a three-dimensional cell assembly in the mixed dispersion 20 applied to the substrate 10 is phase-changed from the sol state to the gel state, and the cells are fixed to form the cell assembly 60. The mixed dispersion 20 is applied onto the cell aggregate 60 in the same manner as described above, and the three-dimensional cell aggregate preparation composition is phase-changed from a sol state to a gel state to form the cell aggregate 60. By repeating a plurality of times, a three-dimensional cell aggregate can be obtained.

  FIG. 3 shows another example of the inkjet apparatus. The principle of the ink jet ejection apparatus of FIG. 3 is the same as that of FIG. 2, but the composition applying means for producing cells and three-dimensional cell aggregates is different. That is, after the cells 120 are applied onto the substrate 100 using the inkjet head 140, the sol-state three-dimensional cell aggregate preparation composition 130 is applied onto the cells 120. As in FIG. 2, the three-dimensional cell aggregate preparation composition heated to a temperature exceeding the lower critical solution temperature of the polymer by the temperature controller 150 is phase-changed from the sol state to the gel state, Aggregates 160 are formed. The cell 120 is provided on the cell aggregate 160 by the same operation as described above, the three-dimensional cell aggregate preparation composition 130 is provided on the cell 120, and the three-dimensional cell aggregate preparation composition is provided. By repeating the phase change from sol to gel and forming the cell aggregate 160 a plurality of times, a three-dimensional cell aggregate can be obtained.

<Other processes>
As the other steps, the cells and the composition for preparing a three-dimensional cell aggregate are applied on a substrate, and then the phase change of the composition for preparing a three-dimensional cell aggregate from a sol state to a gel state is performed. A step of culturing cells at a temperature exceeding the maximum temperature of the lower critical solution temperature of the polymer having the lower critical solution temperature (hereinafter sometimes referred to as “culture step”), and after the incubation step, The temperature of the polymer having a dissolution temperature is lowered to a temperature lower than the lowest critical dissolution temperature, and the composition for preparing a three-dimensional cell aggregate is phase-changed from a gel state to a sol state (hereinafter also referred to as “solation”). ) Step (hereinafter also referred to as a “solation step”) and a step of removing the polymer (hereinafter also referred to as a “removal step”) after the solification step.

<< Culture process >>
In the culturing step, after changing the composition for preparing a three-dimensional cell assembly from a sol state to a gel state, cells are cultured at a temperature exceeding the maximum temperature of the lower critical solution temperature of the polymer having the lower critical solution temperature. It is a process. By culturing the cells, the cells proliferate and expand, and adhere to each other, whereby a three-dimensional cell aggregate can be formed.

  The culture is not particularly limited, and conditions such as culture temperature, culture apparatus, culture time, medium, concentration of carbon dioxide in the medium, medium supply method, medium supply amount, and pH are appropriately selected according to the purpose. be able to.

  The culture temperature is not particularly limited and may be appropriately selected depending on the intended purpose. It is preferably 25 ° C. or higher and 45 ° C. or lower, more preferably 33 ° C. or higher and 40 ° C. or lower, and particularly preferably 36 ° C. or higher and 38 ° C. or lower. preferable. When the culture temperature is 25 ° C. or higher and 45 ° C. or lower, the cells can be suitably cultured.

  The culture apparatus is not particularly limited as long as the cells can be incubated at the optimum culture temperature and can be appropriately selected according to the purpose. Examples thereof include known culture plates and incubators.

There is no restriction | limiting in particular as said culture | cultivation time, Although it can select suitably according to the objective, 12 hours or more and 60 hours or less are preferable, and 15 hours or more and 50 hours or less are more preferable. When the culture time is 12 hours or more and 60 hours or less, the cells can be suitably cultured.
In addition, in order to confirm whether cells are proliferating and spreading and adhere to each other to form a three-dimensional cell aggregate, the tissue is stained with a hematoxylin-eosin method (HE), for example, NHDF By observing the cross-section with the tissue cross-section observation image by the above procedure, it can be confirmed by the void ratio in the tissue and the degree of cell extension.

  The medium is not particularly limited and may be appropriately selected depending on the intended purpose. For example, a medium classified according to the composition of natural medium, semi-synthetic medium, synthetic medium, etc .; semi-solid medium, liquid medium, powder medium (Hereinafter, also referred to as “powder culture medium”) and the like. These may be used alone or in combination of two or more. When the cells are derived from animals, any medium can be used as long as it is a medium used for culturing animal cells.

  There is no restriction | limiting in particular as a culture medium used for culture | cultivation of the said animal cell, According to the objective, it can select suitably, For example, Dulbecco's Modified Eagle's Medium (Dulbecco's Modified Eagles's Medium; D-MEM), ham F12 medium (Ham's Nutrient Mixture F12), D-MEM / F12 medium, McCoy's 5A medium, Eagle's Medium Essential medium; EMEM medium, EMEM s Minimum Essential Medium (αMEM), MEM medium (Minimum Essential Medium), RPMI 1640 medium, Iskov modified Dulbecco's medium (Iscove's Modified Dulbecco's Medium; IMDM), MCDB131 medium, William medium E, IPL41 medium, Fischer's medium, StemPro34 (manufactured by Invitrogen), X-VIVO 10 (manufactured by Cambridge) X-VIVO 15 (manufactured by Cambridge), HPGM (manufactured by Cambridge), StemSpan H3000 (manufactured by Stem Cell Technology), StemSpan SFEM (manufactured by Stem Cell Technology), Stemline II (manufactured by Sigma Aldrich), QBSF-60 (quality bio) Logical Corp.), StemProhESCSFM (Invitrogen Corp.), Essential8 (registered trademark) medium (Gibco Corp.), m eSR1 or 2 medium (manufactured by Stem Cell Technology), Ripro FF or Ripro FF2 (manufactured by Riprocel), PSGro hESC / iPSC medium (manufactured by System Bioscience), NutriStem (registered trademark) medium (manufactured by Biological Industries), CSTI -7 medium (manufactured by Cell Science Laboratory), MesenPRO RS medium (manufactured by Gibco), MF-Medium (registered trademark) mesenchymal stem cell growth medium (manufactured by Toyobo Co., Ltd.), Sf-900II (manufactured by Invitrogen), Opti-Pro (manufactured by Invitrogen) and the like can be mentioned. These may be used alone or in combination of two or more.

  The carbon dioxide concentration in the medium is not particularly limited and may be appropriately selected depending on the intended purpose, but is preferably 2% or more and 5% or less, and more preferably 3% or more and 4% or less. When the carbon dioxide concentration is 2% or more and 5% or less, the cells can be suitably cultured.

  There is no restriction | limiting in particular as the supply method of the said culture medium, For example, it can select suitably according to the objective, The inkjet method, the dispenser method, the pipette method, the aspirator method etc. are mentioned. These may be used alone or in combination of two or more.

The amount of the medium to be supplied is preferably 5 × 10 ⁵ or more and 5 × 10 ⁸ or less, preferably 5 × 10 ⁵ or more and 5 × 10 ^{7 to} 1 mL of medium. It is more preferable to supply so that it may become less. When the number of the cells is 5 × 10 ⁵ or more and 5 × 10 ⁸ or less with respect to 1 mL of the medium, it is suitable for cell growth and extension. The number of cells can be measured using an automatic cell counter (trade name: Countess Automated Cell Counter, manufactured by Invitrogen) in the same manner as in the method for measuring the volume average particle diameter.

  There is no restriction | limiting in particular as said pH, Although it can select suitably according to the objective, 4-7 is preferable and 5-6 is more preferable.

  In the culturing step, the sterilized reagent is preferably used as the reagent to be used, and the sterilized one is preferably used for the culturing operation and the culturing apparatus.

<< Zolization process >>
The solification step is a step of culturing the cells and then lowering the temperature to a temperature below the lower critical solution temperature of the polymer to change the composition for preparing a three-dimensional cell aggregate from a gel state to a sol state. The three-dimensional cell aggregate can be taken out by changing the phase of the composition for preparing a three-dimensional cell aggregate from a gel state to a sol state.

  There is no restriction | limiting in particular as temperature of the said temperature fall, What is necessary is just below the lower critical solution temperature of the said polymer, 0 to 50 degreeC is preferable and 0 to 32 degreeC is more preferable. A known device can be used for the temperature drop. The apparatus is not particularly limited as long as the temperature can be lowered below the lower critical solution temperature of the polymer, and examples thereof include known culture plates and incubators.

<< Removal process >>
The removing step is a step of removing the polymer after culturing the cells and changing the composition for preparing a three-dimensional cell assembly from a gel state to a sol state. By removing the polymer in the sol-state composition for producing a three-dimensional cell assembly from the three-dimensional cell assembly, only the three-dimensional cell assembly can be obtained. In the three-dimensional cell assembly, all the polymers are not removed, and even if the polymer remains in the whole or a part of the three-dimensional cell assembly, it can be used without any problem.

The removal is not particularly limited as long as it is a mild condition that does not break the adhesion between cells, and can be appropriately selected according to the purpose. Examples thereof include washing with a washing solution.
Examples of the washing liquid include a phosphate buffer (hereinafter also referred to as “PBS”), a Tris-HCl buffer, and the like.

(Three-dimensional cell assembly)
The three-dimensional cell aggregate includes cells and the composition for producing a three-dimensional cell aggregate of the present invention. Production of three-dimensional cell aggregates that contain polymer particles whose volume average particle size in the sol composition is smaller than the volume average particle size of cells in the free state, and change in phase between the sol state and the gel state due to temperature changes It contains a composition for use and further has other members as required.
The three-dimensional cell aggregate can be preferably produced by the method for producing a three-dimensional cell aggregate of the present invention.
As the at least two kinds of polymers, the same materials as those for producing a three-dimensional cell assembly of the present invention can be used.

  There is no restriction | limiting in particular as a magnitude | size, a shape, a structure, etc. of the said three-dimensional cell aggregate | assembly, According to the objective, it can select suitably.

  There is no restriction | limiting in particular as average thickness of the said three-dimensional cell aggregate | assembly, Although it can select suitably according to the objective, 0.01 mm or more and 20 mm or less are preferable, 0.05 mm or more and 20 mm or less are more preferable, and 0. 1 mm or more and 20 mm or less are particularly preferable. When the average thickness is 0.01 mm or more and 20 mm or less, it can be suitably used for transplantation to a damaged part of a human body and for a drug screening material. The average thickness is obtained by staining the tissue with HE and using cross-sectional observation, or in the case of non-destructive means, optical coherence tomography (OCT: Optical Coherence Tomography, trade name: Ganymede II, manufactured by Thorlabs) Can be measured.

There is no restriction | limiting in particular as a shape of the said three-dimensional cell aggregate | assembly, According to the objective, it can select suitably, For example, planar shape, such as a film form and a sheet form; Columnar shape; Spherical shape; Blood vessel shape; Complex organ The shape of these is mentioned.
Examples of the complex organ include heart, liver, kidney, lung, small intestine, large intestine and the like.

  The remaining amount of the polymer in the three-dimensional cell aggregate is preferably 3% by mass or less, and more preferably 1% by mass or less with respect to the total amount of the three-dimensional cell aggregate. When the content is 3% by mass or less, toxicity to cells can be reduced.

In order to isolate the polymer in the three-dimensional cell aggregate, the following steps are performed. That is, after removing the culture medium of the cultured tissue and washing with PBS, a collagenase solution (trade name: NB 4G / 0.26 PZ U / mL, manufactured by Cosmo Bio Co., Ltd.) is added to the inside of the incubator (product) Name: KM-CC17RU2, Panasonic Corporation, 37 ° C., 5% CO ₂ atmosphere) is allowed to stand for 2 hours, and cells are isolated from the binding between cells and between ECM (extracellular matrix) / cells. Next, the solution was separated with a mesh filter having a diameter of 20 μm, and the filtered solution was transferred to a centrifuge tube and centrifuged (trade name: H-19FM, 1.2 × 10 ³ rpm (234G), 5 minutes, And collect the supernatant containing polymer particles with a pipette. After collection, 1 mL of PBS is added to the centrifuge tube and gently pipetted to disperse the cells and the remaining polymer. The above-mentioned centrifugation and supernatant recovery are repeated three times, and the supernatant is collected to obtain a polymer dispersion constituting a three-dimensional cell aggregate. Using a concentrated particle size analyzer (trade name: FPAR-1000, manufactured by Otsuka Electronics Co., Ltd.), the residual amount of the polymer in the three-dimensional cell aggregate and the particle size distribution of the dispersion containing the polymer can be measured. it can.

  The three-dimensional cell aggregate is preferably used for transplantation to a damaged site of a human body, transplantation material for tissue regeneration that induces tissue regeneration, in vitro examination material for tissue morphogenesis process, drug screening material, and the like. it can.

Examples of the present invention and comparative examples will be described below, but the present invention is not limited to these examples.
The volume average particle size and the particle size distribution of the polymer having the core structure, the polymer having the shell structure, and the polymer particles having the core / shell structure were measured as follows.

-Volume average particle size and particle size distribution of a polymer having a core structure, a polymer having a shell structure, and a polymer particle having a core-shell structure-
As the volume average particle size and particle size distribution, a sample solution was prepared as follows using a concentrated particle size analyzer (trade name: FPAR-1000, manufactured by Otsuka Electronics Co., Ltd.), and the obtained sample solution was It can be measured under the following measurement conditions.
-Preparation of sample solution-
A powdered polymer synthesized so as to be 0.5% by mass was swollen and dispersed in pure water obtained using a pure water production apparatus (trade name: GSH-2000, manufactured by ADVANTEC). The liquid volume for the measurement was 5 mL, and the dispersion was a sample liquid prepared by stirring a 20 mm rotator at 200 rpm for about one day using a stirrer.
-Measurement conditions-
Solvent: water (refractive index: 1.3314, viscosity at 25 ° C .: 0.884 mPa · s (cP), optimal light amount adjustment is appropriately set by ND filter)
Measurement probe: Concentration probe Measurement routine: Measurement: 180 ° C. at 25 ° C. → Measurement: 600 ° C. at 25 ° C. (The liquid temperature gradually changes from 25 ° C. to 35 ° C. when the body side is changed to 35 ° C. Diameter change is monitored) → Measurement: 35 ° C for 180 seconds

-Particle size change rate-
Using the obtained polymers and the volume average particle size of the polymer particles, the particle size change rate was determined by the following formula.
Change rate of particle size (%) = (Volume average particle size of polymer at temperature B below the lower critical solution temperature / Volume average particle size of polymer at temperature A exceeding the lower critical solution temperature) × 100

Example 1
<Preparation of polymer particle A having core-shell structure>
Gelatin (made by Nitta Gelatin Co., Ltd.) was mixed with pure water so as to be 2% by mass and dissolved in a 60 ° C. hot water bath. 40 g of the warmed 2% by weight gelatin aqueous solution was placed in a 200 mL beaker and stirred, and 60 g of acetone was added all at once. 400 μL of 24 mass% to 26 mass% glutaraldehyde (manufactured by Tokyo Chemical Industry Co., Ltd.) as a crosslinking agent was added to the aqueous solution to which acetone was added, and heated for 30 minutes while stirring at 300 rpm on a 60 ° C. hot plate. After the heating, the temperature was returned to room temperature, and 100 g of acetone was further added to agglomerate and precipitate the polymer particles. In order to remove moisture from the precipitate, removal of the supernatant and washing with acetone were repeated several times. The precipitate was dried on a 60 ° C. hot plate for a while and then dried under reduced pressure on a 50 ° C. hot plate for 3 hours to obtain powdered polymer particles. The yield was 0.58 g, and the yield was about 72%.

  Next, the polymer particles were mixed with pure water so as to be 1% by mass, and dispersed firmly by ultrasonic dispersion. 45 g of the dispersed polymer aqueous solution is put into a 100 mL two-necked flask (manufactured by Shibata Kagaku Co., Ltd.), and N-isopropylacrylamide (lower critical solution temperature: 32 ° C., manufactured by Tokyo Chemical Industry Co., Ltd.) 500 mg as a monomer, a crosslinking agent. As a surfactant, 5 mg of N, N′-methylenebisacrylamide (manufactured by Tokyo Chemical Industry Co., Ltd.) and 50 mg of sodium dodecylbenzenesulfonate (manufactured by Kanto Chemical Co., Ltd.) as a surfactant were added. 1% by mass (93 mmol / L) of N-isopropylacrylamide with respect to the total amount of water, 1% by mass (0.73 mol%) of N, N′-methylenebisacrylamide with respect to the total amount of N-isopropylacrylamide, On the other hand, sodium dodecylbenzenesulfonate was prepared to be 0.1% by mass, and was stirred and completely dissolved by a 2 mm rotator to obtain an aqueous solution.

Next, the aqueous solution was purged with argon gas at room temperature (25 ° C.) at a flow rate of 100 mL / min for 15 minutes. Further, the aqueous solution was flowed with argon gas at a flow rate of 100 mL / min for 15 minutes in a 70 ° C. oil bath. Dissolve 10 mg of 2,2′-azobis (2-methylpropionamidine) dihydrochloride (manufactured by Wako Pure Chemical Industries, Ltd.) as a polymerization initiator in 2.5 g of water, and 2,2′- with respect to the total amount of N-isopropylacrylamide. A solution prepared so that azobis (2-methylpropionamidine) dihydrochloride was 2% by mass (0.83 mol%) was added and polymerized for 1 hour. After the polymerization, the temperature was returned to room temperature, and the polymerization solution was firmly dispersed by ultrasonic dispersion. The polymerization solution was heated and coagulated in a 60 ° C. hot water bath, and the supernatant was removed. The precipitate was placed in a Teflon (registered trademark) beaker and dried with an 80 ° C. dryer until there was no moisture. A small amount of acetone was added to the dried product, the dried product was redispersed by stirring, and hexane was slowly added to cause reprecipitation. After removing the supernatant, hexane addition was repeated several times and dried on a hot plate at 60 ° C., and then powdered polymer particles A having a core-shell structure were prepared. The yield was 0.21 g and the yield was about 20%.
FIG. 4 shows the particle size distribution of the polymer particles A having a core-shell structure. For comparison, the particle size distribution of gelatin particles constituting the core is also shown.
In addition, it was 300% when the particle size change rate was calculated | required similarly to the polymer particle A using N-isopropylacrylamide which is a polymer which comprises a shell.

(Example 2)
<Production of three-dimensional cell aggregate>
-Preparation of composition A for preparing a three-dimensional cell aggregate containing polymer particles A having a core-shell structure-
The polymer particles A having the core-shell structure were dispersed with pure water at 10% by mass and subjected to high-pressure steam sterilization (trade name: MLS-3030, manufactured by Panasonic Corporation) at 121 ° C. for 20 minutes. Thereafter, Dulbecco's modified Eagle's medium (D-) containing 10% by volume fetal bovine serum (FBS, manufactured by Gibco) and 1% by weight antibiotic (Antibiotic-Antilytic Mix Solution (100x), manufactured by Wako Pure Chemical Industries, Ltd.) (MEM: manufactured by Wako Pure Chemical Industries, Ltd.) The polymer particles A having a sterilized core-shell structure are diluted to 0.4% by mass and dispersed using a stirrer (trade name: ReximRS-4A, manufactured by AsOne). A composition A for preparing a three-dimensional cell assembly was obtained.

[Confirmation of phase state accompanying phase change due to temperature change of polymer particle dispersion A having core / shell structure containing polymer particle A having core / shell structure]
The composition A for preparing a three-dimensional cell assembly is subjected to 37 ° C., which is a temperature exceeding the lower critical solution temperature (32 ° C.) of the polymer A having the core / shell structure, and the lower critical solution of the polymer particle A having the core / shell structure. It left still at 25 degrees C which is the temperature or less, and confirmed the phase state of the composition for three-dimensional cell assembly preparation visually. FIG. 5A is a photograph showing a state at 25 ° C. of a composition for preparing a three-dimensional cell aggregate containing the polymer particle A having the core-shell structure containing a polymer constituting a shell having a lower critical solution temperature of 32 ° C. . FIG. 5B is a photograph showing a state at 37 ° C. of a composition for preparing a three-dimensional cell aggregate containing the polymer particles A containing a polymer constituting a shell having a lower critical solution temperature of 32 ° C.

  As shown in FIG. 5A, it can be confirmed that the polymer particle composition A exists in a sol state at 25 ° C. where the lower critical solution temperature of the polymer constituting the shell is 32 ° C. or less. On the other hand, as shown in FIG. 5B, it was confirmed that the polymer particle composition A having the core-shell structure was present in a gel state at 37 ° C., which is lower than the lower critical solution temperature of 32 ° C.

[Confirmation of relationship between shear viscosity of polymer particle A having core-shell structure and temperature]
The relationship between the shear viscosity and the temperature of the composition for preparing a three-dimensional cell assembly containing the polymer particles A having the core-shell structure was measured and confirmed as follows.
1% by mass of polymer particles A having a core-shell structure containing a polymer constituting a shell having a lower critical solution temperature of 32 ° C. (volume average particle size: 0.43 μm), and 10% by volume fetal calf serum at 25 ° C. Disperse in Dulbecco's modified eagle medium (DMEM) and measure the shear viscosity at 25 ° C for 100 seconds, then increase the temperature to 37 ° C and measure for another 100 seconds, then lower the temperature to 25 ° C and measure for 100 seconds did. FIG. 6 is a graph showing the relationship between the shear viscosity of the composition A for preparing a three-dimensional cell assembly and the temperature. As shown in FIG. 6, the shear viscosity of the composition for preparing a three-dimensional cell assembly containing polymer particles A when the temperature is raised from 25 ° C., which is lower than the lower critical solution temperature, to 37 ° C., which exceeds the lower critical solution temperature, It rose about 1,600 times in 40 seconds, and it was confirmed that the temperature response speed related to the phase change between the sol state and the gel state was fast. Further, the shear viscosity of the composition for preparing a three-dimensional cell assembly when the temperature is lowered from 37 ° C. exceeding the lower critical solution temperature to 25 ° C. which is lower than the lower critical solution temperature is about 1 in 30 seconds. It decreased 350 times, and it was confirmed that the temperature response speed related to the phase change between the gel state and the sol state was fast.

-Cell preparation example 1-
A frozen green fluorescent dye (trade name: Cell Tracker Green, manufactured by Life Technology) is thawed to room temperature, dissolved in dimethyl sulfoxide (hereinafter sometimes referred to as “DMSO”) at a concentration of 10 mM, and a serum-free medium. And a serum-free medium containing 5 mL / dish of a green fluorescent dye was prepared. Next, human dermal fibroblasts (trade name: CC2507, manufactured by Lonza, Inc., hereinafter sometimes referred to as “NHDF”) that have been cultured in a 35 mm dish were collected with an aspirator, 2 mL of serum medium was added and cultured in an incubator (37 ° C., 5% CO ₂ environment) for 30 minutes. In addition, the volume average particle diameter in the free state of NHDF before adding to the serum-free medium containing green fluorescent dye was 15 μm. In addition, the volume average particle diameter in the free state of NHDF was measured according to the following measurement conditions. Next, the dish medium was collected with an aspirator, 2 mL of serum-containing medium was added, the cells were washed with an aspirator, and excess green fluorescent dye and DMSO were washed away. Further, 3 mL of serum-containing medium was added and cultured in an incubator (37 ° C., 5% CO ₂ environment) for 30 minutes or more to obtain NHDF stained with a green fluorescent dye so that the cells could be observed.

[Measurement condition of volume average particle diameter of cells in free state]
In an incubator (trade name: KM-CC17RU2, manufactured by Panasonic Corporation, 37 ° C., 5% CO ₂ environment), 1% by mass antibiotic (Antibiotic-Antilytic Coated Solution Solution (100x), manufactured by Wako Pure Chemical Industries, Ltd.) After culturing NHDF (trade name: CC-2509, manufactured by Lonza) in Dulbecco's modified Eagle medium containing WBC (manufactured by Wako Pure Chemical Industries, Ltd., hereinafter also referred to as “D-MEM”), an aspirator (product) Name: VACUSIP (manufactured by INTEGRA), 10 volume% fetal bovine serum (hereinafter also referred to as “FBS”) in a 100 mm dish and the medium were removed. 3 mL of phosphate buffered saline (manufactured by Wako Pure Chemical Industries, Ltd., hereinafter sometimes referred to as “PBS (−)”) was added to the dish, and PBS (−) was removed by suction with an aspirator, and the surface was washed. . After repeating the washing operation with PBS (−) three times, 3 mL of 0.1 mass% trypsin solution (Trypsin, from Porcine Pancreas, manufactured by Wako Pure Chemical Industries, Ltd.) was added, and the mixture was heated in an incubator for 5 minutes. Cells were detached from the dish. After confirming cell detachment with a phase contrast microscope, 4 mL of D-MEM containing 10% by mass FBS and 1% by mass antibiotics was added to inactivate trypsin. The mixture was transferred to a centrifuge tube, centrifuged (trade name: H-19FM, manufactured by KOKUSAN, 1.2 × 10 ³ rpm (234G), 5 minutes, 5 ° C.), and the supernatant was removed using an aspirator. After removal, 1 mL of D-MEM containing 10% by mass FBS and 1% by mass antibiotics was added to the centrifuge tube, and gently pipetting to disperse the cells. 10 μL of the cell suspension was taken out into an Eppendorf tube, and 10 μL of 0.4 mass% trypan blue staining solution was added to perform pipetting. 10 μL was taken out from the stained cell suspension and placed on a PMMA plastic slide, and the volume average particle diameter of the cells was measured using a trade name: Countess Automated Cell Counter (manufactured by Invitrogen).

-Cell preparation example 2-
Cells can be observed in the same manner as in Cell Preparation Example 1 except that a red fluorescent dye (trade name: Cell Tracker Orange, manufactured by Life Technology) is used instead of the green fluorescent dye in Cell Preparation Example 1. Thus, NHDF stained with a red fluorescent dye was obtained.

-Preparation of cell dispersion A-
In a 35 mm dish, NHDF stained with the red fluorescent dye obtained in Preparation Example 2 of the above cells contains 10% by mass FBS and 1% by mass antibiotic in the 35 mm dish with an aspirator (trade name: VACUSIP, manufactured by INTEGRA). D-MEM was removed. 3 mL of phosphate buffered saline (PBS (-), manufactured by Wako Pure Chemical Industries, Ltd.) was added to the dish, and PBS (-) was removed by suction with an aspirator, and the surface was washed. After repeating the washing operation with PBS (−) three times, 3 mL of 0.1 mass% trypsin solution (Trypsin, from Porcine Pancreas, manufactured by Wako Pure Chemical Industries, Ltd.) was added, and the mixture was heated in an incubator for 5 minutes. Cells were detached from the dish. After confirming cell detachment with a phase contrast microscope, 4 mL of D-MEM containing 10 mass% FBS and 1 mass% antibiotics was added to inactivate trypsin. The mixture was transferred to a centrifuge tube, centrifuged (trade name: H-19FM, manufactured by KOKUSAN, 1.2 × 10 ³ rpm (234G), 5 minutes at 5 ° C.), and the supernatant was removed with an aspirator. After the removal, 100 μL of D-MEM containing 10% by mass FBS and 1% by mass antibiotic was added to the centrifuge tube, and then pipetting was performed gently to disperse the cells, whereby a cell dispersion 1 was obtained. The volume average particle size of the cells in the free state measured according to the measurement conditions of the volume average particle size of the cells in the free state in the cell dispersion 1 is 13 μm, the number of cells is 5 × 10 ⁵ , and cell survival The rate was 91% by number.
The number of cells and the number of viable cells were measured using a trade name: Countess Automated Cell Counter (manufactured by Invitrogen) in the same manner as in the measurement conditions of the volume average particle diameter of cells in a free state.

-Production of three-dimensional cell aggregates-
As shown in FIG. 7A, the medium is removed from the 35 mm dish (base material) 300 of NHDF 310 stained with the green fluorescent dye obtained in Preparation Example 1 of the cell with an aspirator, and the three-dimensional cell is used using a pipette. 1 mL of the assembly-forming composition A 350 was added onto NHDF 310 dyed with a green fluorescent dye. Furthermore, in a 35 mm dish placed in a confocal fluorescence microscope (manufactured by Leica) in an incubator (37 ° C., 5% CO ₂ environment) set to a temperature exceeding the lower critical solution temperature (32 ° C.) of the polymer particles A The layer was formed by allowing the composition A for preparing a three-dimensional cell aggregate disposed on the cells to gel for 5 minutes. Next, 0.1 mL (cell number: 5 × 10 ⁶ cells) of the cell dispersion A was gently seeded on the layer using a pipette, and NHDF stained with a green fluorescent dye as shown in FIG. 7B. A laminate 1 was obtained in which 310, a three-dimensional cell aggregate preparation composition A 350, and NHDF 320 stained with a red fluorescent dye were laminated in this order.

Next, the temperature of the dish is lowered from 37 ° C. to 25 ° C., which is lower than the lower critical solution temperature (32 ° C.) of the polymer particles A, and allowed to stand for 10 minutes, and the composition A for preparing a three-dimensional cell aggregate is sol 3D cells in which NHDF 310 stained with a green fluorescent dye and NHDF 320 stained with a red fluorescent dye are stacked as shown in FIG. 7C by allowing NHDF stained with a red fluorescent dye to settle. A collection was obtained. FIG. 8 shows the result of observation of the three-dimensional cell aggregate with a fluorescence microscope. As shown in FIG. 8, NHDF stained with a red fluorescent dye (shown in black in the figure) can be observed on NHDF stained with a green fluorescent dye (shown in gray in the figure). Extension could be confirmed, and adhesion between cells and self-organization could be confirmed.
From the above results, it was found that the polymer particle A having a core-shell structure has little cytotoxicity.

  The average thickness of the three-dimensional cell aggregate was measured by staining the three-dimensional cell aggregate with the hematoxylin-eosin method (HE), confirming the cross section, and using a stereomicroscope. As a result, the average thickness of the three-dimensional cell aggregate was 0.01 mm or more and 0.03 mm or less. The result of the cross-sectional observation image of the three-dimensional cell aggregate measured using the stereomicroscope is shown in FIG. From the void ratio in the tissue and the degree of cell extension, it can be seen that the cells are proliferating and extending.

As an aspect of this invention, it is as follows, for example.
<1> A material for preparing a three-dimensional cell aggregate, comprising at least two kinds of polymers having different particle size change rates.
<2> The at least two kinds of polymers include a polymer constituting a core and a polymer constituting a shell covering the core surface,
The material for producing a three-dimensional cell assembly according to <1>, wherein the polymer constituting the core and the polymer constituting the shell form polymer particles having a core-shell structure.
<3> The material for preparing a three-dimensional cell assembly according to <2>, wherein the particle size change rate of the polymer constituting the shell is larger than the particle size change rate of the polymer constituting the core.
<4> The above <2> to <3>, wherein the polymer constituting the core is at least one selected from collagen, gelatin, proteoglycan, hyaluronic acid, fibronectin, laminin, tenascin, entactin, elastin, and fibrillin. The material for producing a three-dimensional cell aggregate according to any one of the above.
<5> A composition for preparing a three-dimensional cell assembly, comprising the material for preparing a three-dimensional cell assembly according to any one of <1> to <4>.
<6> The composition for producing a three-dimensional cell aggregate according to <5>, wherein the phase changes from a sol state to a gel state due to a temperature change.
<7> The composition for producing a three-dimensional cell assembly according to <6>, wherein the phase change is performed by raising the temperature to a temperature exceeding a lower critical solution temperature of a polymer constituting the shell.
<8> The composition for producing a three-dimensional cell assembly according to any one of <5> to <7>,
A set for producing a three-dimensional cell assembly.
<9> The volume average particle diameter of the polymer particles having the core-shell structure in the composition for preparing a three-dimensional cell assembly in a sol state is smaller than the volume average particle diameter of cells in a free state. A set for preparing a three-dimensional cell aggregate.
<10> The set for producing a three-dimensional cell aggregate according to any one of <8> to <9>, wherein the cells are adhesive cells.
<11> A composition storage container, wherein the composition for preparing a three-dimensional cell assembly according to any one of <5> to <7> is stored in a container.
<12> A composition in which a stimulus is applied to the composition for preparing a three-dimensional cell assembly according to any one of <5> to <7>, and the composition for preparing the three-dimensional cell assembly is provided on a substrate. It is a method for producing a three-dimensional cell aggregate, comprising an object imparting step.
<13> The method for producing a three-dimensional cell aggregate according to <12>, further including a cell imparting step for imparting cells.
<14> The method for producing a three-dimensional cell aggregate according to <13>, wherein an application temperature in the composition application step is equal to or lower than a minimum temperature of a lower critical solution temperature of a polymer having a lower critical solution temperature.
<15> The method for producing a three-dimensional cell aggregate according to any one of <13> to <14>, wherein the composition application step and the cell application step are sequentially repeated.
<16> Culture for culturing cells at a temperature exceeding the maximum lower limit critical dissolution temperature of a polymer having a lower critical dissolution temperature after phase-changing the composition for preparing a three-dimensional cell assembly from a sol state to a gel state The method for producing a three-dimensional cell aggregate according to any one of <13> to <15>, further including a step.
<17> The method according to <16>, further comprising a removal step of removing the at least two types of polymers by culturing the cells and then changing the phase of the composition for preparing a three-dimensional cell assembly from a gel state to a sol state. This is a method for producing a three-dimensional cell assembly.
<18> A three-dimensional cell aggregate produced by the three-dimensional cell aggregate production method according to any one of <12> to <17>.
<19> The three-dimensional cell aggregate according to <18>, wherein the average thickness is 0.01 mm or more and 20 mm or less.
<20> The three-dimensional cell aggregate according to any one of <18> to <19>, wherein the residual amount of the polymer is 3% by mass or less.

  The material for preparing a three-dimensional cell assembly according to any one of <1> to <4>, the composition for preparing a three-dimensional cell assembly according to any one of <5> to <7>, <8 > To <10>, a set for preparing a three-dimensional cell aggregate according to any one of the above, a composition storage container according to <11>, and a three-dimensional cell aggregate according to any one of <12> to <17> According to the method for producing a body and the three-dimensional cell aggregate according to any one of <18> to <20>, the above-described problems can be solved and the object of the present invention can be achieved.

JP 2013-59312 A International Publication No. 2011-059112 Pamphlet

1, 10, 100, 300 Substrate 120, 310, 320 Cell D, 130 Composition for preparing three-dimensional cell aggregate in sol state D ′, 350 Composition for preparing three-dimensional cell aggregate in gel state

Claims (17)

  A material for preparing a three-dimensional cell aggregate, comprising at least two kinds of polymers having different particle size change rates. The at least two polymers include a polymer constituting a core and a polymer constituting a shell covering the surface of the core;
The material for producing a three-dimensional cell aggregate according to claim 1, wherein the polymer constituting the core and the polymer constituting the shell form polymer particles having a core-shell structure.   The material for preparing a three-dimensional cell assembly according to claim 2, wherein the particle size change rate of the polymer constituting the shell is larger than the particle size change rate of the polymer constituting the core.   The tertiary according to any one of claims 2 to 3, wherein the polymer constituting the core is at least one selected from collagen, gelatin, proteoglycan, hyaluronic acid, fibronectin, laminin, tenascin, entactin, elastin, and fibrillin. Original cell aggregate preparation material.   A composition for preparing a three-dimensional cell assembly, comprising the material for preparing a three-dimensional cell assembly according to any one of claims 1 to 4.   The composition for producing a three-dimensional cell aggregate according to claim 5, wherein the phase changes from a sol state to a gel state due to a temperature change.   The composition for producing a three-dimensional cell aggregate according to claim 6, wherein the phase change is performed by raising the temperature to a temperature exceeding a lower critical solution temperature of a polymer constituting the shell. A composition for producing a three-dimensional cell assembly according to any one of claims 5 to 7,
A set for producing a three-dimensional cell aggregate, comprising: a cell.   The volume average particle diameter of the polymer particles in the composition for preparing a three-dimensional cell assembly in a sol state is smaller than the volume average particle diameter of cells in a free state. set.   The three-dimensional cell assembly preparation set according to any one of claims 8 to 9, wherein the cells are adhesive cells.   A composition storage container comprising the composition for preparing a three-dimensional cell assembly according to any one of claims 5 to 7 in a container.   A composition applying step of applying a stimulus to the composition for preparing a three-dimensional cell assembly according to any one of claims 5 to 7 and applying the composition for preparing a three-dimensional cell assembly on a substrate is included. A method for producing a three-dimensional cell aggregate characterized by   The method for producing a three-dimensional cell aggregate according to claim 12, further comprising a cell application step for applying cells.   The method for producing a three-dimensional cell aggregate according to claim 13, wherein an application temperature in the composition application step is not higher than a minimum temperature of a lower critical solution temperature of a polymer constituting the shell.   The method for producing a three-dimensional cell aggregate according to any one of claims 13 to 14, wherein the composition applying step and the cell applying step are sequentially repeated.   It further includes a culturing step of culturing the cells at a temperature exceeding the maximum critical dissolution temperature of the polymer constituting the shell after the phase change of the composition for preparing a three-dimensional cell assembly from a sol state to a gel state. The method for producing a three-dimensional cell aggregate according to any one of claims 13 to 15.   The three-dimensional cell according to claim 16, further comprising a removal step of removing the at least two kinds of polymers by changing the phase of the composition for preparing a three-dimensional cell aggregate from a gel state to a sol state after culturing the cells. A method for producing an aggregate.