Method for producing temperature-responsive beads

Abstract

To provide a method for producing temperature-responsive beads through a process that is easy to perform on an industrial scale.SOLUTION: A method for producing temperature-responsive beads comprises a coating agent containing at least a temperature-responsive polymer having a lower critical solution temperature (LCST) in the range of 0°C or more to 50°C or less, and a carrier, the method comprising at least the steps of: dispersing the carrier in a reaction solution having the coating agent dissolved therein; and applying a poor solvent of the coating agent to a liquid containing the coating agent and the carrier to precipitate the coating agent on the surface of the carrier.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to a method for producing temperature-responsive beads. [Background technology]

[0002] Pluripotent stem cells possess the pluripotency to differentiate into cells of any lineage, and research is underway to establish regenerative medicine and cell therapy. However, reducing the cost of consumables such as culture media and growth factors has become a challenge, necessitating the development of an inexpensive and stable cell supply system. Many animal cells, including human cells, are generally anchorage-dependent and require a scaffold for cell proliferation. To reduce costs, it was necessary to design a scaffold that would increase cell proliferation per unit volume of medium. To solve this problem, microcarriers composed of beads made from synthetic polymers and natural polymers such as polysaccharides have been developed. By culturing microcarriers in a container containing a cell suspension, cells adhere and proliferate not only on the surface of the container but also on the surface of the microcarrier, thereby increasing cell proliferation per unit volume of medium.

[0003] However, after proliferation, cells must be detached from the scaffold and recovered. Cells adhere to the scaffold via adhesive proteins produced by the cells, and proteolytic enzymes such as trypsin are generally used to detach and recover cells that have adhered to the scaffold. However, proteolytic enzymes such as trypsin not only degrade adhesive proteins but also cell-specific surface proteins, which can damage the cells, posing a challenge.

[0004] To solve these problems, for example, Patent Document 1 discloses that beads coated with a temperature-responsive polymer enable the recovery of cultured cells by a cooling process without using enzymes. Patent Document 1 also discloses a method of coating the beads with the polymer by placing polystyrene beads and a polymer solvent in a flask and reducing the pressure while stirring in an evaporator.

[0005] However, this method has problems such as limitations on the amount of temperature-responsive beads that can be produced and the time and effort required to clean and maintain the evaporator, making it unsuitable for industrial production.

[0006] Furthermore, Patent Document 2 also discloses a method for preparing temperature-responsive beads for cell culture, which involves a method of growing a temperature-responsive polymer from an atom transfer radical polymerization initiation point on the surface of a substrate by the atom transfer radical method in the presence of a catalyst, or a method of immobilizing an atom transfer radical polymerization initiator and growing a temperature-responsive polymer from the initiator by the atom transfer radical method in the presence of a catalyst. [Prior art documents] [Patent documents]

[0007] [Patent Document 1] Patent No. 6954047 [Patent Document 2] Patent No. 6313822 Summary of the Invention [Problem to be solved by the invention]

[0008] An object of the present invention is to provide a method for producing temperature-responsive beads by an industrially easy process. [Means for solving the problem]

[0009] As a result of extensive research, the present inventors have found that temperature-responsive beads can be easily produced industrially by adding a poor solvent to precipitate a polymer on the surface of beads and then filtering the beads. [1] A method for producing temperature-responsive beads comprising a carrier and a coating agent containing a temperature-responsive polymer having a lower critical solution temperature (LSCT) in the range of 0°C to 50°C, the method comprising at least the steps of: dispersing the carrier in the coating agent; and applying a poor solvent for the coating agent to a liquid containing the coating agent and the carrier, thereby precipitating the coating agent on the surface of the carrier. [2] The method according to [1], wherein the amount of the coating agent to be deposited is in the range of 0.1% by mass to 3% by mass relative to the mass of the carrier. [3] The method according to [1] or [2], wherein the particle size of the support is in the range of 50 μm to 1000 μm, and the thickness of the coating agent deposited on the surface of the support is in the range of 10 nm to 1000 nm. [4] The method according to any one of [1] to [3], wherein the coating agent further comprises a cell adhesive polymer. [5] The method according to any one of [1] to [4], wherein the temperature-responsive polymer is a block copolymer containing the following blocks (A), (B), and (C): (A) Polymer block having an HLB value (Griffin method) in the range of 7 to 20 (B) Polymer block having an HLB value (Griffin method) in the range of 0 to less than 7 (C) A temperature-responsive polymer block having an LCST in the range of 0°C or more and 50°C or less. [6] The method according to [4] or [5], wherein the cell adhesive polymer is a copolymer of p-carboxystyrene and styrene. [Effects of the Invention]

[0010] The method described in the present invention enables industrial production of temperature-responsive beads. Furthermore, since the method does not involve a heating step, there is no risk of deterioration of the polymer or temperature-responsive beads due to heating. Furthermore, the film thickness of the polymer layer can be controlled by the amount of polymer added. DETAILED DESCRIPTION OF THE INVENTION

[0011] The present invention will be described in detail below.

[0012] In the present invention, the type of the temperature-responsive polymer is not particularly limited, but an example thereof is a block copolymer containing the following blocks (A), (B), and (C). (A) A polymer block having an HLB value (Griffin method) in the range of 7 or more and 20 or less. (B) A polymer block having an HLB value (Griffin method) in the range of 0 or more and less than 7. (C) A temperature-responsive polymer block having a lower critical solution temperature (LCST) in water in the range of 0°C to 50°C.

[0013] The HLB value (Hydrophile-Lipophile Balance: HLB) is a value that represents the degree of affinity for water and oil, as described in W.C. Griffin, Journal of the Society of Cosmetic Chemists, 1, 311 (1949), and takes a value from 0 to 20, with the closer to 0 the higher the hydrophobicity and the closer to 20 the higher the hydrophilicity. Methods for calculating the HLB value using a formula include the Atlas method, the Griffin method, the Davis method, and the Kawakami method. In this specification, the value calculated by the Griffin method was used, and the value was calculated using the following formula based on the formula weight of the hydrophilic moiety in the repeating unit of each block constituting the block copolymer of the present invention and the total formula weight of the repeating units.

[0014] [Number 1] HLB value = 20 × (formula weight of the hydrophilic portion in the repeating unit) / (total formula weight of the repeating units) Examples of the hydrophilic moiety in the repeating unit of each block mentioned above include a sulfone moiety (-SO3-), a phosphono group (-PO3-), a carboxyl group (-COOH), an ester group (-COO-), an amide group (-CONH-), an imide group (-CON-), an aldehyde group (-CHO), a carbonyl group (-CO-), a hydroxyl group (-OH), an amino group (-NH2), an acetyl group (-COCH3), an ethyleneamine group (-CH2CH2N-), an ethyleneoxy group (-CH2CHO-), an alkali metal ion, an alkaline earth metal ion, an ammonium ion, a halide ion, and an acetate ion.

[0015] When calculating the hydrophilic moiety in a repeating unit, the atoms constituting the hydrophilic moiety must not overlap with atoms constituting other hydrophilic moieties. An example of calculating the HLB value in a repeating unit is shown below. For example, in the case of 2-methacryloyloxyethyl phosphorylcholine (molecular weight: 295.27), the hydrophilic moiety consists of one part ester moiety, one part phosphono group moiety, and one part ethyleneamine moiety, and the molecular weight of the hydrophilic moiety is 181.04, so the HLB value is 12.3. In the case of 2-dimethylaminoethyl methacrylate (molecular weight: 157.11), the hydrophilic moiety consists of one part ester moiety and one part ethyleneamine moiety, and the molecular weight of the hydrophilic moiety is 86.07, so the HLB value is 11.0. In the case of methyl methacrylate (molecular weight: 100.12), the hydrophilic moiety consists of one part ester moiety, and the molecular weight of the hydrophilic moiety is 44.01, so the HLB value is 8.8. In the case of n-butyl methacrylate (molecular weight: 142.20), the hydrophilic portion is composed of 1 part of an ester portion, and the molecular weight of the hydrophilic portion is 44.01, so the HLB value is 6.2.

[0016] The block (A) preferably has an HLB value of 8.0 or more and 20.0 or less, in order to shorten the cooling time required to detach cells grown on the temperature-responsive beads. The repeating units constituting the block (A) are not particularly limited as long as they have an HLB value of 7.0 or more and 20.0 or less, and examples thereof include 2-methacryloyloxyethyl phosphorylcholine, polyethylene glycol methacrylate, 2-methoxyethyl acrylate, tetrahydrofurfuryl acrylate, dimethyl(2-methacryloyloxyethyl)(carboxylatomethyl)aminium, dimethyl(2-methacryloyloxyethyl)(2-carboxylatoethyl)aminium, dimethyl(3-methacryloylaminopropyl)(3-sulfonatopropyl)aminium, dimethyl(3-methacryloylaminopropyl)(4-sulfonatobutyl)aminium, and dimethyl(2-methacryloyloxyethyl)(2-sulfonatoethyl)aminium. Examples of the repeating unit include repeating units formed by polymerizing dimethylaminomethyl (meth)acrylate, 2-dimethylaminoethyl (meth)acrylate, dimethyl[(meth)acrylamidomethyl]amine, and dimethyl[(meth)acrylamidoethyl]amine as monomers, and repeating units formed by reacting repeating units formed by polymerizing 2-dimethylaminoethyl (meth)acrylate, 3-dimethylaminopropyl (meth)acrylate, dimethyl[(meth)acrylamidoethyl]amine, and dimethyl[3-(meth)acrylamidopropyl]amine as monomers with a C1 to C4 alkyl halide, ethylene oxide, propylene oxide, 1,2-butylene oxide, or 2-chloroethyl methyl ether. Among these repeating units, from the viewpoints of being able to control cell adhesiveness and shortening the cooling time required for cell detachment, repeating units produced by polymerizing 2-methacryloyloxyethyl phosphorylcholine, polyethylene glycol methacrylate, 2-methoxyethyl acrylate, tetrahydrofurfuryl acrylate, 2-dimethylaminomethyl (meth)acrylate, 2-dimethylaminoethyl (meth)acrylate, dimethyl[(meth)acrylamidomethyl]amine, and dimethyl[(meth)acrylamidoethyl]amine as monomers are preferred.Furthermore, block (A) may be composed of one type of repeating unit or two or more types of repeating units, so long as the HLB value is 7.0 or more and 20.0 or less.

[0017] In order to obtain a stable film when coated onto beads, block (B) more preferably has an HLB value of 0 or more and 6 or less. The repeating units constituting block (B) are not particularly limited as long as they have an HLB value of 0.0 or more and less than 7.0, and examples thereof include repeating units formed by polymerizing styrene, 1-vinylnaphthalene, 2-vinylnaphthalene, 9-vinylanthracene, and 1-vinylpyrene as monomers, and repeating units formed by polymerizing (meth)acrylate compounds such as ethyl (meth)acrylate, n-propyl (meth)acrylate, n-butyl (meth)acrylate, n-pentyl (meth)acrylate, n-hexyl (meth)acrylate, n-heptyl (meth)acrylate, n-octyl (meth)acrylate, and n-tridecyl (meth)acrylate as monomers. Among these repeating units, in order to obtain a stable film when coated on beads, repeating units formed by polymerizing styrene as a monomer or repeating units formed by polymerizing a (meth)acrylate compound such as n-propyl(meth)acrylate, n-butyl(meth)acrylate, n-pentyl(meth)acrylate, n-hexyl(meth)acrylate, n-heptyl(meth)acrylate, or n-octyl(meth)acrylate as a monomer are preferred. Furthermore, block (B) may be composed of one type of repeating unit or two or more types of repeating units, as long as its HLB value is 0.0 or more and less than 7.0.

[0018] The lower critical solution temperature (LCST) is the temperature below which a polymer dissolves in water to form a transparent solution, but above which it becomes insoluble and cloudy, or precipitates and undergoes phase separation. Examples of repeating units contained in block (C) and their LCSTs with respect to water include N-isopropylacrylamide (LCST = 32°C), Nn-propylmethacrylamide (LCST = 22°C), N-tetrahydrofurfurylacrylamide (LCST = 28°C), N-ethoxyethylacrylamide (LCST = 35°C), N,N-diethylacrylamide (LCST = 32°C), N-isopropylmethacrylamide (LCST = 44°C), Nn-propylmethacrylamide (LCST = 28°C), N-tetrahydrofurfurylmethacrylamide (LCST = 35°C), N-methyl-N-isopropylacrylamide (LCST = 23°C), and N-methyl-Nn-propylacrylamide (LCST = 20°C). Block (C) in the present invention may use one type of repeating unit or a combination of two or more types. In addition to the temperature-responsive repeating unit, the polymer may contain a different repeating unit, so long as it has temperature responsiveness.

[0019] An example of a block copolymer containing blocks (A), (B), and (C) is a block copolymer consisting of repeating units of (A) 2-methoxyethyl acrylate (HLB value = 13.5), (B) n-butyl methacrylate (HLB value = 6.2), and (C) N-isopropylacrylamide (LCST = 32°C).

[0020] In order to fix the polymer to the support, it is preferable that the number average molecular weight of the polymer is a certain value or more, and in order to reduce the viscosity of the polymer solution, it is also preferable that the number average molecular weight of the polymer is a certain value or less. For example, the number average molecular weight of the polymer is 1,000 to 1,000,000, preferably 5,000 to 500,000, and more preferably 10,000 to 200,000.

[0021] The solvent for the coating agent containing at least a temperature-responsive polymer is not particularly limited, and can be selected from solvents in which the polymer to be immobilized is soluble and the carrier is insoluble, such as methanol, ethanol, 1-propanol, 2-propanol, 1-methoxy-2-propanol, n-butanol, 2-butanol, t-butanol, acetone, dimethylformamide, and dimethyl sulfoxide.

[0022] The concentration of the polymer solution is not particularly limited, and is, for example, 0.01 to 99.9 wt%. The method for preparing the polymer solution is not particularly limited, but heating or stirring may be used to increase the dissolution rate of the polymer. The solution may also be filtered to remove insoluble matter.

[0023] The carrier used in the present invention is a powder or granule. The particle size of the carrier may be in the range of 50 μm to 1000 μm. The shape of the carrier is not particularly limited, and examples thereof include a sphere, a rectangular parallelepiped, a cylindrical column, a cylinder, and a star shape. The true density of the carrier is not particularly limited, but is preferably higher than the density of the polymer solution, and is, for example, 0.9 to 20.0 g / cm. 2 The carrier may or may not have pores, but it is preferable that the carrier has pores in order to increase the amount of polymer immobilized on the carrier. The specific surface area per dry mass of the carrier is not particularly limited, but for example, it is 10 to 100,000 cm 2 / g. The specific surface area per dry mass is measured by the BET method.

[0024] The carrier material is not particularly limited, and various materials such as metals, metal oxides, inorganic salts, synthetic polymers, biopolymers, and composites thereof can be used, but synthetic polymers are preferred because of their ease of processing. The type of synthetic polymer is not particularly limited, and examples include polystyrene, polyethylene, polypropylene, polyethylene terephthalate, polycarbonate, and polyvinyl chloride. The carrier surface may be coated with a functional group, and examples include acyl groups, acryloyl groups, hydroxy groups, carboxy groups, amino groups, ammonium groups, nitro groups, nitrile groups, nitroso groups, phosphate groups, sulfo groups, thiol groups, and halogens.

[0025] The method for dispersing the carrier in the reaction solution in which the coating agent has been dissolved is not particularly limited, and examples thereof include stirring with a stirring blade (impeller, etc.), end-over-end stirring (rotator, etc.), shaking stirring (shaker, etc.), mixing rotor stirring, vortex mixer stirring, magnetic stirrer stirring, and ultrasonic dispersion. Dispersion by stirring with a stirring blade (impeller, etc.), end-over-end stirring (rotator, etc.), shaking stirring, mixing rotor stirring, or vortex mixer stirring is more preferred in order to avoid crushing the carrier and the temperature-responsive polymer precipitated on the bead surfaces.

[0026] The present invention is characterized by including a step of precipitating a temperature-responsive polymer on the surface of beads by adding a poor solvent to a liquid containing a coating agent and a carrier.

[0027] The poor solvent refers to a solvent that does not dissolve the temperature-responsive polymer, and examples of the poor solvent include hexane, heptane, dimethyl ether, water, pentane, benzene, toluene, acetonitrile, cyclohexane, methyl ethyl ketone, tetrahydrofuran, methyl acetate, and ethyl acetate. More preferred poor solvents include hexane, heptane, dimethyl ether, and water.

[0028] The amount of poor solvent to be applied to the liquid containing the coating agent and the carrier is not particularly limited, but as an example, it is equal to or greater than the volume of the coating agent.

[0029] The method for precipitating the coating agent on the surface of the carrier by allowing a poor solvent for the coating agent to act on a liquid containing the coating agent and the carrier is not particularly limited, and a mixture of the coating agent and polystyrene beads may be dropped into a poor solvent, or conversely, a mixture of the coating agent and polystyrene beads may be dropped. More preferably, dropping the mixture while stirring can prevent the beads from aggregating.

[0030] The amount of coating agent to be precipitated is preferably in the range of 0.1% by mass to 3% by mass, more preferably 1.0% by mass to 3% by mass, relative to the mass of the carrier. The amount of coating agent precipitated can be determined by extracting the temperature-responsive polymer from the coated polystyrene beads with an extraction solvent in which the polymer to be immobilized is soluble and the carrier is insoluble, and quantifying the amount by GPC. Examples of extraction solvents include dimethylformamide.

[0031] The thickness of the coating agent deposited on the surface of the carrier may be in the range of 10 nm to 1000 nm. The thickness of the coating agent can be calculated from the amount of the coating agent deposited and the average particle size of the polystyrene beads.

[0032] It is more preferable that the coating agent contains at least a temperature-responsive polymer having an LSCT in the range of 0°C or more and 50°C or less and a cell adhesive polymer, and it is more preferable that the cell adhesive polymer is a copolymer of p-carboxystyrene and styrene.

[0033] The beads obtained by the production method of the present invention can be used for cell culture. The type of cells to be cultured is not particularly limited. Examples include mesenchymal stem cells, Chinese hamster ovary-derived CHO cells, mouse connective tissue L929 cells, human embryonic kidney-derived HEK293 cells, and human cervical cancer-derived HeLa cells. Furthermore, epithelial and endothelial cells that constitute various tissues and organs in the body, contractile skeletal muscle cells, smooth muscle cells, and cardiac muscle cells, neuronal cells, glial cells, and fibroblasts that constitute the nervous system, hepatic parenchymal cells, non-hepatic parenchymal cells, and adipocytes that are involved in the metabolism of the body, as well as stem cells present in various tissues and cells induced to differentiate from them. Other examples include cells contained in blood, lymph, cerebrospinal fluid, sputum, urine, or feces, as well as microorganisms, viruses, and protozoa present in the body or in the environment. The type of culture medium used is not particularly limited; either serum or serum-free medium can be used, and can be selected appropriately depending on the cell type.

[0034] The cell culture method using the beads obtained by the production method of the present invention is not particularly limited, and may be performed under static conditions or agitated conditions. When culturing under agitated conditions, the agitation speed is not particularly limited, and is, for example, 1 to 1,000 rpm. The culture temperature during culturing is not particularly limited, and is, for example, 10 to 50°C. When the polymer immobilized on the beads is a temperature-responsive polymer exhibiting an LCST, it is preferable to perform the culture at or above the LCST, at which the temperature-responsive polymer becomes hydrophobic and cells easily adhere. The method for recovering cells after culturing can be selected appropriately depending on the function of the polymer immobilized on the beads. Generally, cells can be detached from the beads and recovered using a proteolytic enzyme such as trypsin. When the polymer immobilized on the beads is a temperature-responsive polymer exhibiting an LCST, cells on the beads can be recovered by cooling to a temperature below the LCST. The cooling method is not particularly limited, and may be performed directly using a cooler, or the medium may be replaced with a cooling liquid. The cooling liquid may be either a medium or a phosphate buffer, but selecting a phosphate buffer makes it easier for cells to detach from the microcarrier. Adding a chelating agent such as EDTA or EGTA to the phosphate buffer solution makes it easier for the cells to detach from the beads. There are no particular limitations on the concentration of the chelating agent in the phosphate buffer solution, and it is usually between 1 μM and 1 mM.

[0035] The culture performance of a cell culture method using beads obtained by the production method of the present invention is not particularly limited, but when the polymer immobilized on the beads is a temperature-responsive polymer exhibiting an LCST, it is evaluated based on the total cell count and the cooling detachment rate. The method for evaluating the temperature-responsive cooling detachment rate is not particularly limited, but as an example, (1) the number of cells recovered by cooling is measured, and (2) the number of cells that did not detach by cooling is measured using a protease. The number of cells in (1) is then divided by the sum of the number of cells in (1) and (2), and the result is multiplied by 100 to obtain the cooling detachment rate. The method for measuring the cell number is not particularly limited, but it can be evaluated using a hemocytometer. [Example]

[0036] The present invention will be described in more detail below using examples, but the present invention is not limited to these examples. Unless otherwise specified, commercially available reagents were used.

[0037] <Polymer composition analysis> The values ​​were determined by proton nuclear magnetic resonance spectroscopy (1H-NMR) using a nuclear magnetic resonance analyzer (manufactured by JEOL Ltd., trade name JNM-ECZ400S / L1).

[0038] <Analysis of polymer molecular weight and molecular weight distribution> The weight-average molecular weight (Mw), number-average molecular weight (Mn), and molecular weight distribution (Mw / Mn) were measured by gel permeation chromatography (GPC). The GPC system used was a Tosoh HLC-8320GPC. Two Tosoh TSKgel SuperAWM-H columns were used. The column temperature was set at 40°C, and the eluent was 2,2,2-trifluoroethanol containing 10 mM sodium trifluoroacetate. The measurement sample was prepared at 1.0 mg / mL. A molecular weight calibration curve was prepared using polymethyl methacrylate (Sigma-Aldrich) of known molecular weight.

[0039] <Installation of the evaporator> The equipment consisted of a Tokyo Rikakikai N-1300 rotary evaporator, a Tokyo Rikakikai CCA-1112A small cooling water circulation system, a Tokyo Rikakikai NVP-2100V diaphragm-type vacuum pump, and a Tokyo Rikakikai OSB-2200 water bath. Water was used as the fluid in the small cooling water circulation system, and the temperature was set to 3°C. The temperature of the water bath was set to 50°C.

[0040] Synthesis Example 1: Synthesis of temperature-responsive polymer MBI (1) 0.650 g (5 mmol) of 2-methoxyethyl acrylate (MEA) was added to a 100 mL two-neck flask, and then 31.8 mg (100 μmol) of cyanomethyl dodecyl trithiocarbonate, 1.6 mg (10 μmol) of azobisisobutyronitrile (AIBN), and 10 mL of 1,4-dioxane were added. After purging with argon gas, the mixture was heated and stirred at 62°C for 24 hours.

[0041] (2) After heating and stirring (1), 3.845 g (30 mmol) of n-butyl acrylate (BA) was added, followed by 1.6 mg (10 μmol) of AIBN and 5 mL of 1,4-dioxane. After purging with argon gas, the mixture was heated and stirred at 62°C for 48 hours.

[0042] (3) After heating and stirring (2), 7.355 g (65 mmol) of N-isopropylacrylamide (IPAAm, LCST (lower critical solution temperature) is 32°C) was added, followed by 1.6 mg (10 μmol) of AIBN and 35 mL of 1,4-dioxane. After purging with argon gas, the mixture was heated and stirred at 62°C for 48 hours.

[0043] (4) After heating and stirring (3), water was added to generate a precipitate, which was then dried under reduced pressure to obtain a yellow solid.

[0044] (5) The resulting yellow solid was dissolved in chloroform, and the chloroform phase was collected using a separatory funnel. The collected chloroform phase was concentrated using an evaporator, and then hexane was added to generate a precipitate. The precipitate was collected by filtration and dried under reduced pressure to obtain 5.805 g of the temperature-responsive polymer MBI.

[0045] The resulting MBI had a composition ratio of MEA / BA / IPAAm=5 / 26 / 69 (mol %), a number average molecular weight Mn of 85,000, and a molecular weight distribution Mw / Mn of 1.78.

[0046] Synthesis Example 2: Synthesis of cell adhesive polymer CStS (1) 1.156 g (8 mmol) of p-carboxystyrene (CSt) and 1.271 g (12 mmol) of styrene (St) were added to a 100 mL two-neck flask, and then 3.3 mg (20 μmol) of AIBN and 20 mL of tert-butyl alcohol were added. After the atmosphere was replaced with argon gas, the mixture was heated and stirred at 64°C for 24 hours.

[0047] (2) After heating and stirring (1), n-heptane was added to generate a precipitate, which was then dried under reduced pressure to obtain 0.92 g of the cell adhesive polymer CStS.

[0048] The composition ratio of CStS was CSt / St=33 / 67 (mol %), the number average molecular weight Mn was 106,000, and the molecular weight distribution Mw / Mn was 1.84.

[0049] Example 1: Preparation of temperature-responsive beads (1) 1-methoxy-2-propanol solution of MBI produced in Synthesis Example 1 (MBI concentration 5.0 × 10 -2 % by mass), and 4 g of a 1-methoxy-2-propanol solution of CStS produced in Synthesis Example 2 (CStS concentration: 5.0 × 10 -3 % by mass) was added to 5.6 g of 1-methoxy-2-propanol to prepare a polymer solution, thereby producing a coating agent.

[0050] (2) In a 200 mL flask, polystyrene beads containing quaternary ammonium salts as functional groups (average particle diameter: 130 μm, particle density: 1.09 g / cm) were placed. 3 1.07 g of the polymer solution (1) was added to 10 g of the mixture, and the mixture was allowed to stand for 1 minute.

[0051] (3) 50 mL of water, a poor solvent for the coating agent, was added while stirring to precipitate the coating agent on the surface of the polystyrene beads. After filtering and washing with water, polystyrene beads (temperature-responsive beads) with the coating agent on their surfaces were obtained.

[0052] Example 2 Cell proliferation and cell recovery using temperature-responsive beads (1) 200 mg of the temperature-responsive beads produced in Example 1 and 30 mL of mesenchymal stem cell growth medium (PromoCell) were added to a 30 mL single-use bioreactor (Able), and then 5 × 10 human bone marrow-derived mesenchymal stem cells (Lonza) at passage 2 were added. 5 The cells were added and cultured in an incubator at 37°C with a 5% (v / v) CO2 atmosphere for 6 days with stirring (agitation speed: 100 rpm).

[0053] (2) After the incubation, the bioreactor was transferred to a safety cabinet at room temperature and allowed to stand to allow the temperature-responsive beads to settle. 22.5 mL of the medium was removed from the bioreactor, and 22.5 mL of phosphate-buffered saline (PBS(-)) containing 1 mM EDTA and no calcium or magnesium ions was added, followed by standing at 4°C for 10 minutes.

[0054] The reason for adding EDTA is to detach cells from each other. However, cells could be detached from the temperature-responsive beads by cooling them to room temperature.

[0055] (3) After leaving the mixture to stand, the cells were suspended by stirring at 300 rpm for 3 minutes at room temperature, then passed through a 50 mL tube fitted with a cell strainer with a 70 μm opening. Five mL of PBS(-) was then added twice onto the cell strainer, allowing the cells to be recovered.

[0056] The above results demonstrate that mesenchymal stem cells can be cultured using the temperature-responsive microbeads produced by the method of the present invention, and that the cultured cells can be detached and recovered by cooling. Example 3 Analysis of polymer coating amount and film thickness The polymer coating component of 0.50 g of the temperature-responsive beads obtained in Example 1 was extracted with dimethylformamide to separate it from the beads, and the dimethylformamide was evaporated to remove the polymer coating component. The polymer coating component was dissolved in 1 mL of 2,2,2-trifluoroethanol containing 10 mM sodium trifluoroacetate, and the amount of polymer coated per 0.50 g of polymer-coated beads was analyzed from the GPC peak intensity using a calibration curve with a polymer coating solution of a specified concentration. The analysis result was 1960 μg / g-beads. The amount of coating agent precipitated in the temperature-responsive beads was 2.0 mass% relative to the mass of the polystyrene beads.

[0057] <How to calculate the specific surface area of ​​beads> The calculation was performed with an average particle size of 130 μm for polystyrene beads and a particle density of 1.09.

[0058] The surface area of ​​the beads is calculated using the following formula:

[0059] [Number 2] Surface area of ​​beads = 4 × π × (0.13 (cm) / 2) 2 The mass of the beads is calculated using the following formula:

[0060] [Number 3] Mass of beads = 4 / 3 x π x (0.13 (cm) / 2) 3 ×1.09 The specific surface area of ​​the beads is calculated by dividing the value calculated by Equation 2 by the value calculated by Equation 3. The bead surface area per bead mass is 427.4 cm 2 It turned out to be / g-beads.

[0061] <How to calculate film thickness> The film thickness of the coating agent is calculated using the following formula.

[0062] [Number 4] Film thickness = 1960 (μg / g-beads) / specific gravity of polymer (g / cm 3 ) / specific surface area (cm 2 / g-beads) The specific gravity of the polymer is 1g / cm 3 The film thickness was calculated to be 46 nm.

Claims

1. A method for producing temperature-responsive beads comprising a carrier and a coating agent containing a temperature-responsive polymer having a lower critical solution temperature (LSCT) in the range of 0°C to 50°C, a step of dispersing the carrier in the coating agent; the step of applying a poor solvent for the coating agent to a liquid containing the coating agent and the carrier, thereby precipitating the coating agent on the surface of the carrier.

2. The method according to claim 1, wherein the amount of the coating agent to be deposited is in the range of 0.1% by mass to 3% by mass relative to the mass of the carrier.

3. 3. The method according to claim 2, wherein the particle size of the carrier is in the range of 50 μm to 1000 μm, and the coating agent to be deposited on the carrier surface has a film thickness in the range of 10 nm to 1000 nm.

4. The method according to claim 1 , wherein the coating agent further comprises a cell adhesive polymer.

5. The method according to claim 4, wherein the temperature-responsive polymer is a block copolymer comprising the following blocks (A), (B), and (C): (A) Polymer block having an HLB value (Griffin method) in the range of 7 to 20 (B) Polymer block having an HLB value (Griffin method) in the range of 0 to less than 7 (C) A temperature-responsive polymer block having an LCST in the range of 0°C or more and 50°C or less.

6. The method of claim 4, wherein the cell adhesive polymer is a copolymer of p-carboxystyrene and styrene.

7. The method of claim 5, wherein the cell adhesive polymer is a copolymer of p-carboxystyrene and styrene.