JP6519168B2 - Three-dimensional cell assembly, method for producing the same, and solution for forming the same - Google Patents

Description

  The present invention relates to three-dimensional cell aggregates, as well as methods for their preparation and solutions for formation.

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

  The cell sheet may be a polymer or oligomer having an upper limit critical solution temperature to water in the range of 0 ° C. to 80 ° C., or a polymer or oligomer having a lower limit critical solution temperature to water in the range of 0 ° C. to 80 ° C. The cells are coated on the surface, the cells are cultured on the substrate, and the cells are subjected to the phase change between the sol state and the gel state by setting the temperature above the upper critical solution temperature or below the lower critical solution temperature. Can be peeled off.

  However, since the cell sheet is a single layer having a thickness of less than 0.01 μm and is thin and weak in strength, a cell sheet composed of a three-dimensional aggregate capable of reproducing a living tissue can be prepared. Requires the complicated operation of superposing the monolayer cell sheets layer by layer. In addition, there is a problem that precise and complicated configuration of various cells such as cardiomyocytes and vascular endothelial cells can not be achieved only by stacking the monolayer cell sheets one by one.

Therefore, cells are cultured on a three-dimensional molded body of a volume-changeable hydrophilic polymer gel capable of producing a three-dimensional cell aggregate, and the volume change of the hydrophilic polymer gel is imparted with a three-dimensional structure. A method for producing a three-dimensional cell assembly has been proposed (see, for example, Patent Document 1).
Also, a temperature-responsive cell culture bead has been proposed that uses a polymer whose hydration power changes on the bead surface within a temperature range of 0 ° C. to 80 ° C. (see, for example, Patent Document 2).

However, Patent Document 1 has a problem in that it is difficult to obtain a three-dimensional cell assembly capable of reproducing a living tissue, because precise and complicated configuration of various cells can not be performed.
Further, in Patent Document 2 described above, since the average particle size of the temperature-responsive cell culture beads is larger than the average particle size of the cells, it is difficult to impart a three-dimensional structure to the cells in a state of precise configuration. There is.
Furthermore, in handling cells, it is desirable that the toxicity to cells be low, but none of the prior art has sufficiently been studied from the point of toxicity to cells.

  Therefore, an object of the present invention is to provide a method for producing a three-dimensional cell assembly which has low cell toxicity and can realize precise and complicated three-dimensional configuration of cells.

  The method for producing a three-dimensional cell assembly of the present invention as a means for solving the above problems comprises disposing cells and a composition containing polymer particles phase-changed by temperature change on a substrate, and changing the temperature by temperature change. A phase change from the sol state to the gel state of the composition containing the polymer particles is repeated several times to produce a three-dimensional cell assembly.

  According to the present invention, it is possible to solve the above-mentioned various problems in the prior art and achieve the above object, to produce a three-dimensional cell assembly which can achieve precise and complex three-dimensional configuration of cells with low toxicity to cells. We can provide a way.

FIG. 1 is a schematic view in which a composition containing polymer particles is phase-changed from a sol state to a gel state. FIG. 2 is a schematic view showing an example of the ink jet discharge apparatus. FIG. 3 is a schematic view showing another example of the ink jet discharge apparatus. FIG. 4A is a photograph showing the state of polymer particle dispersion A at 25 ° C., which has a lower critical solution temperature of 32 ° C. FIG. 4B is a photograph showing the state of polymer particle dispersion A at 37 ° C., which has a lower critical solution temperature of 32 ° C. FIG. 5 is a graph showing the relationship between shear viscosity and temperature of a composition containing polymer particles B. FIG. 6 is a graph showing the particle size distribution of the polymer particles A at a liquid temperature of 25 ° C. FIG. 7 is a graph showing the particle size distribution of the polymer particles B at a liquid temperature of 25 ° C. FIG. 8 is a graph showing the particle size distribution of the polymer particles B at a liquid temperature of 37.degree. FIG. 9 is a graph showing the particle size distribution of polymer particles C at a liquid temperature of 25 ° C. FIG. 10 is a graph showing the particle size distribution of the polymer particles D at a liquid temperature of 25 ° C. FIG. 11 is a graph showing the particle size distribution of the polymer particles D at a liquid temperature of 37.degree. FIG. 12 is a graph showing the particle size distribution of the polymer particles E at a liquid temperature of 25 ° C. FIG. 13 is a graph showing the particle size distribution of the polymer particles E at a liquid temperature of 37 ° C. FIG. 14 is a graph showing the particle size distribution of polymer particles F at a liquid temperature of 25 ° C. FIG. 15A is a schematic view of disposing a composition containing polymer particles in a sol state on cells disposed on a substrate. FIG. 15B is a schematic view of arranging a cell on the layer after forming a layer by gelling a composition including a polymer particle in sol state arranged on a cell arranged on a substrate. FIG. 15C is a schematic view of obtaining a three-dimensional cell assembly by solting and removing a composition containing gel-like polymer particles from a cell laminate in which cells, polymer particles and cells are laminated in this order. FIG. 16 is a photograph of three-dimensional cell aggregates observed with a fluorescent confocal microscope. FIG. 17 is a cross-sectional observation image obtained by observing the three-dimensional cell assembly 1 produced in Example 1 using a stereomicroscope. FIG. 18A is a photograph of the three-dimensional cell assembly 1 produced in Example 1 observed with a phase contrast microscope. FIG. 18B is a photograph of the cell assembly 1 produced in Comparative Example 1 as observed with a phase contrast microscope.

  In order to solve the above-mentioned subject, as a result of the present inventor repeating earnest examination, the following knowledge was acquired. That is, by using a composition containing polymer particles that cause a phase change between a sol state and a gel state depending on temperature, the toxicity to cells is low, and the temperature is raised to a temperature higher than the lower critical solution temperature of the polymer particles. The composition containing the polymer particles is phase-changed from the sol state to the gel state, and the cells are cultured in a fixed state at a desired location. By culturing in the above state, the cells adhere to each other, and a three-dimensional cell assembly can be produced. In addition, by lowering the temperature below the lower limit solution temperature of the polymer after the culture, the composition containing the polymer particles is phase-changed from the gel state to the sol state, whereby the dispersibility of the polymer particles is improved. It is a finding that it can be removed from the gap of the adhered cells. The present invention is based on such findings.

(Method for producing three-dimensional cell assembly)
In the method for producing a three-dimensional cell assembly of the present invention, a step of arranging cells and a composition containing polymer particles phase-changed by temperature change on a substrate (hereinafter referred to as "cell and polymer particle application step" In some cases, the composition containing the polymer particles may be phase-changed from a sol state to a gel state (hereinafter referred to as "gelation") due to temperature change (hereinafter referred to as "gelation step") And may include other steps selected as required. In addition, the said cell and polymer particle provision process and the said gelatinization process are repeated several times.
The three-dimensional cell assembly obtained by the method for producing the three-dimensional cell assembly according to the present invention is a three-dimensional cell assembly according to the present invention, and at least the polymer particles on the substrate in the cell and polymer particle application step. The solution used to arrange the composition containing the present invention is the solution for three-dimensional cell assembly formation of the present invention.

<Cell and polymer particle application process>
The cell application step is a step of arranging cells and a composition containing polymer particles phase-changed by temperature change on a substrate.

<< substrate >>
The size, shape, structure, material and the like of the substrate are not particularly limited as long as they do not inhibit cell activity and proliferation, and can be appropriately selected according to the purpose.
Examples of the shape include a three-dimensional shape such as a dish, a multiplate, a flask, and a cell insert;
As said structure, a porous structure etc. are mentioned, for example.
As a material of the said base | substrate, an organic material, an inorganic material, etc. are mentioned, for example. 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), medium density Examples thereof include polyethylene (MDPE), vinyl chloride, vinylidene chloride, polyphenylene sulfide, polyether sulfone, polyethylene naphthalate, polypropylene, acrylic materials such as urethane acrylate, and cellulose. These may be used alone or in combination of two or more.
Examples of the inorganic material include glass and ceramics. These may be used alone or in combination of two or more.

<< Cells >>
The type of cell or the like is not particularly limited as long as it can be disposed on the substrate, and may be appropriately selected depending on the purpose. Taxonomically, for example, eukaryotic cells, prokaryotic cells, etc. It can be used for all cells, whether multicellular or unicellular living cells.
Examples of the eukaryotic cells include animal cells, insect cells, plant cells, fungi and the like. These may be used alone or in combination of two or more. Among these, animal cells are preferable. From the point that the cells form a three-dimensional cell aggregate, the cells adhere to each other, and the cell adhesion is such that it is not isolated unless it is subjected to physicochemical treatment. Cells having are more preferred.

Examples of the adherent cells include differentiated cells, cells before differentiation, and the like.
The differentiated cells include, for example, hepatocytes that are parenchymal cells of the liver; stellate cells; Kupffer cells; vascular endothelial cells; endothelial cells such as endothelium and corneal endothelial cells; fibroblasts; Osteocytes; 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; breast cells; pericytes; smooth muscle cells, Muscle cells such as cardiac muscle cells; renal cells; pancreatic islet cells; nerve cells such as peripheral nerve cells and optic nerve cells; chondrocytes; bone cells and the like. 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 they may be passaged several times.
As cells before differentiation, embryonic stem cells which are undifferentiated cells, pluripotent stem cells such as mesenchymal stem cells having pluripotency; unipotent stem cells such as vascular endothelial precursor cells having unipotency; differentiation Cells that have ended; and 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 archaebacteria. These may be used alone or in combination of two or more.

  The volume average particle size (D50) 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 for the inkjet method. The volume average particle size can be measured, for example, using an automatic cell counter (trade name: Countess Automated Cell Counter, manufactured by Invitrogen). When the cells are in a free state, they have a substantially spherical shape, so that the volume average particle size (D50) can be measured.

The volume average particle diameter (D50) of the cells can be measured by the following measurement method.
-Method of measuring volume average particle diameter (D50)-
1% antibiotics (Antibiotic-Antimycotic Mixed Stock Solution (100x), Wako Pure Chemical Industries, Ltd.) in an incubator (trade name: KM-CC17RU2, manufactured by Panasonic Corporation, 37 ° C., 5% CO ₂ environment) After culturing the cells in Dulbecco's modified Eagle medium (including Wako Pure Chemical Industries, Ltd., sometimes referred to as "D-MEM" below) containing them, inside a 100 mm dish with an aspirator (trade name: VACUSIP, manufactured by INTEGRA) Remove 10% fetal bovine serum (hereinafter sometimes referred to as "FBS") and the medium. Add 3 mL of phosphate buffered saline (sometimes referred to as "PBS (-)" by Wako Pure Chemical Industries, Ltd.) to a dish, aspirate and remove PBS (-) with an aspirator, and wash the surface . The washing operation with PBS (-) is repeated three times, 3 mL of 0.1% trypsin solution (Trypsin, from Porcine Pancreas, Wako Pure Chemical Industries, Ltd.) is added, and the mixture is heated for 5 minutes in an incubator. Detach the cells from the After confirming detachment of the cells with a phase contrast microscope, 4 mL of D-MEM containing 10% FBS and 1% antibiotic is added to inactivate the trypsin. Transfer to a centrifuge tube, centrifugation (trade name: H-19FM, manufactured by KOKUSAN, 1.2 × 10 ³ rpm (234 G), 5 min, 5 ° C.), and remove the supernatant with an aspirator. After removal, 1 mL of D-MEM containing 10% FBS and 1% antibiotic is added to a centrifuge tube, and pipetting is performed gently to disperse the cells. 10 μL of the cell suspension is removed into an eppendorf tube, 10 μL of 0.4% trypan blue staining solution is added, and the cells are stained by pipetting. 10 μL of the stained cell suspension can be taken out, placed on a PMMA plastic slide, and measured using an automatic cell counter (trade name: Countess Automated Cell Counter, manufactured by Invitrogen). The number of cells and the cell viability can also be determined by the same measurement method.

Before arranging the composition containing the cells and the polymer particles on the substrate, it is preferable to apply an organic substance that promotes cell adhesion to the surface of the substrate and the polymer particles according to a known method. By applying the organic substance, adhesion of the cells can be promoted.
The organic substance is not particularly limited, and is a substance involved in adhesion between cells or between cells and extracellular matrix, for example, peptides derived from organisms such as laminin, collagen, fibronectin, gelatin, etc .; peptides derived from the cells And proteins such as arginine-glycine-aspartic acid (RGD) and the like. These may be used alone or in combination of two or more.

There is no restriction | limiting in particular as a cell number in the solution before arrange | positioning on the base | substrate of the said cell, According to the objective, it can select suitably, 5 x 10 ⁵ cells / mL or more 5 x 10 ⁸ cells / mL Or less is preferable, and 5 × 10 ⁵ cells / mL to 5 × 10 ⁷ cells / mL are more preferable. When the number of cells is 5 × 10 ⁵ cells / mL or more and 5 × 10 ⁸ cells / mL or less, cells can be reliably included in the discharged droplets, which is suitable for precise placement of cells.
As an apparatus for measuring the number of cells, an automatic cell counter (trade name: Countess Automated Cell Counter, manufactured by Invitrogen) and the like may be mentioned in the same manner as the method of measuring the volume average particle size (D50).

The cell viability after placing the cells on the substrate is preferably 90% or more, more preferably 95% or more, more preferably 98% or more to the viability in the solvent before placing the cells on the substrate. Particularly preferred. When the cell viability is 90% or more, a load due to placing cells on a substrate can be reduced to form a three-dimensional cell assembly.
As a measuring device of the said cell viability, it is the same as the measuring method of the said volume average particle diameter (D50), An automatic cell counter (brand name: Countess Automated Cell Counter, product made in Invitrogen) etc. is mentioned.

<< Polymer particles >>
As the polymer particles, as long as the composition containing the polymer particles undergoes phase change due to temperature change, there is no particular limitation on the size, shape, structure, material and the like, and it may be appropriately selected according to the purpose. it can.

Here, the phase change of the composition containing the polymer particles will be described below.
As the physical properties of the polymer particles, a composition containing a polymer particle having a volume average particle diameter of 100 μm or less is dispersed in the composition in the form of particles such as colloid by dispersing one component by temperature change. The state and the gel state having a shear viscosity of 500 mPa · s or more and having no fluidity can be phase-changed from the sol state to the gel state or from the gel state to the sol state.
Cells can be fixed by changing the state of the composition containing the polymer particles from the sol state to the gel state. Moreover, it becomes easy to remove the said polymer particle from a three-dimensional cell assembly by phase-changing the state of the composition containing the said polymer particle from a gel state to a sol state.

  As a change point of the phase change between the sol state and the gel state, for example, when the phase change from the sol state to the gel state, the polymer particles shrink or an aggregate network between the polymer particles The point at which the shear viscosity rapidly increases and the fluidity of the polymer particles disappears by forming a; the polymer particles swell and disperse when phase change from the gel state to the sol state The shear viscosity drops sharply, and the polymer particles obtain fluidity.

  The shape of the polymer particle is, for example, a spherical shape such as a spherical shape or a semispherical shape, from the viewpoint of suppressing excessive adsorption of the polymer particles to cells and reducing toxicity to the cells; It can be mentioned. These may be used alone or in combination of two or more.

Compositions comprising polymer particles that undergo a phase change with temperature have a critical solution temperature.
Examples of the polymer particles having the critical solution temperature include polymer particles having an upper critical solution temperature and polymer particles having a lower critical solution temperature. Among these, the cells are not dissolved in an aqueous solution at a temperature higher than the temperature because they are not loaded with cells, they do not dissolve even if they come in contact with an aqueous solution, and they divide into two layers, but the lower limit critical dissolution that dissolves in an aqueous solution below that temperature Polymeric particles having a temperature are preferred.

  FIG. 1 is a schematic view showing a phase change from a sol state to a gel state of a composition containing polymer particles. As shown in FIG. 1, for example, the polymer particles 2 contained in the composition D applied to the substrate 1 are in a sol state at a temperature lower than the lower critical solution temperature of the polymer particles 2 and have a low viscosity and aggregate. It is fluidly present in the solution. When the substrate 1 is heated to a temperature higher than the lower critical solution temperature of the polymer particles 2 with respect to the composition containing the polymer particles 2 disposed on the substrate 1, the composition including the polymer particles 2 in the sol state is gelated. The composition becomes non-flowable and contains the polymer particles 3 in gel state, a quasi-aggregate network is formed, and a layer containing the composition containing the polymer particles 3 in gel state is formed. A composition containing the polymer particle 2 in the sol state is further disposed on the layer, and a layer which is gelated in the same manner as described above and phase-changed to the composition containing the polymer particle 3 in the gel state is laminated. By repeating this a plurality of times, it is possible to form a three-dimensional laminate in which a plurality of layers containing a composition containing polymer particles 3 in a gel state are laminated. By containing the cells in the composition D, a three-dimensional cell aggregate can be formed by forming a three-dimensional laminate in the same manner as described above.

The lower limit critical solution temperature of the polymer particles is preferably 0 ° C. or more and 50 ° C. or less from the viewpoint that the optimal culture temperature of the cells is 37 ° C., and when culturing cells at 37 ° C., 0 ° C. or more and 35 ° C. or less Is more preferred. When the lower limit critical solution temperature is 0 ° C. or more, the cells are not excessively cooled and no load is applied to the cells, and when the temperature is 50 ° C. or less, the cells are not excessively heated; There is no load on the
The lower limit critical solution temperature can be calculated by measuring the cloud point of the polymer particles according to the total light transmittance measurement method based on the JIS K 7105 method.

The volume average particle size (D50) in the composition in the sol state of the polymer particles is preferably smaller than the volume average particle size in the free state of the cells, more preferably 50 μm or less, and particularly preferably 10 μm or less. When the volume average particle size is smaller than the volume average particle size in the free state of the cells, the toxicity to the cells can be lowered, the viscosity can be lowered, and the droplet size can be reduced. Therefore, it can be suitably used in the ink jet method, and when the composition containing the polymer particles is phase-changed from the gel state to the sol state by lowering the temperature below the lower critical solution temperature of the polymer particles after the culture. It becomes easy to remove polymer particles contained in the composition in the sol state from three-dimensional cell aggregates.
As a volume average particle diameter (D50) in the composition in the gel state of the said polymer particle, 50 micrometers or less are preferable, and 0.05 micrometer or more and 5 micrometers or less are more preferable. When the volume average particle size is 50 μm or less, the toxicity to the cells can be lowered, and when a laminate comprising the cells and the polymer particles is prepared and the cells are cultured, The distance between the two can be close and it does not inhibit the adhesion between cells.

The cumulant particle size in the composition in the sol state of the polymer particles is preferably smaller than the cumulant particle size in the free state of the cells, more preferably 50 μm or less, and particularly preferably 10 μm or less. If the cumulant particle size is smaller than the cumulant particle size in the free state of the cells, the toxicity to the cells can be lowered, the viscosity can be lowered, and the droplet size can be reduced, It can be suitably used for the inkjet method, and when the polymer particles are phase-changed from the gel state to the sol state after being cultured and cooled to the lower critical solution temperature of the polymer particles, three-dimensional cell aggregates This facilitates removal of polymer particles in the composition in the sol state.
The cumulant particle size in the composition in the gel state of the polymer particles is preferably 50 μm or less, and more preferably 0.05 μm or more and 5 μm or less. When the cumulant particle size is 50 μm or less, the toxicity to cells can be lowered, and when a laminate comprising the cells and the polymer particles is prepared and the cells are cultured, the cells The distance can be reduced and the adhesion between cells is not inhibited.

The volume average particle diameter (D50) and the cumulant particle diameter were obtained by the following preparation examples of the sample liquid using a concentrated particle diameter analyzer (trade name: FPAR-1000, manufactured by Otsuka Electronics Co., Ltd.) It can measure on the following measurement conditions using a sample solution.
-Preparation example of sample solution-
The synthesized powdery polymer particles are dispersed at a concentration of 0.5 mass% in pure water obtained using a pure water production apparatus (trade name: GSH-2000, manufactured by ADVANTEC). The amount of liquid for measurement is 5 cc, and a sample liquid can be prepared by stirring a 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, optimum light amount adjustment is appropriately set by the ND filter)
Measurement probe: Thickening probe Measurement routine: Measurement: 180 seconds at 25 ° C. Measurement: 600 seconds at 25 ° C. (When the body side is changed to 35 ° C., the liquid temperature gradually becomes 25 ° C. to 35 ° C. Particles in the meantime) Monitor diameter change) → Measurement: 180 seconds at 35 ° C

There is no restriction | limiting in particular as a shear viscosity of the composition containing the polymer particle of the said sol state, According to the objective, it can select suitably, 0.1 Pa.s or less is preferable and 0.01 Pa.s or less is more preferable. . Since the viscosity of the composition containing cells can be lowered when the shear viscosity is 0.01 Pa · s or less, the composition can be suitably used in the inkjet method.
There is no restriction | limiting in particular as a shear viscosity of the composition containing the polymer particle of the said gel state, According to the objective, it can select suitably, 0.2 Pa.s or more is preferable, 1 Pa.s or more is more preferable, 10 Pa S or more is particularly preferable. Since the viscosity of the composition containing cells can be increased when the shear viscosity is 0.2 Pa · s or more, cell immobilization is possible.

The shear viscosity can be determined by the following measurement method.
-Method of measuring shear viscosity-
The polymer particles are dispersed in pure water at a concentration of 10% by mass, and high-pressure steam sterilization (trade name: MLS-3030, manufactured by Panasonic Corporation) at 121 ° C. for 20 minutes is performed. Thereafter, Dulbecco's modified Eagle's medium (Japanese sum) containing 10% fetal bovine serum (FBS, manufactured by Gibco), 1% antibiotic (trade name: Antibiotic-Antimycotic Mixed Stock Solution (100x), manufactured by Wako Pure Chemical Industries, Ltd.) The sterilized polymer particles are diluted to 1% by mass with Optical Pure Chemical Industries, Ltd., and dispersed using a stirrer (trade name: Rexim RS-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 shear rate 0.1 (1 / s) while raising temperature to 37 ° C after measuring for 100 seconds (10 points every 10 seconds) at shear rate 10 (1 / s) at 25 ° C. Can be obtained by measuring for 100 seconds (10 points every 10 seconds).

  The polymer particles are preferably uniformly dispersed in the composition. The polymer particles can be suitably used in the inkjet method by being uniformly dispersed.

  The composition containing the polymer particles generally involves a volume change at a temperature around the lower critical solution temperature of the polymer particles having the lower critical solution temperature of the polymer particles in order to cause the phase change. Examples of the volume change include swelling, which is a phenomenon in which polymer particles absorb a liquid (dispersion medium) to increase the volume. The degree of this volume change can be expressed by the degree of swelling, and as the degree of swelling, the volume of the polymer particles below the lower critical solution temperature of the polymer particles is higher than the lower critical solution temperature of the polymer particles 30 or less is preferable with respect to the volume of particle | grains, and 8 or less is more preferable. When the degree of swelling is 30 or less, removal of polymer particles by solification is facilitated.

When the temperature is lowered from the temperature A higher than the lower limit critical solution temperature to the temperature B lower than the lower limit critical solution temperature, the swelling degree can be expressed by the following equation.
Degree of swelling = (mass of polymer particles in sol state at temperature B / mass of polymer particles in gel state at temperature A)
Here, temperature A> temperature B.

  At the time of the volume change, the composition containing the polymer particles may be isotropically changed as long as it is anisotropically deformed. Among these, from the viewpoint of preventing the cells from being easily damaged at the time of peeling, it is preferable that the composition containing the polymer particles is deformed only in a specific direction at the time of the volume change.

  The light transmittance of the polymer particles is preferably 70% or more, and more preferably 90% or more. It becomes easy to confirm the state of the cell in a cell culture period as the said light transmittance is 70% or more. When the concentration of the crosslinking agent is increased, the light transmittance is lowered. The light transmittance can be measured according to the total light transmittance measurement method based on JIS K 7105 method.

  Examples of the polymer particles include homopolymers formed from a single monomer, random copolymers formed from a plurality of monomers, block copolymers, macromonomers, brush-like polymers obtained by polymerizing them, branched graft polymers, hyperbranched hyper Branch polymer etc. are mentioned. These may be used alone or in combination of two or more.

Examples of the structure of the polymer particle include the structure of a single polymer, and a composite structure in which a plurality of single polymers are aggregated.
Examples of the composite structure in which the plurality of the polymer single bodies are aggregated include a combination use of the polymer particles and a hydrophilic polymer other than the polymer particles. In this case, from the viewpoint of phase change due to temperature change, the surface of the polymer particles is preferably formed of polymer particles whose volume change is due to temperature change.

There is no restriction | limiting in particular as a weight average molecular weight of hydrophilic polymers other than the said polymer particle, According to the objective, it can select suitably, 1,000 or more and 50,000 or less are preferable, and 2,000 or more and 50,000 or less Is more preferable, and 5,000 or more and 50,000 or less is particularly preferable. The hydrophilicity can be improved when the weight average molecular weight is 1,000 or more, and the polymer particles themselves are prevented from becoming bulky when the weight average molecular weight is 50,000 or less, and phase change due to temperature change Becomes easier.
The weight average molecular weight can be determined, for example, by measuring the tetrahydrofuran solubles using gel permeation chromatography (GPC).

  Examples of the polymer particles include alkyl acrylamide polymers such as polyisopropyl acrylamide and polydiethyl acrylamide; polyvinyl methyl ether; polyvinyl ether having oxyethylene in a side chain; methyl cellulose, carboxymethyl cellulose and the like. These may be used alone or in combination of two or more.

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

As a composite in which a plurality of the polymer particles are aggregated, it is preferable that the polymer particles are crosslinked by a crosslinking agent.
As said crosslinking agent, a bifunctional crosslinking agent, a trifunctional crosslinking agent etc. are mentioned, for example. These may be used alone or in combination of two or more.
Examples of the difunctional crosslinking agent include difunctional acrylamides such as N, N′-methylenebis (meth) acrylamide and N, N-propylenebis (meth) acrylamide; ethylene glycol di (meth) acrylates; Meta) acrylate; polyethylene glycol di (meth) acrylate etc. are mentioned. These may be used alone or in combination of two or more. Among these, N, N'-methylenebis (meth) acrylamide is preferable, and N, N'-methylenebisacrylamide is more preferable.
Examples of the trifunctional crosslinking agent include triallyl cyanurate, triallyl isocyanurate and the like. These may be used alone or in combination of two or more.
By changing the type and concentration of the crosslinking agent, it is possible to adjust the lower critical solution temperature, the transparency and the swelling of the polymer particles.
The lower critical solution temperature can be lowered by increasing the concentration of the crosslinking agent.

  It is preferable that the polymer particles have low toxicity to cells.

  A well-known temperature rising apparatus can be used as an apparatus which carries out the phase change of the composition containing the said polymer particle of sol-form to gel-like. The temperature raising device is not particularly limited as long as it can be raised to a temperature higher than the lower limit critical solution temperature of the polymer particles, and examples thereof include known culture plates, incubators, and the like.

The content of the polymer particles is preferably 0.1% by mass to 10% by mass, and more preferably 0.5% by mass to 3% by mass, with respect to the total amount of the composition before being disposed on the substrate. 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.
The remaining amount of the polymer particles when removing the polymer particles from the three-dimensional cell assembly after culture is preferably 3% by mass or less, more preferably 1% by mass or less based on the total amount of the three-dimensional cell assembly. preferable. When the content is 1% by mass or less, the toxicity to cells can be reduced.

<< How to distribute >>
In the cell and polymer particle application step, as a method of arranging the composition containing the cells and the polymer particles, for example, a method of simultaneously arranging cells and a composition containing polymer particles on a substrate (cells and polymers Particle simultaneous application step); after the cells are disposed on the substrate (hereinafter sometimes referred to as "cell application step"), a composition containing polymer particles is disposed on the cells (hereinafter referred to as " And the like) may be mentioned.
In the step of simultaneously applying the cells and the polymer particles, for example, a method of preparing a dispersion containing the cells and a composition containing the polymer particles, and simultaneously arranging on a substrate; the cells, the polymer particles and And the like are prepared in advance, and the mixed dispersion may be disposed on a substrate.
In the method of disposing the mixed dispersion on a substrate, the polymer particles can prevent aggregation of the cells in the mixed dispersion and improve the dispersibility of the cells.

The shear viscosity of the dispersion containing cells, the composition containing the polymer particles, and the mixed dispersion, when the polymer particles are contained, is 0. 0 at a temperature not higher than the lower critical solution temperature of the polymer particles. 1 Pa · s or less is preferable, and 0.01 Pa · s or less is more preferable. It can use suitably for an inkjet method as the said shear viscosity is 0.1 Pa * s or less.
As the shear viscosity of the dispersion containing cells, the composition containing the polymer particles, and the mixed dispersion, when the polymer particles are contained, it is 1 Pa · at a temperature higher than the lower critical solution temperature of the polymer particles. s or more is preferable, and 10 Pa · s or more is more preferable. When the shear viscosity is 10 Pa · s or more, the cells can be immobilized. The shear viscosity is measured for 100 seconds (10 points every 10 seconds) at a shear rate of 10 (1 / s) using a rheometer (trade name: MCR301, manufactured by Anton Paar), and then heated to 40 ° C. It can be determined by measuring for 100 seconds (10 points every 10 seconds) at a shear rate of 0.1 (1 / s).

The number of cells in the dispersion containing cells and the number of cells in the mixed solution in which the cells and the polymer particles are mixed are not particularly limited and can be appropriately selected according to the purpose, 5 × 10 ⁵ cells / mL or more and 5 × 10 ⁸ cells / mL or less are preferable, and 5 × 10 ⁵ cells / mL or more and 5 × 10 ⁷ cells / mL or less are preferable. When the number of cells is 5 × 10 ⁵ cells / mL or more and 5 × 10 ⁸ cells / mL or less, cells can be reliably included in the discharged droplets, which is suitable for precise placement of cells. 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 the method for measuring the volume average particle diameter (D50).

  Examples of the method of arranging the composition containing the cells and the polymer particles include an inkjet method, a dispenser method, a pipetting method, and an aspirator method. These may be used alone or in combination of two or more. Among these, the inkjet method is preferable in that precise and complicated cell placement can be performed at the single cell level.

  The ink jet method is not particularly limited, and known methods can be used. In addition, in order to implement the said inkjet method, a well-known inkjet discharge apparatus can be used suitably as a cell and a polymer particle flight means.

<< Ink jet discharge device >>
The ink jet discharge device is not particularly limited as long as a composition containing cells and polymer particles can be disposed on a substrate, and includes cells and polymer particle flying means, and cells and polymer containing parts. It has other means as needed.
The cell and polymer particle flight means are, for example, means for arranging a composition containing cells and polymer particles on a substrate, and means for simultaneously arranging cells and compositions containing polymer particles on a substrate; After the cells are disposed on the substrate, there may be mentioned means by which a composition containing polymer particles is disposed on the cells.

  The cell and polymer particle flying means have a nozzle capable of flying a composition containing cells and polymer particles onto the substrate by an inkjet method. In addition, as the nozzle, a nozzle (ejection head) in a known inkjet printer can be suitably used, and the inkjet printer can be suitably used as the cell and polymer particle flying means. In addition, as said inkjet printer, SG7100 made from Ricoh Co., Ltd. etc. are mentioned suitably, for example. The ink jet printer is preferable in that the amount of cells and polymer particles that can be dropped from the head at one time is large, and the application area is wide, so that the speed of application can be increased.

  The cell and polymer particle flighting means is a means for applying a stimulus to the composition containing the cell and polymer particles of the present invention, and flying the composition containing the cells and polymer particles to form an image, There is no restriction in particular, and it can choose suitably according to the object, and a subsequent injection type, an on-demand type, etc. are mentioned. Examples of the on-demand type include a piezo type, a thermal type, and an electrostatic type. Among these, the piezo method is particularly preferable.

Specifically, the cell and polymer particle flight means preferably includes a liquid chamber, a fluid resistance unit, a diaphragm, and a nozzle member, and the liquid chamber, fluid resistance unit, diaphragm, and nozzle member. Preferably, at least a part of is formed of a material containing at least one of silicone and nickel.
The nozzle diameter of the nozzle is not particularly limited, and is preferably twice or more the size of the cells in order to prevent the cells from clogging the nozzle portion. Since the size of animal cells, particularly human cells, is generally 5 μm to 30 μm, the nozzle diameter is preferably 10 μm to 200 μm according to the cells used. On the other hand, 60 μm or less is more preferable because it becomes difficult to achieve the original purpose of forming microdroplets if the droplets become too large. Among these, when it is 10 μm or more, the dispersion containing cells can be suitably discharged, and from the point of being suitably used for the inkjet method, 10 μm or more and 60 μm or less is particularly preferable.

  In the present invention, even when the ink jet printer capable of providing the cells and the polymer particles accurately and efficiently is used, clogging or the like does not occur in the nozzle or the head thereof to cause corrosion or the like. It is advantageous in that three-dimensional cell aggregates with high dimensional accuracy can be obtained easily and efficiently in a short time.

The cell and polymer particle storage unit is a member in which the cell and the polymer particle are stored, and the size, shape, material, and the like thereof are not particularly limited, and can be appropriately selected according to the purpose. Storage tanks, bags, cartridges, tanks, etc.
In the case of using means for simultaneously arranging the cells and the composition containing the polymer particles on a substrate, it is preferable to accommodate a mixed dispersion of cells and polymer particles in the member.
In the case of using means in which a composition containing polymer particles is disposed on the cells after the cells are disposed on a substrate, the dispersion containing the cells and the composition containing the polymer particles are accommodated. Preferably, the members to be separated are separate.

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

  The inkjet discharge device uses the inkjet head 40 to distribute the mixed dispersion liquid 20 in which cells and polymer particles are mixed on the substrate 10. At this time, the temperature control device 50 is used to raise the temperature of the substrate 10 to a temperature higher than the lower critical solution temperature of the polymer particles. Preferably, the temperature is raised before the mixed dispersion is placed on the substrate 10, and the substrate 10 is warmed and kept at a temperature higher than the lower critical solution temperature of the polymer particles. As a result, the composition containing the polymer particles in the mixed dispersion disposed on the substrate 10 is phase-changed from the sol state to the gel state to fix the cells, and the cell aggregate 60 is formed. The mixed dispersion is disposed on the cell aggregate 60 in the same manner as described above, and the composition containing the polymer particles is phase-changed from the sol state to the gel state to form the cell aggregate 60 a plurality of times. A three-dimensional cell assembly can be obtained by repeating.

  FIG. 3 shows another example of the ink jet discharge apparatus. The ink jet discharge device of FIG. 3 is basically the same as FIG. 2, but the cell and polymer particle flight means are different. That is, after the cells 120 are disposed on the substrate 100 using the inkjet head 140, the composition 130 including the polymer particles is disposed on the cells 120. Similar to FIG. 2, the composition containing the polymer particles warmed to a temperature higher than the lower critical solution temperature of the polymer particles by the temperature control device 150 is phase-changed from the sol state to the gel state to form a cell aggregate. 160 are formed. The cells 120 are arranged on the cell aggregate 160 by the same operation as described above, the composition 130 containing polymer particles is arranged on the cells 120, and the composition containing the polymer particles is in the form of sol to gel. The three-dimensional cell assembly can be obtained by repeating the phase change to form the cell assembly 160 multiple times.

<Other process>
In the other steps, after arranging the cells and the composition containing the polymer particles on a substrate, the composition containing the polymer particles is phase-changed from the sol state to the gel state, A step of culturing cells at a temperature higher than the lower limit critical solution temperature (hereinafter sometimes referred to as "culturing step"), after culturing the cells, the temperature is lowered below the lower limit critical solution temperature of the polymer particles, A step of phase-converting a composition containing a gel from a gel state to a sol state (hereinafter sometimes referred to as "solization") (hereinafter sometimes referred to as "solization step"), culturing the cells, It is preferable to have a step of removing polymer particles (hereinafter sometimes referred to as “removing step”) after phase-changing the composition containing polymer particles from the gel state to the sol state.

<< Culturing process >>
The culture step is a step of culturing the cells at a temperature higher than the lower critical solution temperature of the polymer particles after phase-changing the composition containing the polymer particles from the sol state to the gel state. By the culture, cells grow, spread, and adhere to each other to form a three-dimensional cell assembly.

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

  The culture temperature is not particularly limited and may be appropriately selected according to the purpose. The culture temperature is preferably 25 ° C to 45 ° C, more preferably 33 ° C to 40 ° C, and 36 ° C to 38 ° C. Is particularly preferred. A cell can be suitably cultured as the said culture temperature is 25 degreeC or more and 45 degrees C or less.

  The culture apparatus is not particularly limited as long as the cells can be kept warm and cultured at the optimum culture temperature, and examples thereof include known culture plates and incubators.

There is no restriction | limiting in particular as said culture | cultivation time, According to the objective, it can select suitably, 12 to 60 hours are preferable, and 15 to 50 hours are preferable. Cells can be suitably cultured when the culture time is 12 hours or more and 60 hours or less.
In addition, in order to confirm whether cells form three-dimensional cell aggregates by proliferating, spreading, and adhering between cells, hematoxylin-eosin method (HE) staining of tissue bodies, for example, NHDF By observing the cross section according to the tissue body cross-sectional observation image by the procedure described above, it is possible to confirm the void ratio in the tissue body and the degree of cell development.

  There is no restriction | limiting in particular as said culture medium, According to the objective, it can select suitably, For example, the culture medium classified by composition, such as a natural culture medium, a semi-synthetic culture medium, and a synthetic culture medium; The culture media classified by the shape of (It may be said that a powder culture medium may be hereafter mentioned) etc. are mentioned. These may be used alone or in combination of two or more. When cells are of animal origin, any medium can be used as long as it is a medium used for culturing animal cells.

  Examples of media used for culturing the animal cells include Dulbecco's Modified Eagles Medium (D-MEM), Ham's Nutrient Mixture F12, and D-MEM / F12. Medium, McCoy's 5A medium (McCoy's 5A medium), Eagle's MEM medium (Eagles's Minimum Essential Medium; EMEM), αMEM medium (alpha Modified Eagles's Minimum Essential Medium; αMEM), MEM medium (Minimum Essential Medium), RPMI 1640 medium, Iscove's modified Dulbecco's medium (Iscove's Mod) fied Dulbecco's Medium; IMDM), MCDB 131 medium, William medium E, IPL 41 medium, Fischer's medium, StemPro 34 (manufactured by Invitrogen), X-VIVO 10 (manufactured by Kenbrex), X-VIVO 15 (Kenbrex) ), HPGM (manufactured by Kenbrex), StemSpan H3000 (manufactured by Stemcell Technology), StemSpanSFEM (manufactured by Stemcell Technology), Stemline II (manufactured by Sigma Aldrich), QBSF-60 (manufactured by Quality Biological), StemProhESCSFM (Invitrogen) , Essential 8 (registered trademark) medium (Gibco), mTeSR 1 or 2 medium (Stemcell Technology) ReproFF or ReproFF2 (Reprocell), PSGro hESC / iPSC Medium (System Biosciences), NutriStem® Medium (Biological Industries), CSTI-7 Medium (Cell Science Laboratories) MesenPRO RS medium (Gibco), MF-Medium (registered trademark) mesenchymal stem cell growth medium (Toyobo Co., Ltd.), Sf-900 II (Invitrogen), Opti-Pro (Invitrogen), etc. Be These may be used alone or in combination of two or more.

  There is no restriction | limiting in particular as a carbon dioxide density | concentration in the said culture medium, According to the objective, it can select suitably, 2% or more and 5% or less are preferable, and 3% or more and 4% or less are more preferable. A cell can be suitably cultured as the said carbon dioxide concentration is 2% or more and 5% or less.

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

The supply amount of the medium, the cell number to a medium 1mL is, it is preferable to supply such that of 5 × 10 ⁵ cells or more 5 × 10 ⁸ or less, of 5 × 10 ⁵ cells or more 5 × 10 ⁷ It is preferable to supply so that it may become less than one. The cell number of 5 × 10 ⁵ or more and 5 × 10 ⁸ or less per 1 mL of medium is suitable for cell growth and spreading. 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 the method for measuring the volume average particle diameter (D50).

  There is no restriction | limiting in particular as said pH, According to the objective, it can select suitably, 4 or more and 7 or less are preferable, and 5 or more and 6 or less are more preferable.

  In the culture step, it is preferable to use a reagent that has been sterilized, and it is preferable to use a culture operation and culture apparatus that have been subjected to aseptic treatment.

<< Solization process >>
The solation step is a step of cooling the temperature below the lower limit critical solution temperature of the polymer particles after culturing the cells to change the phase of the composition containing the polymer particles from the gel state to the sol state. The phase change from the gel state to the sol state of the composition containing the polymer particles makes it possible to take out the three-dimensional cell assembly.

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

<< Removal process >>
The removing step is a step of culturing the cells, removing the polymer particles after phase-changing the composition containing the polymer particles from the gel state to the sol state. By removing the polymer particles in the composition in the sol state from the three-dimensional cell assembly, only the three-dimensional cell assembly can be obtained. In the three-dimensional cell assembly, all the polymer particles may not be removed, and the polymer particles may be used without problems even if the polymer particles remain in whole or in part.

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 solution include phosphate buffer (hereinafter sometimes referred to as "PBS"), Tris-HCl buffer and the like. The removal method is described in detail below.

-Removal method-
After removing the culture medium of the cultured tissue body in order to isolate the cells from the produced three-dimensional cell assembly and washing with PBS, collagenase solution (trade name: NB 4G / 0.26 PZ U) / Ml, manufactured by Cosmo Bio Inc., and left for 2 hours in an incubator (trade name: KM-CC17RU2, manufactured by Panasonic Corp., 37 ° C., 5% CO ₂ environment) for 2 hours, between cells, and ECM (cells) Cells are isolated from external matrix) / intercellular binding. Next, the solution is separated by a mesh filter (trade name: CellTrics Fillter, Ikeda Rika Co., Ltd.) with a diameter of 20 μm, and the filtered solution is transferred to a centrifuge tube and centrifuged (trade name: H-19FM, KOKUSAN CO., LTD.) , 1.2 × 10 ³ rpm (234 G), 5 min, 5 ° C.), and remove the supernatant with an aspirator. After removal, 1 mL of Dulbecco's modified Eagle medium containing 10% FBS and 1% antibiotic is added to a centrifuge tube, and pipetting is performed gently to disperse the cells. The above-mentioned centrifugation and supernatant removal can be repeated three times to remove the polymer particles.

(Three-dimensional cell assembly)
The three-dimensional cell assembly of the present invention comprises polymer particles having a volume average particle size smaller in the composition in the sol state than the volume average particle size in the free state of the cells, and changes in temperature between the sol state and the gel state The composition contains a phase-changeable composition, and further comprises other components as required.
The three-dimensional cell assembly of the present invention can be suitably produced by the method for producing a three-dimensional cell assembly of the present invention.
The said polymer particle can use the thing similar to the manufacturing method of the three-dimensional cell assembly of this invention.

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

  There is no restriction | limiting in particular as an average thickness of the said three-dimensional cell assembly, 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, 0.1 mm or more and 20 mm or less are especially preferable. When the thickness is 0.05 mm or more and 20 mm or less, it can be suitably used for transplantation to a damaged site of the human body and for a drug screening material. As the thickness, the tissue body is HE-stained and cross-sectional observation is used, or in the case of nondestructive means, optical coherence tomography (OCT: Optical Coherence Tomography, optical coherence tomography, trade name: Ganymede II, Thorlabs). It can be used to measure.

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

The content of the polymer particles is preferably 0.1% by mass to 10% by mass, and more preferably 0.5% by mass to 3% by mass, with respect to the total amount of the composition before being disposed on the substrate. 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.
The remaining amount of the polymer particles when removing the polymer particles from the three-dimensional cell assembly after culture is preferably 3% by mass or less, more preferably 1% by mass or less based on the total amount of the three-dimensional cell assembly. preferable. When the content is 1% by mass or less, the toxicity to cells can be reduced.

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

-Application-
The three-dimensional cell assembly of the present invention is suitably used for transplantation to a damaged site of the human body, a graft material for tissue regeneration inducing tissue regeneration, a material for in vitro examination of tissue morphogenesis process, a drug screening material, etc. be able to.

(Solution for three-dimensional cell assembly)
The solution for three-dimensional cell assembly formation of the present invention contains a composition containing the polymer particles, or a mixed dispersion of the cells and the polymer particles, which is used in the method for producing a three-dimensional cell assembly of the present invention. And, if necessary, further contain other ingredients.

  There is no restriction | limiting in particular as a composition containing the said polymer particle, and the other component contained in the said mixed dispersion liquid, According to the objective, it can select suitably, For example, deionized water; ultrapure water; physiological salt Water; phosphate buffered saline (PBS); the above medium; 5% dextrose aqueous solution; electrolyte; suspension agent; soothing agent; albumin, a stabilizer of Prionex (registered trademark, Pentafarm Japan) (albumin or Prionex ( (Registered trademark, Pentafarm Japan) and the like) preservatives, preservatives, natural compatible materials having ecological compatibility including natural polysaccharides, or polymeric materials mimicking a structure. There is no restriction | limiting in particular as content of the said other component, According to the objective, it can select suitably.

  EXAMPLES The present invention will be described below by way of Examples and Comparative Examples, but the present invention is not limited to these Examples. Moreover, unless otherwise indicated, "part" and "%" shown in an Example and a comparative example show a "mass part" and "mass%." In addition, in the Example and comparative example which are shown below, content of each component shows a pure part conversion value.

<Example of preparation of polymer particles>
<< Preparation Example 1 of Polymer Particle A >>
In a 100 mL two-necked flask (manufactured by Shibata Scientific Co., Ltd.), N-isopropylacrylamide (lower limit critical solution temperature: 32 ° C., manufactured by Tokyo Chemical Industry Co., Ltd.) 2.5 g as a monomer, N, N'-methylene bis as a crosslinking agent 25 mg of acrylamide (manufactured by Tokyo Kasei Kogyo Co., Ltd.) and 12.5 mg of sodium dodecylbenzenesulfonate (manufactured by Kanto Chemical Co., Ltd.) as a surfactant were added to 45 g of water as a solvent.
5% by mass (0.47 mol / L) of N-isopropylacrylamide relative to the total amount of water, 1% by mass (0.73 mol%) of N, N'-methylenebisacrylamide relative to the total amount of N-isopropylacrylamide, water The sodium dodecylbenzene sulfonate was adjusted to the value shown in Table 1 with respect to the total amount, and was completely dissolved by stirring with a 2 mm rotor 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 seconds. After the argon purge, 10 mg of 2,2'-azobis (2-methylpropionamidine) dihydrochloride (manufactured by Wako Pure Chemical Industries, Ltd.) as a polymerization initiator is dissolved in 2.5 g of water at 70 ° C., N-isopropylacrylamide A solution of 0.4% by mass (0.17 mol%) of 2,2′-azobis (2-methylpropionamidine) dihydrochloride with respect to the total amount was added and polymerized for 1 hour. About 48 g of a polymerization solution containing N-isopropylacrylamide polymer particles obtained by the above polymerization was placed in a 100 cc beaker, coagulated with 28 g of methanol, and spontaneously filtered to separate aggregates. Next, the separated aggregate was washed with a mixed solution of 30 g of water and 18 g of methanol, and then dried in a thermostat at 80 ° C. for 5 hours to remove water sufficiently to obtain a mass of polymer particles.

  Next, a mass of the polymer particles is put into a 150 mL screw cap tube (trade name: SV-150, manufactured by Nichiden-Rika Glass Co., Ltd.), 25 g of acetone is added, and uniformly dispersed by a 40 mm rotor. Was added to reprecipitate. The reprecipitated precipitate was crushed with a glass rod and made into granules. Next, the hexane was decanted, and similarly, 50 cc hexane was added to reprecipitate. This operation was repeated three times. Next, after predrying on a hot plate at 50 ° C. for 3 hours, it was dried under reduced pressure at 50 ° C. to prepare powdery polymer particles A. The yield is 2.25 g, the yield is about 90%, the volume average particle size (D50) at 25 ° C. is 2.41 μm, the cumulant particle size at 25 ° C. is 2.24, and 5% by mass dispersion The shear viscosity at 25 ° C. was 0.01 mPa · s, and at 37 ° C. was 2,740 mPa · s. The volume average particle size (D50) and the cumulant particle size of the polymer particles were measured by the following measurement methods. The shear viscosity of the polymer particles was measured under the following measurement conditions.

-Method of measuring volume average particle size (D50) of polymer particles and cumulant particle size-
The volume average particle diameter (D50) and the cumulant particle diameter can be obtained by preparing a sample liquid as described below using a concentrated particle diameter analyzer (trade name: FPAR-1000, manufactured by Otsuka Electronics Co., Ltd.) The sample solution obtained can be measured under the following measurement conditions.
-Preparation of sample solution-
Powdered polymer particles A synthesized so as to be 0.5% by mass are swollen and dispersed in pure water obtained using a pure water production apparatus (trade name: GSH-2000, manufactured by ADVANTEC). The amount of liquid for measurement was 5 cc, and a sample liquid was prepared by stirring a 20 mm rotor with a stirrer at 200 rpm for about one day for dispersion.
-Measurement conditions-
Solvent: water (refractive index: 1.3314, viscosity at 25 ° C .: 0.884 cP, optimum light amount adjustment is appropriately set by the ND filter)
Measurement probe: Thickening probe Measurement routine: Measurement: 180 seconds at 25 ° C. Measurement: 600 seconds at 25 ° C. (When the body side is changed to 35 ° C., the liquid temperature gradually becomes 25 ° C. to 35 ° C. Particles in the meantime) Monitor diameter change) → Measurement: 180 seconds at 35 ° C

-Method of measuring shear viscosity-
The sterilized polymer particles A were diluted to 5% by mass with a 0.4% by mass aqueous solution of calcium chloride (manufactured by Wako Pure Chemical Industries, Ltd.), and a stirrer (trade name: Rexim RS-4A, manufactured by AsOne) was used. Dispersed. After confirming the dispersion, shear viscosity was measured with a rheometer (manufactured by Anton Paar / MCR301). Measurement with a rheometer is shear rate 0.1 (1 / s) while raising temperature to 37 ° C after measuring for 100 seconds (10 points every 10 seconds) at shear rate 10 (1 / s) at 25 ° C. It asked by measuring for 100 seconds (10 points every 10 seconds) with.

<< Production Examples 2 to 6 of Polymer Particles B to F >>
Polymer particles B to F were produced in the same manner as in Production Example 1 of polymer particle A, except that the content of the crosslinking agent and the content of the surfactant were changed to the content described in Table 1. In Table 1, the content of the crosslinking agent, the content of the surfactant, the volume average particle diameter at a liquid temperature of 25 ° C., the cumulant particle diameter at a liquid temperature of 25 ° C., the volume average particle diameter at a liquid temperature of 37 ° C., the liquid temperature 37 The cumulant particle size in ° C. and the shear viscosity at 25 ° C. and 37 ° C. are shown. Moreover, the particle size distribution of the said polymer particle in each temperature is shown to FIGS. The volume average particle diameter (D50) of the polymer particles B to F, the cumulant particle diameter, and the shear viscosity were measured in the same manner as the polymer particles A.

In Table 1, in the polymer particles A, C, and F, the volume average particle size at a liquid temperature of 37 ° C. and the cumulant particle size were not measured.

Preparation Example 1 of Polymer Particle Dispersions A to F
The polymer particles A to F were dispersed in pure water at 10% by mass, and high-pressure steam sterilization (trade name: MLS-3030, manufactured by Panasonic Corporation) at 121 ° C. for 20 minutes. Then, Dulbecco's modified Eagle's medium (D-MEM: 10% fetal bovine serum (FBS, Gibco), 1% antibiotic (Antibiotic-Antimycotic Mixed Stock Solution (100x), Wako Pure Chemical Industries, Ltd.) The sterilized polymer particles are diluted to 0.4% by mass with Wako Pure Chemical Industries, Ltd., dispersed using a stirrer (trade name: Rexim RS-4A, manufactured by AsOne), and polymer particle dispersion solution A to I got F.

<Confirmation of Phase State Associated with Phase Change Due to Temperature Change of Polymer Particle Dispersion A Containing Polymer Particle A>
The polymer particle dispersion A is allowed to stand at 37 ° C., which is a temperature higher than the lower critical solution temperature (32 ° C.) of the polymer particles A, and 25 ° C., which is lower than the lower critical solution temperature of the polymer particles A, The phase state of the particle dispersion A was visually confirmed. FIG. 4A is a photograph showing the state of the polymer particle dispersion A at 25 ° C., which has a lower critical solution temperature of 32 ° C. FIG. 4B is a photograph showing the polymer particle dispersion A at 37 ° C., which has a lower critical solution temperature of 32 ° C.

  As shown in FIG. 4A, it can be confirmed that the polymer particle dispersion A exists in a sol state at 25 ° C. where the lower limit critical solution temperature is 32 ° C. or less. On the other hand, as shown in FIG. 4B, at 37 ° C. where the lower critical solution temperature is higher than 32 ° C., it can be confirmed that the polymer particle dispersion A exists in a gel state.

<Confirmation of relationship between shear viscosity and temperature of composition containing polymer particles B>
The relationship between the shear viscosity of the composition containing the polymer particles B and the temperature was measured and confirmed as follows.
1% by mass of polymer particles B (volume average particle diameter: 2.41 μm) having a lower critical solution temperature of 32 ° C. is dispersed at 20 ° C. in Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal bovine serum, After measuring the shear viscosity at 20 ° C. for 100 seconds, the temperature was further raised to 37 ° C. for 100 seconds. FIG. 5 is a graph showing the relationship between the shear viscosity of the composition containing polymer particles B and the temperature. As shown in FIG. 5, the shear viscosity of the composition containing the polymer particle B when the temperature is raised from 20 ° C., which is lower than the lower critical solution temperature, to 37 ° C., which exceeds the lower critical solution temperature, is about 2,860 in 90 seconds. It can be confirmed that the temperature response speed related to the phase change between the sol state and the gel state is fast.

Example 1
<Example of cell preparation>
-Preparation Example of Cell 1-
A cryopreserved green fluorescent dye (trade name: Cell Tracker Green, manufactured by LifeTechnology) is thawed to room temperature, dissolved in dimethyl sulfoxide (hereinafter sometimes referred to as "DMSO") at a concentration of 10 mM, and serum-free medium And mixed with it to prepare a 10 .mu.M solution in 5 mL / dish of a serum-free medium containing green fluorescent dye. Next, human skin fibroblasts (trade name: CC2507, manufactured by Lonza, hereinafter sometimes referred to as "NHDF") cultured in a 35 mm dish are recovered with an aspirator, and the green fluorescent dye-containing non-containing 2 mL of serum medium was added and cultured for 30 minutes in an incubator (37 ° C., 5% CO ₂ environment). In addition, the volume average particle diameter in the free state of NHDF before adding to the green fluorescent dye-containing serum-free medium 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 culture medium of the dish was collected with an aspirator, 2 mL of a medium containing serum was added, the collection was carried out with an aspirator, and the cells were washed to wash away excess green fluorescent dye and DMSO. Furthermore, 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 cells could be observed.

-Measurement conditions of volume average particle size (D50) in free state of cells-
1% antibiotics (Antibiotic-Antimycotic Mixed Stock Solution (100x), Wako Pure Chemical Industries, Ltd.) in an incubator (trade name: KM-CC17RU2, manufactured by Panasonic Corporation, 37 ° C., 5% CO ₂ environment) After culturing NHDF (trade name: CC-2509, manufactured by Lonza) in Dulbecco's modified Eagle medium (including Wako Pure Chemical Industries, Ltd., sometimes referred to as "D-MEM"), aspirator (trade name) : 10% fetal bovine serum (hereinafter sometimes referred to as "FBS") in a 100 mm dish with VACUSIP (manufactured by INTEGRA), and the medium was removed. To the dish, 3 mL of phosphate buffered saline (made by Wako Pure Chemical Industries, Ltd., hereinafter sometimes referred to as "PBS (-)") was added, the PBS (-) was removed by aspiration with an aspirator, and the surface was washed . The washing operation with PBS (-) is repeated three times, 3 mL of 0.1% trypsin solution (Trypsin, from Porcine Pancreas, Wako Pure Chemical Industries, Ltd.) is added, and the mixture is heated for 5 minutes in an incubator. The cells were detached from the After confirming detachment of the cells with a phase contrast microscope, 4 mL of D-MEM containing 10% FBS and 1% antibiotics was added to inactivate the trypsin. The mixture was transferred to a centrifuge tube, centrifuged (trade name: H-19FM, manufactured by KOKUSAN, 1.2 × 10 ³ rpm (234 G), 5 minutes, 5 ° C.), and the supernatant was removed with an aspirator. After removal, 1 mL of D-MEM containing 10% FBS and 1% antibiotic was added to a centrifuge tube, and pipetting was performed gently to disperse the cells. 10 μL of the cell suspension was removed into an eppendorf tube, and 10 μL of 0.4% trypan blue staining solution was added to perform pipetting. 10 μL of the stained cell suspension was taken out and placed on a PMMA plastic slide, and the volume average particle size (D50) of the cells was measured using a trade name: Countess Automated Cell Counter (manufactured by Invitrogen).

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

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

<Preparation of three-dimensional cell assembly>
As shown in FIG. 15A, the medium is removed from the 35 mm dish 300 of NHDF 310 stained with the green fluorescent dye obtained in Preparation Example 1 of the cells with an aspirator, and the polymer particle dispersion B 350 is subjected to green fluorescence using a pipette. 1 mL was added onto the dye-stained NHDF 310. Furthermore, in an incubator (37 ° C., 5% CO ₂ environment) set to a temperature higher than the critical critical dissolution temperature (32 ° C.) of the polymer particles B in which a 35 mm dish is placed in a confocal fluorescence microscope (manufactured by Leica) After standing for a minute, a layer was formed by gelling the polymer particle dispersion B disposed on the cells. Next, gently pipette 0.1 mL (cell number: 5 × 10 ⁶ ) of the cell dispersion 1 onto the layer using a pipette, and stain it with a green fluorescent dye as shown in FIG. 15B. Polymer particle dispersion B350 and NHDF 320 dyed with a red fluorescent dye were laminated in this order to obtain a laminate 1.

  Next, the temperature of the dish is lowered to 37 ° C. to 25 ° C. which is equal to or lower than the lower critical solution temperature (32 ° C.) of the polymer particles B, and left for 10 minutes to sol the polymer particle dispersion B to red fluorescent light By precipitating the dye-stained NHDF, a three-dimensional cell aggregate 1 was obtained in which the green fluorescent dye-stained NHDF 310 and the red fluorescent dye-stained NHDF 320 were stacked as shown in FIG. 15C. The result of observing the three-dimensional cell laminate 1 with a fluorescence microscope is FIG. As shown in FIG. 16, on the NHDF stained with the green fluorescent dye (gray in the figure), the NHDF stained with the red fluorescent dye (black in the figure) can be observed, and the spreading of the cells can be confirmed. It was confirmed that adhesion between itself and self-organization had occurred.

  The average thickness of the three-dimensional cell assembly was obtained by staining the three-dimensional cell assembly 1 with hematoxylin-eosin method (HE), confirming the cross section, and measuring it using a stereomicroscope. As a result, the average thickness of the three-dimensional cell assembly 1 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 assembly 1 measured using a stereomicroscope is shown in FIG. The percentage of voids in the tissue and the degree of cell development indicate that the cells are proliferating and developing.

(Examples 2 to 6)
Three-dimensional cell aggregates 2 to 6 were obtained in the same manner as in Example 1 except that the polymer particle dispersion B was changed to polymer particle dispersions A and C to F. As a result of observing the three-dimensional cell assemblies 2 to 6 with a fluorescence microscope, it is possible to confirm cell spreading as in the three-dimensional cell assembly 1 and confirm that adhesion between cells and self-organization occur. did it. The average thickness of the three-dimensional cell assembly 2 to 6 was 0.01 mm or more.

(Comparative example 1)
A cell aggregate 1 was obtained in the same manner as in Example 1 except that the polymer particle dispersion B was not used. As a result of observing the cell assembly 1 with a confocal fluorescence microscope, the cells did not form a three-dimensional laminate, and the thickness of the cell assembly 1 was a thin cell sheet of less than 0.01 mm.

(Evaluation of cytotoxicity of polymer particles)
The three-dimensional cell assembly 1 prepared in Example 1 was observed with a phase contrast microscope. The results are shown in FIG. 18A. Moreover, the cell assembly 1 produced in the said comparative example 1 was observed with the phase-contrast microscope. The results are shown in FIG. 18B.

  From the results of FIGS. 18A and 18B, compared to the NHDF after culture of Comparative Example 1 of FIG. 18B, the three-dimensional cell assembly 1 after culture of Example 1 of FIG. It can be seen that the polymer particles are not toxic to cells. Although not shown in the figure, even when the polymer particle dispersions A and C to F are used instead of the polymer particle dispersion B, cell proliferation and spreading can be confirmed, and toxicity to the cells is It has been confirmed that there is no.

As an aspect of this invention, it is as follows, for example.
<1> A plurality of cells and a composition containing polymer particles phase-changed by temperature change are disposed on a substrate, and the composition containing the polymer particles is phase-changed from sol state to gel state by temperature change. A method for producing a three-dimensional cell assembly, characterized in that three-dimensional cell assemblies are produced repeatedly.
<2> The method for producing a three-dimensional cell assembly according to <1>, wherein the temperature change is performed by raising the temperature to a temperature higher than the lower critical solution temperature of the polymer particles.
<3> The three-dimensional cell assembly according to any one of <1> to <2>, wherein the volume average particle size in the composition in the sol state of the polymer particles is smaller than the volume average particle size in the free state of cells. It is a manufacturing method.
<4> The phase according to any one of <1> to <3>, wherein the cells are cultured at a temperature higher than the lower critical solution temperature of the polymer particles after phase-changing the composition containing the polymer particles from the sol state to the gel state. A method of producing a three-dimensional cell assembly of
It is a manufacturing method of the three-dimensional cell assembly in any one of said <1> to <4> whose volume average particle diameter in the composition of the sol state of <5> polymer particle is 50 micrometers or less.
<6> The method for producing a three-dimensional cell assembly according to any one of <1> to <5>, wherein the cells are adherent cells.
<7> The method for producing a three-dimensional cell aggregate according to any one of <1> to <6>, wherein the polymer particles are uniformly dispersed in the composition.
It is a manufacturing method of the three-dimensional cell assembly in any one of said <1> to <7> in which a <8> cell and the composition containing a polymer particle are simultaneously arrange | positioned on a base | substrate.
<9> The method for producing a three-dimensional cell assembly according to any one of <1> to <7>, wherein the composition containing the polymer particles is disposed on the cells after the cells are disposed on the substrate. is there.
<10> The three-dimensional structure according to any one of <1> to <9>, wherein the composition containing at least the polymer particles is disposed on the substrate by at least one selected from an inkjet method, a dispenser method, and a pipette method. It is a manufacturing method of a cell assembly.
<11> After culturing the cells, the temperature is lowered below the lower critical solution temperature of the polymer particles, and the composition containing the polymer particles is phase-changed from the gel state to the sol state according to any one of the above <4> to <10> A method of producing a three-dimensional cell assembly of
<12> The method for producing a three-dimensional cell assembly according to <11>, wherein the polymer particles are removed after culturing the cells and changing the composition including the polymer particles from the gel state to the sol state. .
<13> A composition containing polymer particles having a volume average particle size smaller in the sol state than a volume average particle size in the free state of cells, and containing a phase change between the sol state and the gel state by temperature change It is a feature of three-dimensional cell assembly.
<14> The three-dimensional cell assembly according to <13>, wherein the average thickness is 0.01 mm or more and 20 mm or less.
<15> The three-dimensional cell assembly according to any one of <13> to <14>, wherein a volume average particle diameter of the polymer particles in a gel state is 50 μm or less.
<16> A solution for forming a three-dimensional cell aggregate, comprising a composition comprising polymer particles that undergo a phase change between a sol state and a gel state due to a temperature change.

JP, 2013-116045, A JP, 2013-59312, A

1, 10, 100: substrate 2, 130: composition containing polymer particles in sol state 3: composition containing polymer particles in gel state

Claims (16)

  Place cells and a composition containing polymer particles that undergo phase change with temperature change on a substrate, and repeat the phase change from sol state to gel state of the composition containing polymer particles with temperature change several times. A method of producing a three-dimensional cell assembly characterized in that a three-dimensional cell assembly is produced.   The method for producing a three-dimensional cell assembly according to claim 1, wherein the temperature change is carried out by raising the temperature to a temperature higher than the lower critical solution temperature of the polymer particles.   The method for producing a three-dimensional cell aggregate according to any one of claims 1 to 2, wherein the volume average particle size in the composition in the sol state of the polymer particles is smaller than the volume average particle size in the free state of cells.   The three-dimensional cell assembly according to any one of claims 1 to 3, wherein the cells are cultured at a temperature higher than the lower critical solution temperature of the polymer particles after phase-changing the composition containing the polymer particles from the sol state to the gel state. Manufacturing method.   The method for producing a three-dimensional cell aggregate according to any one of claims 1 to 4, wherein the volume average particle size in the composition in the sol state of the polymer particles is 50 μm or less.   The method for producing a three-dimensional cell aggregate according to any one of claims 1 to 5, wherein the cells are adherent cells.   The method for producing a three-dimensional cell aggregate according to any one of claims 1 to 6, wherein the polymer particles are uniformly dispersed in the composition.   The method for producing a three-dimensional cell aggregate according to any one of claims 1 to 7, wherein the cells and the composition containing polymer particles are simultaneously disposed on a substrate.   The method for producing a three-dimensional cell assembly according to any one of claims 1 to 7, wherein a composition containing polymer particles is disposed on the cells after the cells are disposed on a substrate.   The method for producing a three-dimensional cell aggregate according to any one of claims 1 to 9, wherein the composition containing at least polymer particles is disposed on the substrate by at least one selected from the inkjet method, the dispenser method, and the pipetting method. .   The three-dimensional cell assembly according to any one of claims 4 to 10, wherein after the cells are cultured, the temperature is lowered below the lower critical solution temperature of the polymer particles to change the composition containing the polymer particles from the gel state to the sol state. Manufacturing method.   The method for producing a three-dimensional cell aggregate according to claim 11, wherein the cells are cultured, and the composition containing the polymer particles is phase-changed from the gel state to the sol state, and then the polymer particles are removed.   Containing a polymer particle having a smaller volume average particle size in the composition in the sol state than the volume average particle size in the free state of cells, and containing a composition that changes phase between the sol state and the gel state by temperature change Three-dimensional cell assembly characterized by   The three-dimensional cell assembly according to claim 13, wherein the average thickness is 0.01 mm or more and 20 mm or less.   The three-dimensional cell aggregate according to any one of claims 13 to 14, wherein a volume average particle size of the polymer particles in a gel state composition is 50 μm or less.   What is claimed is: 1. A solution for forming a three-dimensional cell aggregate, comprising: a composition comprising polymer particles that undergo a phase change between a sol state and a gel state by temperature change.