Method for culturing cells using temperature-responsive microcarriers, and microbeads for culturing temperature-responsive cells

Abstract

The present invention provides: a method for culturing adherent cells in a culture vessel using a microcarrier covered with a polymer showing a lower critical solution temperature, said method being characterized by comprising the following steps (1)-(4): (1) culturing adherent cells in a culture solution having a temperature equal to or higher than the lower critical solution temperature in a culture medium having a temperature equal to or higher than the lower critical solution temperature; a step for culturing the cells on the surface of the microcarrier; (2) a step in which, after step (1), the culture solution is cooled to a temperature equal to or lower than the lower critical solution temperature; (3) a step in which, after step (2), the culture solution in the culture vessel is stirred to peel the cells from the surface of the microcarrier; and (4) a step for recovering the cells peeled off from the surface of the microcarrier after step (3), the temperature-responsive microbeads comprising a polymer coating film and a carrier, the polymer coating film comprising a temperature-responsive polymer having a lower critical solution temperature at 0-50 DEG C, and the carrier comprising a polymer having a lower critical solution temperature at 0-50 DEG C, and the polymer coating film comprising a polymer having a lower critical solution temperature at 0-50 DEG C and a polymer having a lower critical solution temperature at 0-50 DEG C and a polymer having a lower critical solution temperature at 0-50 DEG C. The surface of the carrier has positive charges, and the film thickness of the temperature-responsive polymer is 10-1000 nm.

Description

Technical Field

[0001] The present invention relates to a cell culture method using a temperature-responsive microcarrier, and a temperature-responsive microbead for cell culture and a culture method thereof. Background Art

[0002] With the development of biopharmaceuticals and regenerative medicine using cells as raw materials, it is required to cultivate cells efficiently, in large quantities and with high quality. Cells are classified into suspension cells and adherent cells, but many useful cells are anchorage-dependent cells such as mesenchymal stem cells and pluripotent stem cells, and the support that cells can adhere to is needed for proliferation. In the past, plastic dishes and flasks were used, but as cell demand expanded, the culture method using microcarriers suitable for mass culture has attracted much attention. Moreover, microcarriers as microbead particles composed of natural macromolecules such as synthetic polymers and polysaccharides have been developed. By placing microcarriers in a container equipped with a cell suspension and cultivating, the surface area that cells can adhere to increases, and the cell proliferation per unit of culture medium can be improved.

[0003] The culture method using microcarriers is a method of stirring the culture solution to suspend cells and microcarriers, and proliferating cells adhered to the surface of the microcarriers. The culture method using microcarriers has the following characteristics: by stirring, the cell density per unit volume can be increased compared with the previous culture method using a plate or a flask.

[0004] On the other hand, the adherent cells after the proliferation need to be peeled off and recovered from the support. Usually, in order to peel off and recover the cells adhered to the support, a protease such as trypsin is used. In particular, in order to recover the cells propagated on the microcarrier surface, a protease such as trypsin is usually used to peel off and recover from the microcarrier. However, there is the following problem with protease such as trypsin: due to the protein on the decomposition cell surface, the cell is damaged and the complicated operation of removing trypsin is required.

[0005] As a cell recovery method for suppressing cell damage, for example, Patent Document 1 discloses a method using a microcarrier covered with a temperature-responsive polymer having a lower critical solution temperature. At a culture temperature of 37°C, cells adhere and proliferate, and by cooling to 20°C, the temperature-responsive polymer becomes hydrophilic, and cells can be recovered without using protease. However, temperature-responsive microcarriers have the problem of low cell proliferation, and improvement is needed.

[0006] In addition, about the problem caused by peeling off from microcarrier using proteolytic enzyme in the culture method using microcarrier, in order to solve this problem, disclose the microcarrier that is covered with the polymer that shows lower critical solution temperature in patent documentation 2, patent documentation 3.According to patent documentation 2, patent documentation 3, by cooling, the polymer that shows lower critical solution temperature is carried out sol transformation, thereby can weaken the adhesive force on microcarrier surface, cell is peeled off from microcarrier, reclaim cell.When reclaiming cell, only by cooling, be difficult to cell fully peeled off from microcarrier surface, therefore under the situation of laboratory scale, usually use as patent documentation 3 records and use the recovery method of liquid transfer operation.But liquid transfer operation is difficult to be applied to mass cultivation, when utilizing the cell recovery method of patent documentation 2, patent documentation 3 records, not yet realize the provision of mass cultivation technology, require to be more suitable for the cell culture method of mass cultivation.

[0007] Prior art literature

[0008] Patent Literature

[0009] Patent Document 1: Japanese Patent No. 6313822

[0010] Patent Document 2: Japanese Patent No. 6954047

[0011] Patent Document 3: Japanese Patent Application Publication No. 2021-106543 Summary of the invention

[0012] Problem that the invention aims to solve

[0013] The first object of the present invention is to provide a cell culture method suitable for mass culture in a method for recovering cells after cell culture on microcarriers covered with a polymer showing a lower critical solution temperature. The second object of the present invention is to provide a temperature-responsive microcarrier with high cell proliferation.

[0014] Solutions for solving problems

[0015] Regarding the first object, the present inventors have repeatedly conducted in-depth studies in view of the problems in the culture method using microcarriers, and found that in a cell culture method after cell culture on microcarriers covered with a polymer showing a lower critical solution temperature, cells can be efficiently and less invasively recovered from the microcarriers by a method including a cooling step and a stirring step, thereby completing the present invention. That is, the present invention includes the following aspects.

[0016] <1-1>

[0017] A method for culturing adherent cells in a culture container using a microcarrier covered with a polymer showing a lower critical solution temperature, characterized in that the method comprises the following steps (1) to (4):

[0018] Step (1), culturing the cells on the surface of the microcarriers in a culture solution at a temperature above the lower critical solution temperature;

[0019] Step (2), after step (1), cooling the culture solution to a temperature below the lower critical solution temperature;

[0020] Step (3), after step (2), stirring the culture solution in the culture container to peel the cells off the surface of the microcarrier;

[0021] Step (4), after step (3), recovering the cells detached from the surface of the microcarrier.

[0022] <1-2>

[0023] The method according to <1-1>, characterized in that in the step (3), stirring is performed at a stirring Reynolds number in the range of 50 to 2000.

[0024] <1-3>

[0025] The method according to <1-1> or <1-2> is characterized in that 10 (v / v)% to 90 (v / v)% of the culture medium used in step (1) is replaced with a culture medium cooled to a temperature below the lower critical solution temperature, thereby performing step (2).

[0026] <1-4>

[0027] The method according to any one of <1-1> to <1-3>, wherein step (4) is performed by removing the microcarriers using a mesh.

[0028] <1-5>

[0029] The method according to any one of <1-1> to <1-4>, wherein the particle size of the microcarrier is 50 μm to 1000 μm.

[0030] <1-6>

[0031] The method according to any one of <1-1> to <1-5>, wherein the lower critical solution temperature is 0°C to 50°C.

[0032] <1-7>

[0033] The method according to any one of <1-1> to <1-6>, wherein the adherent cells are stem cells.

[0034] In addition, regarding the second purpose, the inventors have repeatedly conducted in-depth research in view of the problem that the cell proliferation of temperature-responsive microcarriers is low, and found that microbeads formed by covering microbeads with positive charges with a temperature-responsive polymer having a lower critical solution temperature of 0°C to 50°C can adhere to cells well, and cells can be peeled off and recovered by cooling after the culture is completed, thereby completing the present invention. That is, the present invention also includes the following aspects.

[0035] <2-1> A temperature-responsive microbead, characterized in that it is a temperature-responsive microbead composed of a polymer coating film and a carrier, the polymer coating film is composed of a temperature-responsive polymer having a lower critical solution temperature at 0°C to 50°C, the carrier surface has a positive charge, and the film thickness of the temperature-responsive polymer is 10nm to 1000nm.

[0036] <2-2> The temperature-responsive microbeads according to <2-1>, wherein the component having a positive charge on the surface of the carrier comprises any one of a tertiary amine, a quaternary ammonium salt, and an alkaline earth metal salt.

[0037] <2-3> The microbeads according to <2-1> or <2-2>, wherein the specific gravity of the carrier is 1.0 to 1.1.

[0038] <2-4> The temperature-responsive microbead according to any one of <2-1> to <2-3>, wherein the material of the carrier is polystyrene.

[0039] <2-5> The temperature-responsive microbeads according to any one of <2-1> to <2-4>, wherein the particle size of the carrier is 50 μm to 1000 μm.

[0040] <2-6> A cell culture method using the temperature-responsive microbeads according to any one of <2-1> to <2-5>.

[0041] Effects of the Invention

[0042] In a cell culture method using microcarriers covered with a polymer showing a lower critical solution temperature, cells can be efficiently and low-invasively recovered from the microcarriers by a method including a cooling step and a stirring step.

[0043] In addition, the present invention is characterized in that the temperature-responsive microbeads are composed of a polymer coating film and a carrier, wherein the polymer coating film is composed of a temperature-responsive polymer having a lower critical solution temperature at 0°C to 50°C, and the temperature-responsive microbeads have positive charges on the surface of the carrier, and the cell proliferation is high, and the cells can be recovered by cooling. DETAILED DESCRIPTION

[0044] Hereinafter, the mode for carrying out the present invention will be described in detail, but the present invention is not limited to the following contents. The present invention can be carried out by appropriately changing it within the scope of the gist thereof.

[0045] <Method for culturing adherent cells in a culture vessel using microcarriers covered with a polymer showing a lower critical solution temperature>

[0046] In a first aspect, the present invention provides a cell culture method using a temperature-responsive microcarrier. The present invention is a method for culturing adherent cells in a culture container using a microcarrier covered with a polymer showing a lower critical solution temperature, characterized in that the method comprises the following steps (1) to (4):

[0047] Step (1), culturing cells on the surface of the microcarrier in a culture solution at a temperature above the lower critical solution temperature;

[0048] Step (2), after step (1), cooling the culture solution to a temperature below the lower critical solution temperature;

[0049] Step (3), after step (2), stirring the culture solution in the culture container to peel the cells off the surface of the microcarrier;

[0050] Step (4), after step (3), recovering the cells detached from the surface of the microcarrier.

[0051] The lower critical solution temperature (LCST; Lower Critical Solution Temperature, hereinafter sometimes referred to as LCST) refers to a temperature at which a polymer dissolves in water to form a transparent solution at a temperature below this temperature, but does not dissolve at a temperature above this temperature and becomes cloudy or precipitates, and phase separation occurs. The LCST is not particularly limited, but is preferably near the culture temperature, and as an example, is in the range of 0°C to 50°C, preferably 20°C to 40°C, and more preferably 25°C to 35°C.

[0052] Examples of the repeating unit of the homopolymer having a lower critical solution temperature and its lower critical solution temperature in water include N-isopropylacrylamide (LCST=32°C), N-n-propylmethacrylamide (LCST=22°C), N-tetrahydrofurfurylacrylamide (LCST=28°C), N-ethoxyethylacrylamide (LCST=35°C), N,N-diethylacrylamide (LCST=32°C), N-n-propylmethacrylamide (LCST=28°C), N-tetrahydrofurfurylmethacrylamide (LCST=35°C), N-methyl-N-isopropylacrylamide (LCST=23°C), and N-methyl-N-n-propylacrylamide (LCST=20°C). The lower critical solution temperature fluctuates depending on the concentration of the aqueous solution, and N-isopropylacrylamide is preferred because the lower critical solution temperature of N-isopropylacrylamide has low concentration dependence.

[0053] The repeating unit of the polymer showing the lower critical solution temperature in the present invention may be only one kind, or two or more kinds may be combined. In addition, if it has LCST, in addition to the repeating unit of the polymer showing LCST, the repeating unit of the polymer not showing LCST may also be included. In addition, in addition to the polymer showing LCST, other polymer compounds may also be introduced for the purpose of improving the adhesion to cells and microcarriers. As an example, in an embodiment of the present invention, in order to improve the adhesion of cells, a polymer compound formed by carboxyl styrene and styrene is introduced. As an example, the composition of N-isopropylacrylamide showing LCST in the polymer is 10 mol% or more and 95 mol% or less, preferably 30 mol% or more and 80 mol% or less, and more preferably 65 mol% or more and 70 mol% or less.

[0054] The material of the microcarrier is not particularly limited, and examples thereof include polystyrene, polymethyl methacrylate, polyethylene terephthalate, polycarbonate, cellulose, cyclodextrin, acrylamide, alginate, dextran, gelatin, glass, or a mixture of two or more thereof.

[0055] The shape of the microcarrier is not particularly limited, and examples thereof include spherical, ellipsoidal, flat, and tubular microcarriers. In addition, the microcarrier may be a porous microcarrier or may not be a porous microcarrier. In the case of a porous microcarrier, the pore size is not limited.

[0056] The diameter of the microcarrier is not particularly limited, and the major diameter of the microcarrier is preferably 50 μm to 1000 μm, more preferably 100 to 700 μm. If the major diameter is smaller than the above range, it is difficult to separate from the cells, and the recovery rate is reduced. In addition, when the major diameter is larger than the above range, the culture area per unit volume becomes smaller.

[0057] In this specification, the temperature-responsive microcarrier refers to a microcarrier covered with a polymer showing a lower critical solution temperature.

[0058] The method for covering the microcarrier with a polymer showing a lower critical solution temperature is not particularly limited, and examples thereof include a method of chemically covering the repeating units showing a lower critical solution temperature by electron beam irradiation; and a method of physically covering the microcarrier by applying a surface treatment agent prepared by dissolving the polymer showing a lower critical solution temperature in a solvent.

[0059] The microcarrier surface covered with a polymer showing LCST can be further covered with an extracellular matrix. The type of the extracellular matrix is ​​not particularly limited, and for example, a basement membrane (matrigel) containing collagen, telomerase, hyaluronic acid, elastin, proteoglycan, glycosaminoglycan, fibronectin, laminin, vitronectin, gelatin or laminin, collagen IV, heparan sulfate proteoglycan, nidogen / nidogen1,2, etc. as the main component can be used, and they can be only one kind, and two or more kinds can also be combined. In addition, it can also be a fragment of these extracellular matrixes.

[0060] Adherent cells are cells that are attached to the surface of a cell culture substrate such as a microcarrier. The source of the cells is not particularly limited, and examples thereof include humans, monkeys, dogs, cats, rabbits, rats, nude mice, mice, guinea pigs, pigs, sheep, Chinese hamsters, and cattle. Specific cells include, for example, various cultured cell lines such as CHO cells derived from Chinese hamster ovaries, Vero cells derived from African green monkey kidneys, mouse connective tissue L929 cells, HEK293 cells derived from human fetal kidneys, and HeLa cells derived from human cervical cancer. Examples of specific cells include epithelial cells, endothelial cells, skeletal muscle cells showing contractility, smooth muscle cells, cardiomyocytes, neuronal cells constituting the nervous system, glial cells, fibroblasts, macrophages and dendritic cells related to the immunity of the organism, hepatic parenchymal cells and non-parenchymal liver cells related to the metabolism of the organism, adipocytes, induced pluripotent stem cells (iPS cells) as cells with differentiation ability, embryonic stem cells (ES cells), embryonic germ cells (EG cells), embryonic carcinoma cells (EC cells), mesenchymal stem cells, liver stem cells, pancreatic stem cells, skin stem cells, muscle stem cells, germ stem cells, and other stem cells, or precursor cells of various tissues, and cells induced by differentiation thereof. Mesenchymal stem cells are particularly preferably used. Mesenchymal stem cells refer to a group of stem cells and their precursor cells that can differentiate into all or several mesenchymal cells such as chondrocytes, osteoblasts, adipocytes, etc. The source of mesenchymal stem cells is not particularly limited, and can include tissue sources such as bone marrow, fat, dental pulp, umbilical cord blood, placenta, synovium, and pluripotent stem cell sources such as ES cells and iPS cells.

[0061] In step (1), by making the temperature of the culture solution in the culture container above the lower critical solution temperature, the polymer gelation showing the lower critical solution temperature is performed, and the adherent cells can adhere to the microcarrier and proliferate. In the case of using human-derived cells, in order to obtain high culture efficiency, it is recommended to carry out near body temperature, as an example, preferably in a temperature range of 30°C to 40°C, and more preferably in a temperature range of 36°C to 38°C. Conditions other than the lower critical solution temperature are not particularly limited, for example, it can be carried out by static culture, it can also be carried out by stirring culture, preferably stirring culture that can increase the culture area per unit volume.

[0062] The cell culture density is not particularly limited as long as the cells adhere and proliferate. In the case of human-derived mesenchymal stem cells, for example, the microcarrier density per unit surface area is preferably 1.0×10 1 cells / cm 2 ~1.0×10 5 cells / cm 2 , more preferably 1.0×10 2 cells / cm 2 ~1.0×10 4 cells / cm 2 Other culture conditions are not particularly limited, and the culture can be carried out under conditions commonly performed in the art.

[0063] The temperature of the culture solution during cooling in step (2) is preferably a temperature that is 1°C or more lower than the lower critical solution temperature. As a method for lowering the temperature of the microcarrier (also referred to as "cooling treatment"), examples include replacing the liquid in the culture container with a cooled liquid and storing it in a cold place, but in order to shorten the time spent on cooling, it is preferably replaced with a cooled liquid. The replacement amount when replacing with a cooled liquid is not particularly limited, and as an example, it is preferably 10% to 90%, and in order to increase the cooling rate, it is more preferably 50% to 90%. The cooled liquid is not particularly limited, and the culture solution, other culture medium solution, isotonic solution, etc. can be selected according to the purpose. In addition, the cooling time is preferably 5 minutes to 60 minutes.

[0064] The stirring method in step (3) is not particularly limited, and an example thereof is a method of stirring the culture solution by central stirring by rotating a stirring blade vertically inserted into the center of the culture container. The degree of stirring is not particularly limited, and stirring is preferably performed in a range of 50 to 2000 at a stirring Reynolds number, which is one of the indicators representing the degree of stirring, more preferably in a range of 100 to 1800, and most preferably in a range of 300 to 1500. If the stirring Reynolds number is less than 50, the cells are not completely peeled off from the microcarriers, and the recovery rate is reduced. In addition, if the stirring Reynolds number exceeds 2000, the cells are damaged and the viable cell rate is reduced. The stirring Reynolds number is represented by the following formula (1).

[0065] Stirring Reynolds number = fluid density × stirring speed × stirring diameter / fluid viscosity (1)

[0066] In addition, in order to prevent the peeled cells from re-adhering to the microcarriers, the temperature of the liquid in the culture container in step (3) is preferably 1°C or more lower than the lower critical solution temperature. The liquid used during stirring is not particularly limited, and a culture solution, other culture medium solution, isotonic solution, etc. may be selected according to the purpose. In consideration of the recovery rate and damage of the cells, the stirring time is preferably 1 to 60 minutes.

[0067] The method for separating microcarriers and cells in step (4) is not particularly limited, and examples thereof include a method for separation by difference in sedimentation velocity and a method for separation by difference in particle size. As a method for separation by difference in particle size, examples thereof include a method for separating microcarriers from a cell suspension using a filter. The material of the filter is not particularly limited, and as an example, it is nylon. The pore size of the filter is not particularly limited, and as an example, it is 20 μm to 100 μm, preferably 30 μm to 80 μm, and more preferably 40 μm to 60 μm. If the pore size of the filter is small, the cells do not pass through the filter, and the recovery rate of the cells is reduced. In addition, if the pore size of the filter is large, the microcarriers also pass through the filter, and separation becomes difficult.

[0068] In the present invention, the cell culture container is not particularly limited as long as it can stir and suspend microcarriers. In the examples of the present invention, a 30 mL disposable bioreactor (Able, product number: BWV-S03A) was used as an example.

[0069] The composition of the culture medium used in the culture of the present invention is not particularly limited as long as the cells adhere and proliferate, and includes a basic culture medium and serum, and may also contain antibiotics. The type of basic culture medium is not particularly limited, for example, MEM, αMEM, DMEM, EMEM, GMEM, DMEM / Ham'sF-12, Ham'sF-12, Ham'sF-10, Medium199, RPMI1640, etc. can be used. The type of serum is not particularly limited, for example, fetal bovine serum (Fetal Bovine Serum: FBS), calf serum, adult bovine serum, horse serum, sheep serum, goat serum, pig serum, chicken serum, rabbit serum, and human serum are used. From the perspective of ease of acquisition, FBS is usually often used. The serum concentration in the culture medium is not particularly limited. From the perspective of cost-effectiveness, it is usually used at a concentration of less than 20vol%, but it can also be a concentration of more than 20vol%. In addition, it can also be a serum-free culture medium that does not contain any of untreated or unpurified serum, but contains components derived from purified blood or components derived from animal tissues (growth factors, etc.).

[0070] There is no particular limitation on the method for measuring the viability rate, and as an example, there is a known method for determining the life or death of cells by staining the cytoplasm of dead cells with trypan blue. There is no particular limitation on the method for determining the life or death of cells and counting the number of cells, and there are methods in which an operator manually determines using a hemocytometer and methods in which an automatic cell counting device automatically determines. However, since the measurement can be performed independently of proficiency, the method using an automatic cell counting device is preferred.

[0071] <Temperature-responsive cell culture microbeads and culture method thereof>

[0072] In a second aspect, the present invention provides temperature-responsive microbeads for cell culture and a culture method thereof. The temperature-responsive microbeads of the present invention are characterized in that a temperature-responsive polymer having a critical solution temperature of 0°C to 50°C is covered with a microbead carrier having a positive charge.

[0073] The composition of the microbead carrier with positive charge of the present invention is not particularly limited. As an example, a compound with positive charge can be chemically fixed or physically fixed on the carrier surface. As an example of a chemical fixation method, a method of copolymerizing with a monomer containing a positive charge when manufacturing a carrier and a method of chemically modifying the particle surface with a functional group that becomes a positive charge can be illustrated. The functional group that becomes a positive charge is not particularly limited. As an example, a tertiary amino group, a quaternary ammonium group, and an alkaline earth metal salt can be cited. From the aspect of being particularly easy to modify, a trimethylammonium group, a dimethylamino group, a diethylamino group, and calcium phosphate can be illustrated. In addition, as an example of a method for fixing by physical adsorption, a method of covering a cationic polymer on a carrier and a method of covering an inorganic compound with low solubility in water on the surface can be illustrated.

[0074] The composition of the microbead carrier with a positive charge is not particularly limited. From the aspect of being able to slowly settle in the culture medium, the specific gravity is preferably 1.0 to 1.1, and more preferably 1.01 to 1.06. When the specific gravity is less than 1.0, it becomes difficult to culture because it floats on the culture medium. When the specific gravity is greater than 1.1, it becomes difficult to disperse in the culture medium. The material of the carrier is not particularly limited. Examples include synthetic polymers such as resins of polyethylene, polypropylene, polystyrene, poly(meth) alkyl acrylates, polyalkyl (meth) acrylamides, polyesters, polyurethanes, polyvinyl chloride, polycarbonates or their copolymers, polymers derived from plants such as dextran and cellulose, or wood chips and ceramics. The specific gravity of the microbeads can be designed to be close to the specific gravity of the culture solution. In terms of being able to suppress the sedimentation of the microbeads during cell culture with stirring, it is preferably a microbead carrier made of synthetic polymers such as polystyrene, poly(meth) alkyl acrylates, poly(meth) acrylamides, polyesters, polyurethanes, and more preferably a microbead carrier made of polystyrene. In addition, in order to suppress the dissolution of the material constituting the microbead carrier into the culture medium, it is preferably cross-linked. The shape of the microbead carrier is not particularly limited, and it can be plate-shaped or spherical, or it can be a porous body. In the case where the microbead carrier is spherical, the particle size is not particularly limited, but when the particle size is large, it becomes difficult to disperse in the culture solution, and when the particle size is small, it becomes difficult for cells to adhere, so it is preferably 50μm to 1000μm, more preferably 150μm to 600μm, and further preferably 150μm to 500μm.

[0075] In this specification, the lower critical solution temperature (LCST) refers to the temperature at which the solubility of a certain polymer in water changes. A polymer having an LCST dehydrates on the higher temperature side than the LCST, and the hydrophobic interaction becomes stronger, and hydrates, swells or dissolves on the lower temperature side than the LCST.

[0076] The temperature-responsive polymer of the present invention refers to a polymer containing, as a repeating unit, a monomer whose homopolymer has an LCST of 0° C. to 50° C. with respect to water.

[0077] The structure of the temperature-responsive polymer of the present invention can include repeating units that show LCST as other structures. From the aspect of showing good temperature responsiveness, the repeating unit showing LCST is preferably a block structure. When the temperature-responsive microbeads of the present invention are used for cell culture, they are usually cultured at around 37°C, so the LCST of the repeating unit having LCST is preferably 0°C to 50°C, more preferably 10°C to 40°C, and further preferably 20°C to 35°C. When the LCST is lower than 0°C or exceeds 50°C, damage to the cells is caused, so it is difficult to perform low-invasive cell peeling. As an example of a repeating unit having an LCST at 0°C to 50°C, there can be exemplified a repeating unit produced by polymerizing N-isopropylmethacrylamide (LCST = about 44°C), N-ethoxyethylacrylamide (LCST = about 35°C), N-tetrahydrofurfurylmethacrylamide (LCST = about 35°C), N-isopropylacrylamide (LCST = about 32°C), N,N-diethylacrylamide (LCST = about 32°C), N-n-propylmethacrylamide (LCST = about 28°C), N-tetrahydrofurfurylacrylamide (LCST = about 28°C), N-methyl-N-isopropylacrylamide (LCST = about 22°C), N-n-propylacrylamide (LCST = about 22°C), and N-methyl-N-n-propylacrylamide (LCST = about 20°C) as monomers. The temperature-responsive polymer is not particularly limited. As an example, there can be exemplified (N-isopropylacrylamide)-(n-butyl methacrylate) block copolymers and (N-isopropylacrylamide)-(n-butyl acrylate) block copolymers described in Japanese Patent No. 5846584, (2-dimethylaminoethyl methacrylate)-(n-butyl methacrylate)-(N-isopropylacrylamide) block copolymers described in Japanese Patent No. 6954047, and (N-isopropylacrylamide)-(n-butyl methacrylate)-(2-methoxyethyl acrylate) block copolymers described in Japanese Patent No. 7127330.

[0078] The film thickness of the temperature-responsive polymer of the temperature-responsive microbeads is not particularly limited, but is preferably 10 nm to 1000 nm, and more preferably 50 nm to 500 nm. When it is less than 10 nm, a strong positive charge effect is produced, resulting in poor temperature responsiveness, and when it exceeds 1000 nm, the positive charge effect is weakened.

[0079] The method for fixing the temperature-responsive polymer coating film to the carrier is not particularly limited, and the aforementioned polymer can be chemically fixed or physically adsorbed. In the case of immobilization by physical adsorption, the method is not particularly limited, and as an example, a method of drying after spraying the polymer solution on the microbead carrier can be illustrated. The drying method is not particularly limited, and air drying, reduced pressure drying, etc. can be listed. In the case where the carrier has pores, it is preferably immersed in a solvent for several hours before covering the temperature-responsive polymer to exhaust.

[0080] The temperature-responsive microbeads of the present invention can be used as microcarriers for cell culture. The type of cells is not particularly limited as long as they are cells that adhere to the temperature-responsive microbeads before cooling for cell detachment, and examples thereof include cells derived from humans, monkeys, dogs, cats, rabbits, rats, nude mice, mice, guinea pigs, pigs, sheep, Chinese hamsters, and cattle. Specific cells include, for example, various cultured cell lines such as CHO cells derived from Chinese hamster ovaries, Vero cells derived from African green monkey kidneys, mouse connective tissue L929 cells, HEK293 cells derived from human fetal kidneys, and HeLa cells derived from human cervical cancer. Examples of specific cells include epithelial cells, endothelial cells, skeletal muscle cells showing contractility, smooth muscle cells, cardiomyocytes, neuronal cells constituting the nervous system, glial cells, fibroblasts, macrophages and dendritic cells related to the immunity of the organism, hepatic parenchymal cells and non-parenchymal liver cells related to the metabolism of the organism, adipocytes, induced pluripotent stem cells (iPS cells) as cells with differentiation ability, embryonic stem cells (ES cells), embryonic germ cells (EG cells), embryonic carcinoma cells (EC cells), mesenchymal stem cells, liver stem cells, pancreatic stem cells, skin stem cells, muscle stem cells, germ stem cells, and other stem cells, or precursor cells of various tissues, and cells induced by differentiation thereof. Mesenchymal stem cells are particularly preferably used. Mesenchymal stem cells refer to a group of stem cells and their precursor cells that can differentiate into all or several mesenchymal cells such as chondrocytes, osteoblasts, adipocytes, etc. The source of mesenchymal stem cells is not particularly limited, and can include tissue sources such as bone marrow, fat, dental pulp, umbilical cord blood, placenta, synovium, and pluripotent stem cell sources such as ES cells and iPS cells.

[0081] Regarding the composition of the culture medium used in the cell culture using the temperature-responsive microbeads of the present invention, there is no particular limitation as long as the cells adhere and proliferate, and it includes a basal culture medium and serum, and may also contain antibiotics. The type of basal culture medium is not particularly limited, for example, MEM, αMEM, DMEM, EMEM, GMEM, DMEM / Ham'sF-12, Ham'sF-12, Ham'sF-10, Medium199, RPMI1640, etc. can be used. The type of serum is not particularly limited, for example, fetal bovine serum (Fetal Bovine Serum: FBS), calf serum, adult bovine serum, horse serum, sheep serum, goat serum, pig serum, chicken serum, rabbit serum, and human serum can be used. From the perspective of ease of acquisition, FBS is often used. The serum concentration in the culture medium is not particularly limited. From the perspective of cost-effectiveness, it is usually used at a concentration of less than 20vol%, but it can also be a concentration of more than 20vol%. Alternatively, the medium may contain no untreated or unpurified serum and may contain purified blood-derived components or animal tissue-derived components (growth factors, etc.).

[0082] In the cell culture using the temperature-responsive microbeads of the present invention, cells are cultured on the temperature-responsive microbeads at a temperature higher than the LCST of the temperature-responsive polymer, and after cell proliferation, the proliferated cells are peeled off from the microbeads by setting the temperature below the LCST of the temperature-responsive polymer. The cell culture method is not particularly limited. For example, the temperature-responsive microbeads of the present invention can be added to a cell culture container equipped with a culture medium, and after inoculating the cells, the temperature-responsive microbeads of the present invention can be left to stand, stirred continuously, or stirred or vibrated for a certain period of time, thereby culturing the cells. The cooling method when the cells are cooled and peeled off from the temperature-responsive microbeads of the present invention is not particularly limited. The culture medium can be cooled in a cold place, or the culture medium can be replaced with a cooled culture medium. In addition, in order to efficiently peel off the cells, the culture substrate can be gently tapped, or the culture substrate can be shaken, or the culture solution can be stirred, or pipetting can be used in combination.

[0083] The seeding density of cells is not particularly limited as long as the cells adhere and proliferate. In the case of human-derived mesenchymal stem cells, for example, the seeding density of microcarriers per unit surface area is preferably 1.0×10 1 cells / cm 2 ~1.0×10 5 cells / cm 2 , more preferably 1.0×10 2 cells / cm 2 ~1.0×10 4 cells / cm 2 Other culture conditions are not particularly limited, and the culture can be carried out under conditions commonly performed in the art.

[0084] Example

[0085] The following is an explanation of the examples of the present invention, but the present invention is not limited by these examples. It should be noted that, unless otherwise specified, commercially available reagents were used.

[0086] <Method for culturing adherent cells in a culture vessel using microcarriers covered with a polymer showing a lower critical solution temperature>

[0087] Reference Example 1 Synthesis of Temperature Responsive Polymer 1

[0088] In a 200 mL two-necked flask, 0.650 g (5 mmol) of 2-methoxyethyl acrylate (MEA) was added, and further 31.8 mg (100 μmol) of cyanomethyldodecyl trithiocarbonate, 1.6 mg (10 μmol) of azobisisobutyronitrile and 10 mL of tert-butanol were added. After argon replacement, the mixture was heated and stirred at 62°C for 24 hours.

[0089] After the first heating and stirring, 3.845 g (30 mmol) of n-butyl acrylate (BA) was added, and further 1.6 mg (10 μmol) of azobisisobutyronitrile and 5 mL of tert-butanol were added. After argon replacement, the mixture was heated and stirred at 62° C. for 24 hours.

[0090] After the second heating and stirring, 7.355 g (65 mmol) of N-isopropylacrylamide (IPAAm LCST = 32°C) was added to the above, and 1.6 mg (10 μmol) of azobisisobutyronitrile and 85 mL of tert-butanol were further added. After argon replacement, the mixture was heated and stirred at 62°C for 24 hours.

[0091] After the third heating and stirring, the reaction solution was reprecipitated and purified with water and dried under reduced pressure to obtain a yellow solid. The obtained yellow solid was dissolved in chloroform, and the chloroform phase was recovered using a separatory funnel. The recovered chloroform phase was concentrated with an evaporator and reprecipitated and purified with heptane. The precipitate was recovered by filtration and dried under reduced pressure to obtain 8.295 g of temperature-responsive polymer 1poly (MEA-BA-IPAAm). The composition of the obtained temperature-responsive polymer 1 was MEA: BA: IPAAm = 5: 30: 65 (mol%), the lower critical solution temperature was 32°C, and Mn was 11.8×10 4 , Mw / Mn is 1.45.

[0092] Reference Example 2 Synthesis of Temperature Responsive Polymer 2

[0093] In a 200 mL two-necked flask, 3.845 g (30 mmol) of n-butyl acrylate (BA) was added, and further 31.8 mg (100 μmol) of cyanomethyldodecyl trithiocarbonate, 1.6 mg (10 μmol) of azobisisobutyronitrile and 15 mL of tert-butanol were added. After argon replacement, the mixture was heated and stirred at 62°C for 24 hours.

[0094] After heating and stirring, 7.355 g (65 mmol) of N-isopropylacrylamide (IPAAm LCST = 32°C) was added to the above, and 1.6 mg (10 μmol) of azobisisobutyronitrile and 85 mL of tert-butanol were further added. After argon replacement, the mixture was heated and stirred at 62°C for 24 hours.

[0095] After the second heating and stirring, the reaction solution was reprecipitated and purified with water and dried under reduced pressure to obtain a yellow solid. The obtained yellow solid was dissolved in chloroform, and the chloroform phase was recovered using a separatory funnel. The recovered chloroform phase was concentrated with an evaporator and reprecipitated and purified with heptane. The precipitate was recovered by filtration and dried under reduced pressure to obtain 7.591 g of temperature-responsive polymer 2poly (BA-IPAAm). The composition of the obtained temperature-responsive polymer 2 was BA:IPAAm=32:68 (mol%), the lower critical solution temperature was 32°C, and Mn was 10.8×10 4 , Mw / Mn is 1.35.

[0096] Reference Example 3 Synthesis of Polymer Compound 1 (St / CSt)

[0097] In a 100 mL two-necked flask, 1.156 g (8 mmol) of p-carboxystyrene (CSt, pKa = 4.20) as component (A) and 1.271 g (12 mmol) of styrene (St, HLB value = 0) as component (B) were added, and 3.3 mg (20 μmol) of azobisisobutyronitrile and 20 mL of tert-butyl alcohol were further added. After nitrogen substitution, heating and stirring were performed at 64°C for 24 hours. Purification was performed by the same method as in Example 1 to obtain 0.92 g of polymer compound 1poly (CSt / St). The composition of the obtained polymer compound 1 was CSt:St = 33:67 (mol%), and the number average molecular weight Mn was 10.6×10 4 , the molecular weight distribution Mw / Mn is 1.84.

[0098] Reference Example 4 Preparation of Surface Treatment Agent 1

[0099] 1.00 g of temperature responsive polymer 1, 0.01 g of polymer compound 1 and 48.99 g of 1-methoxy-2-propanol were added to a glass container and left to dissolve overnight.

[0100] Reference Example 5 Preparation of Surface Treatment Agent 2

[0101] 1.00 g of temperature-responsive polymer 2, 0.01 g of polymer compound 1, and 48.99 g of 1-methoxy-2-propanol were added to a glass container and left to dissolve overnight.

[0102] Reference Example 6 Preparation of Surface Treatment Agent 3

[0103] 1.00 g of temperature-responsive polymer 1, 0.05 g of polymer compound 1, and 48.95 g of 1-methoxy-2-propanol were added to a glass container and left to dissolve overnight.

[0104] Reference Example 7 Preparation of temperature-responsive microcarrier 1

[0105] 5 g of untreated microcarriers (Corning, product number: 4625, particle size: 125-212 μm) and 10 g of surface treatment agent 1 were added to a 25 mL eggplant flask and allowed to stand for 1 hour. The solvent was then distilled off by reducing the pressure using an evaporator to obtain temperature-responsive microcarriers 1.

[0106] Reference Example 8 Preparation of Temperature Responsive Microcarrier 2

[0107] 5 g of untreated microcarriers (Corning, product number: 4625, particle size: 125-212 μm) and 10 g of surface treatment agent 2 were added to a 25 mL eggplant flask and allowed to stand for 1 hour. The solvent was then distilled off by reducing the pressure using an evaporator to obtain temperature-responsive microcarriers 2.

[0108] Reference Example 9 Preparation of Temperature Responsive Microcarrier 3

[0109] 5 g of AmberChrom (registered trademark) 1×8 chloride foam, 100-200 mesh (Sigma-Aldrich, product number: 217425, particle size: 54-154 μm) and 10 g of surface treatment agent 3 were added to a 25 mL eggplant-shaped flask and allowed to stand for 1 hour. The solvent was then distilled off by reducing the pressure using an evaporator to obtain temperature-responsive microcarriers 3.

[0110] Example 1

[0111] 0.6 g of temperature-responsive microcarrier 1 was added to a 30 mL disposable bioreactor (Able, product number: BWV-S03A) and 8.64 × 10 5 Human mesenchymal stem cells derived from bone marrow (Lonza Japan Ltd., product number: PT-2501, Lot Number: 0000603525) were cultured at 37°C and 5% CO2 concentration (standing for 59 minutes → stirring at 55 rpm for 1 minute) × 10 times, and then cultured at 55 rpm for 4 days. 30 mL of mesenchymal stem cell proliferation medium 2 (PromoCell, product number: C-28009) was used as the culture medium.

[0112] After culturing for 4 days, 24 mL of the culture solution was withdrawn, 24 mL of the culture solution cooled to 4°C was added, and the mixture was allowed to stand at room temperature (23°C) for 10 minutes. Then, the culture solution was stirred at 200 rpm (stirring Reynolds number: 1330) × 5 minutes to peel off the cells from the microcarriers. After stirring, the microcarriers were removed from the culture solution using a cell filter with a 100 μm filter to obtain a cell suspension. The recovered cell suspension was centrifuged at 160 rcf, 25°C, and 5 minutes, the supernatant was removed, and PBS (-) was added for suspension. The obtained cell suspension was mixed with a 0.4 w / v% trypan blue solution in a ratio of 1:1, and 10 μL was added to a slide for cell number determination (manufactured by Thermo Fisher Scientific, trade name: Countess Cell Counting Chamber Slid), and the cell number A and viability rate of the cell suspension were measured using an automatic cell counter (manufactured by Thermo Fisher Scientific, trade name: Countess II). As a result, the cell number A was 1.12×10 7 The number of cells removed by the cell filter was 95%, and the viable cell rate was 95%. In addition, the microcarriers removed by the cell filter were treated with trypsin-EDTA solution, and the number of undetached cells B was measured in the same manner as above by cooling and stirring. Using these values, the cell recovery rate was calculated by formula (2).

[0113] A / (A+B)×100(%) formula (2)

[0114] As a result, the cell number B was 9.74×10 5 The cell recovery rate was 92%.

[0115] Example 2

[0116] The same method as in Example 1 was used except that the temperature-responsive microcarrier 2 was used. As a result, the cell number A was 6.12×10 6 cells, the viable cell rate was 92%, and the cell number B was 6.80×10 5 The cell recovery rate was 90%.

[0117] Example 3

[0118] After culturing for 4 days, 24 mL of the culture solution was withdrawn, 24 mL of the culture solution cooled to 4°C was added, and the culture solution was allowed to stand at room temperature (23°C) for 10 minutes, and then the culture solution was stirred at 15 rpm (stirring Reynolds number: 100) × 5 minutes to remove the cells from the microcarriers. The same procedure as in Example 1 was used. As a result, the cell number A was 7.84 × 10 6 cells, the viable cell rate was 98%, and the cell number B was 3.05×10 6 The cell recovery rate was 72%.

[0119] Example 4

[0120] After culturing for 4 days, 24 mL of the culture solution was withdrawn, 24 mL of the culture solution cooled to 4°C was added, and the culture solution was allowed to stand at room temperature (23°C) for 10 minutes, and then the culture solution was stirred at 270 rpm (stirring Reynolds number: 1800) for 5 minutes to remove the cells from the microcarriers. The same procedure as in Example 1 was used. As a result, the cell number A was 1.25×10 7 cells, the viable cell rate was 89%, and the cell number B was 1.55×10 6 The cell recovery rate was 98%.

[0121] Example 5

[0122] 0.4 g of temperature-responsive microcarrier 3 was added to a 30 mL disposable bioreactor and 8.64 × 10 5 The same procedure as in Example 1 was followed except that the culture was performed at 37°C and 5% CO2 concentration (standing for 175 minutes → stirring at 80 rpm for 5 minutes) × 8 times and then stirred at 80 rpm for 4 days. As a result, the cell number A was 7.75 × 10 6 cells, the viable cell rate was 96%, and the cell number B was 8.60×10 5 The cell recovery rate was 90%.

[0123] Comparative Example 1

[0124] The culture solution cooled to 4°C was added, and after standing at room temperature (23°C) for 10 minutes, the culture solution was stirred at 5 rpm (stirring Reynolds number: 33) for 5 minutes to remove the cells from the microcarriers. The same procedure as in Example 1 was followed. As a result, the cell number A was 2.39×10 6 cells, the viable cell rate was 88%, and the cell number B was 8.47×10 6 cells, the cell recovery rate was 22%, and the cell recovery rate was greatly reduced.

[0125] Comparative Example 2

[0126] The culture solution cooled to 4°C was added, and after standing at room temperature (23°C) for 10 minutes, the culture solution was stirred at 350 rpm (stirring Reynolds number: 2324) for 5 minutes to remove the cells from the microcarriers. The same procedure as in Example 1 was followed. As a result, the cell number A was 1.29×10 7 cells, the viable cell rate was 68%, and the cell number B was 8.23×10 5 The cell recovery rate was 94%, and the viable cell rate was significantly reduced.

[0127] Comparative Example 3

[0128] The same method as in Example 1 was used except that untreated microcarriers (Corning, product number: 4625, particle size: 125-212 μm) were used. As a result, the cell number A was 4.98×10 5 cells, the viable cell rate was 92%, and the cell number B was 9.47×10 6 The cell recovery rate was 5%.

[0129] Comparative Example 4

[0130] The same method as in Example 5 was used except that untreated microcarrier 2 (AmberChrom (registered trademark) 1×8 chloride foam, 100-200 mesh (Sigma-Aldrich, product number: 217425, particle size: 54-154 μm)) was used. As a result, the cell number A was 2.75×10 5 cells, the viable cell rate was 85%, and the cell number B was 9.68×10 6 The cell recovery rate was 3%.

[0131] [Table 1]

[0132]

[0133] <Temperature-responsive cell culture microbeads and culture method thereof>

[0134] [Analysis of monomer addition ratio and composition of temperature-responsive polymer]

[0135] By using Fourier transform nuclear magnetic resonance (NMR) 1 The H-NMR measurement was performed using JNM-ECZ400S / L1 (manufactured by JEOL Ltd.) as an NMR apparatus, and 10 mg of the temperature-responsive polymer was dissolved in 0.75 mL of deuterated chloroform for measurement.

[0136] [Analysis of Molecular Weight and Molecular Weight Distribution of Temperature Responsive Polymers]

[0137] The weight average molecular weight (Mw), number average molecular weight (Mn) and molecular weight distribution (Mw / Mn) of the temperature-responsive polymer were measured by GPC. The GPC apparatus used was HLC-8320GPC (manufactured by TOSOH CORPORATION). Two TSKgelSuper AWM-H (manufactured by TOSOH CORPORATION) were used as chromatographic columns, the column temperature was set to 40°C, a differential refractometer was used as the concentration detector, and a 10mM sodium trifluoroacetate / 2,2,2-trifluoroethanol solution was used as the eluent. The measurement was carried out under the conditions of a sample concentration of 1 mg / mL, a sample injection volume of 0.1 mL, and an eluent flow rate of 0.6 mL / min. In addition, the standard curve for molecular weight calculation was made using polymethyl methacrylate (manufactured by PSS Polymer Standards Service GmbH) with a known molecular weight, and the measurement was carried out under the same conditions.

[0138] [Measurement of polymer coating film thickness]

[0139] The temperature-responsive microbeads were exposed to a ruthenium oxide vapor atmosphere for 2 hours to dye the polymer coating film. The dyed temperature-responsive microbeads were embedded in a room temperature curing epoxy resin and sliced ​​using an ultrathin slicer. The slices were observed using a transmission electron microscope (device name JEM-2100F, manufactured by JEOL Ltd.), and the film thickness was measured by image analysis.

[0140] Example 6

[0141] [Synthesis of Temperature Responsive Polymer 4]

[0142] In a 500 mL cylindrical flask (inner diameter 80 mm), 1.952 g (15 mmol) of 2-methoxyethyl acrylate (MEA) was added, and 95.1 mg (300 μmol) of cyanomethyl dodecyl trithiocarbonate, 4.8 mg (30 μmol) of azobisisobutyronitrile and 30 mL of tert-butyl alcohol were further added, replaced with argon gas, and reacted at 62° C. for 24 hours. The monomer addition rate of MEA after the reaction was 96%.

[0143] After the first heating and stirring, 11.535 g (90 mmol) of n-butyl acrylate (BA) was added to the reaction solution, and 4.8 mg (30 μmol) of azobisisobutyronitrile and 10 mL of tert-butyl alcohol were further added, and the atmosphere was replaced with argon gas, and the reaction was carried out at 62° C. for 24 hours. The monomer addition rate of BA after the reaction was 95%.

[0144] After heating and stirring, 22.066 g (195 mmol) of N-isopropylacrylamide (IPAAm: LCST = 32°C) was added to the reaction solution, and 4.8 mg (30 μmol) of azobisisobutyronitrile and 255 mL of tert-butyl alcohol were further added, and the atmosphere was replaced with argon gas, and the reaction was carried out at 62°C for 24 hours. The monomer addition rate of IPAAm after the reaction was 99%.

[0145] The total amount of the reaction solution was dripped into a 3L beaker to which 2L of pure water was added, and the precipitated yellow viscous substance was recovered. After the viscous substance was immersed in 2L of pure water for 12 hours, it was heated to 40°C, the solid was recovered, and vacuum dried at 100°C for 12 hours. After the solid was dissolved in 300mL of chloroform, 5g of magnesium sulfate was added, stirred at room temperature for 1 hour, and the filtrate was recovered by filtration. The filtrate was dripped into a 3L beaker to which 2L of heptane was added, the precipitated white solid was recovered, and it was dried under reduced pressure at 100°C for 12 hours to obtain 17.8g of temperature-responsive polymer 4.

[0146] The Mn, Mw / Mn, and composition ratio of the temperature responsive polymer 4 were MEA / BA / IPAAm=5 / 30 / 65 mol %.

[0147] [Preparation of surface treatment agent 4]

[0148] 0.1 g of temperature-responsive polymer 4 and 49.9 g of 1-methoxy-2-propanol were added to a glass container and left to dissolve overnight, and then filtered through a 0.22 μm filter (Millipore, hydrophilic filter) to obtain a surface treatment agent 4 having a polymer concentration of 0.2 wt %.

[0149] [Preparation of Temperature Responsive Microbeads 1]

[0150] 5 g of calcium phosphate-modified nonporous polystyrene particles and 10 g of surface treatment agent 4 were added to a 25 mL eggplant-shaped flask and allowed to stand for 2 hours. The solvent was then distilled off by reducing the pressure using an evaporator to obtain temperature-responsive microbeads 1. The coating film thickness of the temperature-responsive polymer was about 58 nm.

[0151] [Cultivation Evaluation]

[0152] 160 mg of temperature-responsive microbeads washed with PBS(-) were added to a Costar(R) ultra-low adhesion surface 6-well plate (Corning, product number 3471) and 1.0×10 5 Mesenchymal stem cells derived from human bone marrow (Lonza, product number PT-2501, batch No. 21TL046615) were cultured at 37°C and a CO2 concentration of 5%. The culture medium used was 5 mL of mesenchymal stem cell proliferation medium 2 (PromoCell, product number C-28009). After 4 days of culture, 4 mL of the culture medium was extracted, and 4 mL of the culture medium cooled to 4°C was added. The culture medium was allowed to stand at room temperature for 30 minutes, and the cell suspension was passed through a cell filter with a pore size of 100 μm to recover the cells. The filter was washed twice with 4 mL of PBS (-), centrifuged at 200g × 5 minutes together with the cell suspension, and after removing the supernatant, the number of cells suspended in the culture medium and recovered by cooling treatment was measured. Furthermore, the cells (residual cells) that were not peeled off during the cooling treatment were recovered by enzyme treatment with trypsin, and the number of cells was measured as the number of residual cells. The cell proliferation rate was calculated from the sum of the number of cells recovered during the cooling treatment and the number of residual cells, and the cell recovery rate was calculated from the sum of the number of cells recovered during the cooling treatment and the number of residual cells and the number of cells recovered during the cooling treatment. The cell proliferation rate was 590%, and the cell recovery rate achieved by the cooling treatment was 86%.

[0153] Example 7

[0154] [Preparation of temperature-responsive microbeads 2]

[0155] In a 25 mL eggplant-shaped flask, 5 g of quaternary ammonium chloride-modified polystyrene particles having fine pores (Organo, product name Amberlite IRA900J Cl, specific gravity 1.06) and 10 g of surface treatment agent 4 were added and allowed to stand for 2 hours. The solvent was then distilled off by reducing the pressure using an evaporator to obtain temperature-responsive microbeads 2. The temperature-responsive polymer coating film thickness was 62 nm.

[0156] [Cultivation Evaluation]

[0157] The same procedure as in Example 6 was performed except that 200 mg of the temperature-responsive microbeads 2 were used. The cell proliferation rate was 710%, and the cell recovery rate was 89%.

[0158] Example 8

[0159] [Preparation of Temperature Responsive Microbeads 3]

[0160] 5 g of tertiary amine-modified polystyrene particles having fine pores (Organo, product name Amberlyst A21, specific gravity 1.07) and 10 g of surface treatment agent 4 were added to a 25 mL eggplant-shaped flask and allowed to stand for 2 hours. The solvent was then distilled off by reducing the pressure using an evaporator to obtain temperature-responsive microbeads 3. The temperature-responsive polymer coating film thickness was 66 nm.

[0161] [Cultivation Evaluation]

[0162] The same procedure as in Example 6 was performed except that 90 mg of the temperature-responsive microbeads 3 were used. The cell proliferation rate was 430%, and the cell recovery rate was 91%.

[0163] Example 9

[0164] [Preparation of surface treatment agent 5]

[0165] 0.025 g of temperature-responsive polymer 4 and 49.975 g of 1-methoxy-2-propanol were added to a glass container and left to dissolve overnight, and then filtered through a 0.22 μm filter (Millipore, hydrophilic filter) to prepare a surface treatment agent 5 having a polymer concentration of 0.05 wt%.

[0166] [Preparation of temperature-responsive microbeads 4]

[0167] 5 g of quaternary ammonium chloride-modified polystyrene particles having fine pores (Organo, product name Amberlite IRA900J Cl, specific gravity 1.06) and 10 g of surface treatment agent 5 were added to a 25 mL eggplant-shaped flask and allowed to stand for 2 hours. The solvent was then distilled off by reducing the pressure using an evaporator to obtain temperature-responsive microbeads 4. The temperature-responsive polymer coating film thickness was 16 nm.

[0168] [Cultivation Evaluation]

[0169] The same procedure as in Example 6 was performed except that 200 mg of the temperature-responsive microbeads 4 were used. The cell proliferation rate was 750%, and the cell recovery rate was 83%.

[0170] Example 10

[0171] [Preparation of surface treatment agent 6]

[0172] 0.5 g of temperature-responsive polymer 4 and 49.5 g of 1-methoxy-2-propanol were added to a glass container and left to dissolve overnight, and then filtered through a 0.22 μm filter (Millipore, hydrophilic filter) to prepare a surface treatment agent 6 having a polymer concentration of 1 wt%.

[0173] [Preparation of Temperature Responsive Microbeads 5]

[0174] 5 g of quaternary ammonium chloride-modified polystyrene particles having fine pores (Organo, product name Amberlite IRA900J Cl, specific gravity 1.06) and 10 g of surface treatment agent 6 were added to a 25 mL eggplant-shaped flask and allowed to stand for 2 hours. The solvent was then distilled off by reducing the pressure using an evaporator to obtain temperature-responsive microbeads 5. The temperature-responsive polymer coating film thickness was 253 nm.

[0175] [Cultivation Evaluation]

[0176] The same procedure as in Example 6 was performed except that 200 mg of the temperature-responsive microbeads 5 were used. The cell proliferation rate was 720%, and the cell recovery rate was 90%.

[0177] Embodiment 11

[0178] [Preparation of surface treatment agent 7]

[0179] 1 g of temperature-responsive polymer 4 and 49 g of 1-methoxy-2-propanol were added to a glass container and left to dissolve overnight, and then filtered through a 0.22 μm filter (Millipore, hydrophilic filter) to prepare a surface treatment agent 7 having a polymer concentration of 2 wt %.

[0180] [Preparation of Temperature Responsive Microbeads 6]

[0181] In a 25 mL eggplant-shaped flask, 5 g of quaternary ammonium chloride-modified polystyrene particles having fine pores (Organo, product name Amberlite IRA900J Cl, specific gravity 1.06) and 10 g of surface treatment agent 7 were added and allowed to stand for 2 hours. The solvent was then distilled off by reducing the pressure using an evaporator to obtain temperature-responsive microbeads 6. The temperature-responsive polymer coating film thickness was 698 nm.

[0182] [Cultivation Evaluation]

[0183] The same procedure as in Example 6 was performed except that 200 mg of the temperature-responsive microbeads 6 were used. The cell proliferation rate was 680%, and the cell recovery rate was 92%.

[0184] Example 12

[0185] [Synthesis of Temperature Responsive Polymer 5]

[0186] In a 500 mL cylindrical flask (inner diameter 80 mm), 5.165 g (60 mmol) of n-butyl acrylate (BA), 95.1 mg (300 μmol) of cyanomethyl dodecyl trithiocarbonate, 4.8 mg (30 μmol) of azobisisobutyronitrile and 30 mL of tert-butyl alcohol were added, replaced with argon gas, and reacted at 62° C. for 24 hours. The monomer addition rate of BA after the reaction was 97%.

[0187] After heating and stirring, 27.158 g (240 mmol) of N-isopropylacrylamide (IPAAm: LCST = 32°C) was added to the reaction solution, and 4.8 mg (30 μmol) of azobisisobutyronitrile and 255 mL of tert-butyl alcohol were further added, and the atmosphere was replaced with argon gas, and the reaction was carried out at 62°C for 24 hours. The monomer addition rate after the reaction was 98%.

[0188] The total amount of the reaction solution was dripped into a 3L beaker to which 2L of pure water was added, and the precipitated yellow viscous substance was recovered. After the viscous substance was immersed in 2L of pure water for 12 hours, it was heated to 40°C, the solid was recovered, and vacuum dried at 100°C for 12 hours. After the solid was dissolved in 300mL of chloroform, 5g of magnesium sulfate was added, stirred at room temperature for 2 hours, and the filtrate was recovered by filtration. The filtrate was dripped into a 3L beaker to which 2L of heptane was added, the precipitated white solid was recovered, and reduced pressure drying was performed at 100°C for 12 hours to obtain 21.2g of temperature-responsive polymer 5.

[0189] The Mn, Mw / Mn, and composition ratio of the temperature responsive polymer 5 were BA / IPAAm=25 / 75 mol%.

[0190] [Preparation of surface treatment agent 8]

[0191] 0.1 g of temperature-responsive polymer 5 and 49.9 g of 1-methoxy-2-propanol were added to a glass container and left to dissolve overnight, and then filtered through a 0.22 μm filter (Millipore, hydrophilic filter) to obtain a surface treatment agent 8 having a polymer concentration of 0.2 wt %.

[0192] [Preparation of Temperature Responsive Microbeads 7]

[0193] 5 g of quaternary ammonium chloride-modified polystyrene particles having fine pores (Organo, product name Amberlite IRA900J Cl, specific gravity 1.06) and 10 g of surface treatment agent 8 were added to a 25 mL eggplant-shaped flask and allowed to stand for 2 hours. The solvent was then distilled off by reducing the pressure using an evaporator to obtain temperature-responsive microbeads 7. The temperature-responsive polymer coating film thickness was 52 nm.

[0194] [Cultivation Evaluation]

[0195] The same procedure as in Example 6 was performed except that 200 mg of the temperature-responsive microbeads 7 were used. The cell proliferation rate was 720%, and the cell recovery rate was 71%.

[0196] Comparative Example 5

[0197] [Preparation of Temperature Responsive Microbeads 8]

[0198] 5 g of a polystyrene carrier (specific gravity 1.05) and 10 g of a surface treatment agent 4 were added to a 25 mL eggplant-shaped flask and allowed to stand for 2 hours. The solvent was then distilled off by reducing the pressure using an evaporator to obtain temperature-responsive microbeads 8. The temperature-responsive polymer coating film thickness was 52 nm.

[0199] [Cultivation Evaluation]

[0200] The same procedure as in Example 6 was performed except that 60 mg of the temperature-responsive microbeads 8 were used. The cell proliferation rate was 250%, and the cell recovery rate was 80%.

[0201] Comparative Example 6

[0202] [Preparation of surface treatment agent 9]

[0203] 0.05 g of temperature-responsive polymer 1 and 499.95 g of 1-methoxy-2-propanol were added to a glass container and left to dissolve overnight, and then filtered through a 0.22 μm filter (Millipore, hydrophilic filter) to obtain a surface treatment agent 9 having a polymer concentration of 0.01 wt%.

[0204] [Preparation of Temperature Responsive Microbeads 9]

[0205] In a 25 mL eggplant-shaped flask, 5 g of quaternary ammonium chloride-modified polystyrene particles having fine pores (Organo, product name Amberlite IRA900J Cl, specific gravity 1.06) and 10 g of surface treatment agent 9 were added and allowed to stand for 2 hours. The solvent was then distilled off by reducing the pressure using an evaporator to obtain temperature-responsive microbeads 9. The temperature-responsive polymer coating film thickness was 5 nm.

[0206] [Cultivation Evaluation]

[0207] The same procedure as in Example 6 was performed except that 200 mg of the temperature-responsive microbeads 9 were used. The cell proliferation rate was 770%, and the cell recovery rate was 4%.

[0208] Comparative Example 7

[0209] [Preparation of Temperature Responsive Microbeads 10]

[0210] 5 g of sulfonic acid-modified polystyrene particles having fine pores (Organo, product name Amberlite 200CT Na, specific gravity 1.04) and 10 g of surface treatment agent 4 were added to a 25 mL eggplant-shaped flask and allowed to stand for 2 hours. The solvent was then distilled off by reducing the pressure using an evaporator to obtain temperature-responsive microbeads 10. The temperature-responsive polymer coating film thickness was 55 nm.

[0211] [Cultivation Evaluation]

[0212] The same procedure as in Example 6 was performed except that 120 mg of the temperature-responsive microbeads 10 were used. After the culture, the cells did not adhere to the microbeads, and the cell proliferation rate was 90%.

[0213] Comparative Example 8

[0214] [Preparation of Temperature Responsive Microbeads 11]

[0215] 5 g of polystyrene particles modified with carboxylic acid and having fine pores (Organo, product name Amberlite IRC76, specific gravity 1.04) and 10 g of surface treatment agent 4 were added to a 25 mL eggplant-shaped flask and allowed to stand for 2 hours. The solvent was then distilled off by reducing the pressure using an evaporator to obtain temperature-responsive microbeads 11. The temperature-responsive polymer coating film thickness was 59 nm.

[0216] [Cultivation Evaluation]

[0217] The same procedure as in Example 6 was performed except that 140 mg of the temperature-responsive microbeads 11 were used. After the culture, the cells did not adhere to the microbeads, and the cell proliferation rate was 90%.

[0218] Comparative Example 9

[0219] [Cultivation Evaluation]

[0220] The same procedure as in Example 6 was carried out except that 60 mg of non-porous polystyrene particles modified with calcium phosphate were used as microbeads. The cell proliferation rate was 590%, and the cell recovery rate was 3%.

[0221] Comparative Example 10

[0222] [Cultivation Evaluation]

[0223] The same procedure as in Example 6 was carried out except that 200 mg of Amberlite IRA900J Cl (manufactured by Organo, specific gravity 1.06) having pores modified with quaternary ammonium chloride was used as microbeads. The cell proliferation rate was 730%, and the cell recovery rate was 4%.

[0224] Comparative Example 11

[0225] [Cultivation Evaluation]

[0226] The same procedure as in Example 6 was carried out except that 90 mg of Amberlyst A21 (manufactured by Organo, specific gravity 1.07) having pores modified with tertiary amine was used as microbeads. The cell proliferation rate was 460%, and the cell recovery rate was 2%.

[0227] Embodiment 13

[0228] [Preparation of Temperature Responsive Microbeads 12]

[0229] 5 g of quaternary ammonium chloride-modified polystyrene particles with fine pores (manufactured by Sigma-Aldrich, product name AmberChrom1×8 200-400 mesh, specific gravity 1.09) and 10 g of surface treatment agent 4 were added to a 25 mL eggplant-shaped flask and allowed to stand for 2 hours. The solvent was then distilled off by reducing the pressure using an evaporator to obtain temperature-responsive microbeads 12. The temperature-responsive polymer coating film thickness was 50 nm.

[0230] [Cultivation Evaluation]

[0231] The same procedure as in Example 6 was performed except that 20 mg of the temperature-responsive microbeads 12 were used. The cell proliferation rate was 790%, and the cell recovery rate was 88%.

[0232] Embodiment 14

[0233] [Preparation of Temperature Responsive Microbeads 13]

[0234] 5 g of quaternary ammonium chloride-modified polystyrene particles with fine pores (manufactured by Sigma-Aldrich, product name AmberChrom1×8 100-200 mesh, specific gravity 1.09) and 10 g of surface treatment agent 5 were added to a 25 mL eggplant-shaped flask and allowed to stand for 2 hours. The solvent was then distilled off by reducing the pressure using an evaporator to obtain temperature-responsive microbeads 13. The temperature-responsive polymer coating film thickness was 12 nm.

[0235] [Cultivation Evaluation]

[0236] The same procedure as in Example 6 was performed except that 35 mg of the temperature-responsive microbeads 13 were used. The cell proliferation rate was 830%, and the cell recovery rate was 80%.

[0237] Embodiment 15

[0238] [Preparation of Temperature Responsive Microbeads 14]

[0239] 5 g of quaternary ammonium chloride-modified polystyrene particles with fine pores (manufactured by Sigma-Aldrich, product name AmberChrom1×8 100-200 mesh, specific gravity 1.09) and 10 g of surface treatment agent 4 were added to a 25 mL eggplant-shaped flask and allowed to stand for 2 hours. The solvent was then distilled off by reducing the pressure using an evaporator to obtain temperature-responsive microbeads 14. The thickness of the temperature-responsive polymer coating film was 61 nm.

[0240] [Cultivation Evaluation]

[0241] The same procedure as in Example 6 was performed except that 35 mg of the temperature-responsive microbeads 14 were used. The cell proliferation rate was 750%, and the cell recovery rate was 86%.

[0242] Example 16

[0243] [Preparation of Temperature Responsive Microbeads 15]

[0244] 5 g of quaternary ammonium chloride-modified polystyrene particles with fine pores (manufactured by Sigma-Aldrich, product name AmberChrom1×8 50-100 mesh, specific gravity 1.09) and 10 g of surface treatment agent 4 were added to a 25 mL eggplant-shaped flask and allowed to stand for 2 hours. The solvent was then distilled off by reducing the pressure with an evaporator to obtain temperature-responsive microbeads 15. The thickness of the temperature-responsive polymer coating film was 44 nm.

[0245] [Cultivation Evaluation]

[0246] The same procedure as in Example 6 was performed except that 100 mg of the temperature-responsive microbeads 15 were used. The cell proliferation rate was 790%, and the cell recovery rate was 85%.

[0247] Comparative Example 12

[0248] [Preparation of Temperature Responsive Microbeads 16]

[0249] 5 g of quaternary ammonium chloride-modified polystyrene particles with fine pores (manufactured by Sigma-Aldrich, product name AmberChrom1×8 100-200 mesh, specific gravity 1.09) and 10 g of surface treatment agent 9 were added to a 25 mL eggplant-shaped flask and allowed to stand for 2 hours. The solvent was then distilled off by reducing the pressure with an evaporator to obtain temperature-responsive microbeads 16. The temperature-responsive polymer coating film thickness was 5 nm.

[0250] [Cultivation Evaluation]

[0251] The same procedure as in Example 6 was performed except that 35 mg of the temperature-responsive microbeads 16 were used. The cell proliferation rate was 750%, and the cell recovery rate was 6%.

[0252] [Table 2]

[0253]

[0254] [Table 3]

[0255]

[0256] Cell proliferation rate = (number of cells recovered after cooling + number of residual cells) / initial inoculation number × 100 Cell recovery rate = number of cells recovered after cooling / (number of cells recovered after cooling + number of residual cells) × 100

Claims

1. A method for culturing adherent cells in a culture vessel using a microcarrier covered with a polymer showing a lower critical solution temperature, characterized in that The method comprises the following steps (1) to (4): Step (1), culturing the cells on the surface of the microcarrier in a culture solution at a temperature above the lower critical solution temperature; Step (2), after step (1), cooling the culture solution to a temperature below the lower critical solution temperature; Step (3), after step (2), stirring the culture solution in the culture container to peel the cells off the surface of the microcarrier; Step (4), after step (3), recovering the cells detached from the surface of the microcarrier.

2. The method according to claim 1, characterized in that In the step (3), stirring is performed at a stirring Reynolds number in the range of 50 to 2,000.

3. The method according to claim 2, characterized in that Step (2) is performed by replacing 10 (v / v)% to 90 (v / v)% of the culture solution used in step (1) with a culture solution cooled to a temperature not higher than the lower critical solution temperature.

4. The method according to claim 3, characterized in that Step (4) is performed by removing the microcarriers using a filter.

5. The method according to claim 4, characterized in that The particle size of the microcarrier is 50 μm to 1000 μm.

6. The method according to claim 5, characterized in that The lower critical solution temperature is 0℃~50℃.

7. The method according to claim 6, characterized in that Adherent cells are stem cells.

8. A temperature-responsive microbead, characterized in that: The invention relates to temperature-responsive microbeads composed of a polymer coating film and a carrier. The polymer coating film is composed of a temperature-responsive polymer having a lower critical solution temperature at 0°C to 50°C. The carrier surface has a positive charge. The film thickness of the temperature-responsive polymer is 10nm to 1000nm.

9. The temperature-responsive microbead according to claim 8, characterized in that: The component having a positive charge on the surface of the carrier includes any one of a tertiary amine, a quaternary ammonium salt, and an alkaline earth metal salt.

10. The microbead according to claim 9, characterized in that The specific gravity of the carrier is 1.0 to 1.

1.

11. The temperature-responsive microbead according to claim 10, characterized in that: The material of the carrier is polystyrene.

12. The temperature-responsive microbead according to claim 11, characterized in that: The particle size of the carrier is 50 μm to 1000 μm. 13 . A cell culture method using the temperature-responsive microbeads according to claim 12 .

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