Cell culture method using temperature-responsive microcarrier, and beads for temperature-responsive cell culture

Abstract

The present invention provides: a method for culturing adhesive cells using a microcarrier coated with a polymer that exhibits a lower critical temperature within a culture vessel, wherein the method is characterized by including the following steps (1) to (4): (1) a step that cultures the cells on the microcarrier surface in a culture broth of a temperature at or above the lower critical temperature; (2) a step that, after step (1), cools the culture broth to or below the lower critical temperature; (3) a step that, after step (2), stirs the culture broth in the culture vessel, and detaches the cells from the microcarrier surface; and (4) a step that, after step (3), recovers the cells detached from the microcarrier surface; and temperature-responsive beads composed of a carrier and a polymer coat film comprising a temperature-responsive polymer having a lower critical solution temperature of 0°C to 50°C, wherein the carrier surface has a positive charge and the film thickness of the temperature-responsive polymer is 10 nm to 1000 nm.

Description

Technical Field

[0001] The present invention relates to a cell culture method using temperature-responsive microcarriers, temperature-responsive beads for cell culture, and a culture method. Used thereby

Background Art

[0002] With the development of biopharmaceuticals and regenerative medicine using cells as raw materials, there is a demand for a method for efficiently culturing cells in large quantities and with high quality. Floating Many useful cells classified into suspension cells and adherent cells Among the cells, those used in such a method are 、 anchorage-dependent cells such as mesenchymal stem cells and pluripotent stem cells, Such cells are and (Normally) require a substrate to which cells can adhere for proliferation. Conventionally, plastic dishes and flasks have been used, but as the demand for cells expands, attention has been focused on a culture method using microcarriers suitable for large-scale culture. And microcarriers, which are bead-like particles composed of natural polymers such as synthetic polymers and polysaccharides, have been developed. By introducing microcarriers into a container containing a cell suspension and culturing When it is possible to increase cell growth per unit culture medium volume. So that the surface area to which the cells can adhere increases

[0003] In the culture method using microcarriers In cells grow while adhering to the surface of the microcarriers. Are Therefore, by stirring the culture solution, the cells and the microcarriers can be suspended The culture method using microcarriers 、 is Conditions performed When under stirring Conventional and has the characteristic that it can achieve a higher cell density per unit volume compared to 、 the culture method using dishes and flasks. ​​On the one hand, it is necessary to detach and recover the adherent cells after proliferation from the scaffold. Generally, proteolytic enzymes such as trypsin are used to detach and recover the cells adhered to the scaffold. In particular, for the recovery of cells grown on the surface of microcarriers, it is common to use proteolytic enzymes such as trypsin to detach and recover them from the microcarriers. However, proteolytic enzymes such as trypsin Use of are 、 a problem in that they damage the cells by degrading the proteins on the cell surface and From the recovered cells (suspension) require complicated operations to remove trypsin.

[0004] As a method for cell recovery with reduced damage to cells, for example, Patent Document 1 discloses a method using microcarriers coated with a temperature-responsive polymer having a lower critical temperature. At a culture temperature of 37 °C, the cells adhere and proliferate, and by cooling to 20 °C, the temperature-responsive polymer becomes hydrophilic, and the cells can be recovered without using proteolytic enzymes. However, the temperature-responsive microcarriers have the problem of low cell growth and need to be improved.

[0005] Regarding the problems caused by the detachment from microcarriers using proteolytic enzymes in the culture method using microcarriers, microcarriers coated with a polymer showing a lower critical temperature are disclosed in Patent Document 2 and Patent Document 3 to solve the problem. According to Patent Document 2 and Patent Document 3, by sol-gel transition of the polymer showing a lower critical temperature by cooling, the adhesion force on the surface of the microcarrier is weakened, the cells are detached from the microcarrier, and the cells can be recovered. When recovering the cells, it is difficult to completely detach the cells from the surface of the microcarrier only by cooling. Therefore, in the case of a laboratory scale, a recovery method using a pipetting operation in combination as described in Patent Document 3 is usually used. However, the pipetting operation is difficult to apply to large-scale culture Of difficult For the sake of and the cell culture methods described in Patent Document 2 and Patent Document 3 have not led to the provision of large-scale culture techniques, and a cell culture method more suitable for large-scale culture has been demanded.

Prior Art Documents

Patent Documents

[0006]

Patent Document 1

Patent Document 2

Patent Document 3

Summary of the Invention

Problems to be Solved by the Invention

[0007] The first object of the present patent is to provide a cell culture method suitable for large-scale culture in a method for recovering cells after cell culture on microcarriers coated with a polymer exhibiting a lower critical temperature. The second object of the present invention is to provide a temperature-responsive microcarrier with high cell proliferation.

Means for Solving the Problems

[0008] Regarding the first object, the present inventors conducted intensive research in view of the problems in the culture method using microcarriers, and as a result, in the method for recovering cells after cell culture on microcarriers coated with a polymer exhibiting a lower critical temperature, it was found that cells can be recovered from the microcarriers with high efficiency and low invasiveness by a method including a cooling step and a stirring step, and the present invention was completed. That is, the present invention includes the following aspects. Recovery

[0009] <1-1> A method for culturing adherent cells using microcarriers coated with a polymer exhibiting a lower critical temperature in a culture vessel, the method being characterized by including the following steps (1) to (4). (1) A step of culturing the cells on the surface of the microcarriers in a culture solution at a temperature equal to or higher than the lower critical temperature. (2) After step (1), a step of cooling the culture solution to a temperature equal to or lower than the lower critical temperature. (3) After step (2), a step of stirring the culture solution in the culture vessel to detach cells from the surface of the microcarrier; (4) After step (3), a step of collecting the cells detached from the surface of the microcarrier.

[0010] <1-2> The method according to <1-1>, wherein in step (3), stirring is performed in a range where the stirring Reynolds number is 50 to 2000.

[0011] <1-3> The method according to <1-1> or <1-2>, wherein step (2) is performed by replacing the culture solution used in step (1) with a culture solution cooled to a temperature below the lower critical temperature and being 10 (v / v)% to 90 (v / v)% of the culture solution used in step (1).

[0012] <1-4> The method according to any one of <1-1> to <1-3>, wherein step (4) is performed by removing the microcarrier using a mesh.

[0013] <1-5> 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.

[0014] <1-6> The method according to any one of <1-1> to <1-5>, wherein the lower critical temperature is 0 °C to 50 °C.

[0015] <1-7> The method according to any one of <1-1> to <1-6>, wherein the adherent cells are stem cells.

[0016] Regarding the second object, the inventors have conducted extensive research in view of the problem that the temperature-responsive microcarriers have low cell growth ability. As a result, they have found that beads coated with a temperature-responsive polymer having a lower critical solution temperature of 0°C to 50°C on beads having a positive charge can adhere to cells well and the cells can be detached and recovered by cooling after the culture is completed, thus completing the present invention. That is, the present invention also includes the following aspects. <2-1>Temperature-responsive beads composed of a polymer coat film made of a temperature-responsive polymer having a lower critical solution temperature of 0°C to 50°C and a carrier, wherein the surface of the carrier has a positive charge and the film thickness of the temperature-responsive polymer is 10 nm to 1000 nm. <2-2>The temperature-responsive beads according to <2-1>, wherein the component having a positive charge on the surface of the carrier contains any one of a tertiary amine, a quaternary ammonium salt, and an alkaline earth metal salt. <2-3>The beads according to <2-1> or <2-2>, wherein the specific gravity of the carrier is 1.0 to 1.1. <2-4>The temperature-responsive beads according to any one of <2-1> to <2-3>, wherein the material of the carrier is polystyrene. <2-5>The temperature-responsive beads according to any one of <2-1> to <2-4>, wherein the particle size of the carrier is 50 μm to 1000 μm. <2-6>A cell culture method using the temperature-responsive beads according to any one of <2-1> to <2-5>.

Effects of the Invention

[0017] In a cell culture method of a microcarrier coated with a polymer showing a lower critical temperature, cells can be recovered from the microcarrier with high efficiency and low invasiveness by a method including a cooling step and a stirring step. Also, temperature-responsive beads composed of a polymer coat film made of a temperature-responsive polymer having a lower critical solution temperature of 0°C to 50°C and a carrier, wherein the surface of the carrier has a positive charge, have high cell growth ability and the cells can be recovered by cooling.

Modes for Carrying Out the Invention

[0018] Hereinafter, embodiments for carrying out the present invention will be described in detail, but the present invention is not intended to be limited to the following content. The present invention can be implemented with appropriate modifications within the scope of its gist.

[0019] <Method for culturing adherent cells using a microcarrier coated with a polymer showing a lower critical temperature in a culture vessel> In a first aspect, the present invention provides a method for culturing cells using a temperature-responsive microcarrier. The present invention is a method for culturing adherent cells using a microcarrier coated with a polymer showing a lower critical temperature in a culture vessel, and is characterized by including the following steps (1) to (4). (1) A step of culturing cells on the surface of the microcarrier in a culture solution at a temperature equal to or higher than the lower critical temperature. (2) After step (1), a step of cooling the culture solution to a temperature equal to or lower than the lower critical temperature. (3) After step (2), a step of stirring the culture solution in the culture vessel to detach the cells from the surface of the microcarrier. (4) After step (3), a step of collecting the cells detached from the surface of the microcarrier.

[0020] The lower critical temperature (LCST; Lower Critical Solution Temperature, sometimes hereinafter referred to as LCST) is a temperature at which a polymer dissolves in water to form a transparent solution at a temperature lower than this temperature, but at a temperature higher than this temperature Into water it becomes insoluble and turbid or precipitation occurs, and it is the temperature at which phase separation occurs. The LCST is not particularly limited, but it is preferably near the culture temperature, for example, in the range of 0°C to 50°C, preferably in the range of 20°C to 40°C, and more preferably in the range of 25°C to 35°C.

[0021] The repeating unit of the homopolymer showing the lower critical temperature and its lower critical temperature with respect to water are, for example, 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), or N-methyl-N-n-propylacrylamide (LCST = 20 °C), etc. can be exemplified. The lower critical temperature fluctuates with the temperature at which the concentration of the aqueous solution is expressed, but N-isopropylacrylamide is preferable because the concentration dependence of the lower critical temperature expression is low.

[0022] The repeating unit of the polymer showing the lower critical temperature in the present invention may be only one type, or may be a combination of two or more types. Also, as long as it has an LCST, in addition to the repeating unit of the polymer showing the LCST, it may contain a repeating unit of a polymer not showing the LCST. Also, depending on the purpose such as improving the adhesiveness to cells or microcarriers, other polymer compounds may be introduced in addition to the polymer showing the LCST. As an example, in the examples of the present invention, a polymer compound composed of carboxystyrene and styrene is introduced to improve the adhesiveness of cells. As an example, the composition of N-isopropylacrylamide showing LCST in the polymer is preferably 10 mol% or more and 95 mol% or less, more preferably 30 mol% or more and 80 mol% or less, and even more preferably 65 mol% or more and 70 mol% or less.

[0023] Carrier The material is not particularly limited, and examples include polystyrene, polymethyl methacrylate, polyethylene terephthalate, polycarbonate, cellulose, cyclodextrin, acrylamide, alginate, dextran, gelatin, glass, or a mixture of two or more of these.

[0024] The shape of the microcarriers is not particularly limited, and examples include spherical, ellipsoidal, flat plate-shaped, and tubular ones. Also, the microcarriers may or may not be porous. In the case of being porous, there is no limitation on the pore diameter.

[0025] The diameter of the microcarriers is not particularly limited, but the major axis of the microcarriers is preferably 50 μm to 1000 μm, more preferably 100 to 700 μm. If the major axis is smaller than the above range, it becomes difficult to separate from cells and the recovery rate decreases. Also, if the major axis is larger than the above range, the culture area per volume becomes smaller.

[0026] In this specification, the temperature-responsive microcarriers refer to microcarriers coated with a polymer showing a lower critical solution temperature.

[0027] The method for coating the microcarriers with a polymer showing a lower critical solution temperature is not particularly limited, and examples include a method of chemically coating repeating units showing a lower critical solution temperature by electron beam irradiation, and a method of physically coating the microcarriers by applying a surface treatment agent in which a polymer showing a lower critical solution temperature is dissolved in a solvent.

[0028] The surface of the microcarriers coated with a polymer showing LCST may be further coated with an extracellular matrix. The type of the extracellular matrix is not particularly limited, and for example, collagen, atelocollagen, hyaluronic acid, elastin, proteoglycan, glucosaminoglycan, fibronectin, laminin, vitronectin, gelatin, or Matrigel containing laminin, collagen IV, heparan sulfate proteoglycan, entactin / nidogen 1,2, etc. as main components may be used. These may be used alone or in combination of two or more. Also, segments of these extracellular matrices may be used.

[0029] An adherent cell is a cell that attaches to the surface of a cell culture substrate such as a microcarrier. The origin of the cell is not particularly limited, and examples include human, monkey, dog, cat, rabbit, rat, nude mouse, mouse, guinea pig, pig, sheep, Chinese hamster, cow, etc. Specific cells include, for example, various cultured cell lines such as CHO cells derived from Chinese hamster ovary, Vero cells derived from African green monkey kidney, mouse connective tissue L929 cells, HEK293 cells derived from human fetal kidney, and HeLa cells derived from human cervical cancer. In addition, for example, epithelial cells, endothelial cells that make up each tissue and organ in vivo, skeletal muscle cells, smooth muscle cells, cardiomyocytes that show contractility, neuron cells, glial cells that make up the nervous system, fibroblasts, macrophages and dendritic cells involved in the body's immunity, hepatocytes, non-parenchymal liver cells and adipocytes involved in the body's metabolism, and cells with differentiation ability such as induced pluripotent stem (iPS) cells, embryonic stem (ES) cells, embryonic germ (EG) cells, embryonic carcinoma (EC) cells, mesenchymal stem cells, hepatic stem cells, pancreatic stem cells, skin stem cells, muscle stem cells, germ stem cells and other various stem cells, or progenitor cells of each tissue, and further cells differentiated therefrom, etc. are mentioned, and mesenchymal stem cells are particularly preferably used. Mesenchymal stem cells mean a population of stem cells capable of differentiating into all or some mesenchymal cells such as chondrocytes, osteoblasts, adipocytes, etc. and their progenitor cells. The origin of mesenchymal stem cells is not particularly limited, and examples include tissue-derived such as bone marrow, adipose tissue, dental pulp, umbilical cord blood, placenta, synovium, etc. and pluripotent stem cell-derived such as ES cells, iPS cells, etc.

[0030] In step (1), since the temperature of the culture solution in the culture vessel is at a temperature equal to or higher than the lower critical temperature, the polymer showing the lower critical temperature gels, and adherent cells can adhere to and proliferate on the microcarrier. When using human-derived cells, it is encouraged to perform the culture near body temperature for the purpose of obtaining high culture efficiency. As an example, a temperature range of 30°C to 40°C is preferred, and it is more preferred to perform the culture in a temperature range of 36°C to 38°C. There is no particular limitation on the conditions other than the lower critical temperature. For example, it may be performed by static culture or agitation culture, but agitation culture that can increase the culture area per unit volume is preferred.

[0031] The culture density of the cells is not particularly limited as long as the cells adhere and proliferate. In the case of human-derived mesenchymal stem cells, for example, it is 1.0×10 1 cells / cm 2 ~1.0×10 5 cells / cm 2 is preferable, and 1.0×10 2 cells / cm 2 ~1.0×10 4 cells / cm 2 is more preferable. Other culture conditions are not particularly limited, and the cells may be cultured under conditions commonly used in the art.

[0032] The temperature of the culture solution during cooling in step (2) is preferably 1°C or more lower than the lower critical temperature. Examples of the method for lowering the temperature of the microcarrier (also referred to as "cooling treatment") include replacing the liquid in the culture vessel with a cooled liquid and storing it in a cold place. However, in order to shorten the time required for cooling, it is preferable to replace it with a cooled liquid. The replacement amount when replacing with a cooled liquid is not particularly limited, but as an example, 10% to 90% is preferable, and 50% to 90% is more preferable in order to increase the cooling rate. The cooled liquid is not particularly limited, and it can be selected according to the purpose, such as a culture solution, other culture medium solutions, and isotonic solutions. Also, the cooling time is preferably 5 minutes to 60 minutes.

[0033] The stirring method in step (3) is not particularly limited, and an example is a method of stirring the culture solution by central stirring in which a stirring blade vertically inserted into the center of the culture vessel is rotated. The degree of stirring is not particularly limited, but it is preferable to stir within a range where the stirring Reynolds number, which is one of the indices representing the degree of stirring, is 50 to 2000, more preferably within a range of 100 to 1800, and most preferably within a range of 300 to 1500. When the stirring Reynolds number is less than 50, the cells are not completely detached from the microcarrier and the recovery rate decreases. Also, when the stirring Reynolds number exceeds 2000, the cells are damaged and the viable cell rate decreases. The stirring Reynolds number is represented by the following formula (1). Stirring Reynolds number = density of fluid × stirring speed × stirring diameter / viscosity of fluid ··· Formula (1) Also, the temperature of the liquid in the culture vessel in step (3) is preferably 1°C or more lower than the lower critical temperature in order to prevent the detached cells from re - adhering to the microcarriers. There is no particular limitation on the liquid during stirring, and it can be selected according to the purpose, such as a culture solution, other medium solutions, isotonic solutions, etc. The stirring time is preferably 1 minute to 60 minutes in consideration of the cell recovery rate and damage.

[0034] The method for separating the microcarriers and cells in step (4) is not particularly limited, and examples include a method of separating based on the difference in sedimentation rate and a method of separating based on the difference in particle size. As a method of separating based on the difference in particle size, for example, a method of separating the microcarriers and the cell suspension with a mesh can be mentioned. The material of the mesh is not particularly limited, and as an example, it is nylon. There is no particular limitation on the mesh opening, but as an example, it is 20μm to 100μm, preferably 30μm to 80μm, and more preferably 40μm to 60μm. If the mesh opening is small, the cells cannot pass through the mesh and the cell recovery rate decreases. Also, if the mesh opening is large, the microcarriers also pass through the mesh and separation becomes difficult.

[0035] In the present invention, the cell culture vessel is not particularly limited as long as it can stir and suspend the microcarriers. In the examples of the present invention, a 30 - mL single - use bioreactor (ABLE Corporation, product number: BWV - S03A) is used as an example.

[0036] Regarding the composition of the culture medium used in the culture of the present invention, there is no particular limitation as long as the cells adhere and proliferate. It consists of a basal medium and serum, and may contain antibiotics. The type of basal medium is not particularly limited. For example, MEM, αMEM, DMEM, EMEM, GMEM, DMEM / Ham’s F-12, Ham’s F-12, Ham’s F-10, Medium 199, RPMI1640, etc. can be used. The type of serum is not particularly limited. For example, fetal bovine serum (FBS), calf serum, adult bovine serum, horse serum, sheep serum, goat serum, pig serum, chicken serum, rabbit serum, human serum are used, but FBS is generally used frequently due to its easy availability. The serum concentration in the culture medium is not particularly limited. Generally, it is often used at a concentration of 20 vol% or less from the perspective of cost-effectiveness, but a concentration exceeding 20 vol% may also be acceptable. Also, a serum-free medium that does not contain any untreated or unpurified serum and contains purified blood-derived components or animal tissue-derived components (such as growth factors) may be used.

[0037] The method for measuring the viable cell rate is not particularly limited. As an example, a method for determining the life and death of cells by trypan blue staining, which stains the cytoplasm of dead cells blue, is known. The method for determining the life and death of cells and the method for counting the number of cells are not particularly limited. There are methods such as manual determination by an operator using a hemocytometer and automatic determination by an automatic cell measurement device. Since the measurement can be performed regardless of the proficiency level, the method using an automatic cell measurement device is preferred.

[0038] <Temperature-responsive beads for cell culture and its culture method> In a second aspect, the present invention provides temperature-responsive beads for cell culture and its culture method. The temperature-responsive beads of the present invention are characterized in that a temperature-responsive polymer having a lower critical solution temperature of 0°C to 50°C is coated on a bead carrier having a positive charge.

[0039] The configuration of the bead carrier having a positive charge of the present invention is not particularly limited. As an example, it may be a compound having a positive charge chemically fixed on the carrier surface or physically fixed. As an example of a method of chemical fixation, a method of copolymerizing with a monomer containing a positive charge when producing the carrier, or a method of chemically modifying the particle surface with a functional group that becomes a positive charge can be exemplified. There is no particular limitation on the functional group that becomes a positive charge, but examples include a tertiary amino group, a quaternary ammonium group, and an alkaline earth metal salt. In particular, due to the ease of modification, trimethylammonium group, dimethylamino group, diethylamino group, and calcium phosphate can be exemplified. Further, as an example of a method of fixing by physical adsorption, a method of coating the carrier with a cationic polymer or a method of coating the surface with an inorganic compound having low solubility in water can be exemplified.

[0040] The composition of the bead carrier having a positive charge is not particularly limited, but preferably has a specific gravity of 1.0 to 1.1, more preferably 1.01 to 1.06, since it can be gently sedimented in the medium. When the specific gravity is less than 1.0, it floats in the medium, making culturing difficult, and when it is greater than 1.1, dispersion in the medium becomes difficult. The material of the carrier is not particularly limited, and examples include synthetic polymers such as polyethylene, polypropylene, polystyrene, polyalkyl (meth)acrylate, polyalkyl (meth)acrylamide, polyester, polyurethane, polyvinyl chloride, polycarbonate, or copolymers thereof, plant-derived polymers such as dextran and cellulose, or wood chips and ceramics. In terms of being able to design the specific gravity of the beads close to that of the culture solution and suppressing the sedimentation of the beads during cell culture with stirring, bead carriers made of synthetic polymers such as polystyrene, polyalkyl (meth)acrylate, polyalkyl (meth)acrylamide, polyester, and polyurethane are preferred, and more preferably, bead carriers made of polystyrene. Also, in order to suppress the elution of the material constituting the bead carrier into the medium, it is preferably crosslinked. The shape of the bead carrier is not particularly limited and may be plate-shaped, spherical, or a porous body. When the bead carrier is spherical, there is no particular limitation on the particle size, but if the particle size is large, dispersion in the culture solution becomes difficult, and if it is small, cell adhesion becomes difficult. Therefore, it is preferably 50 μm to 1000 μm, more preferably 150 μm to 600 μm, and even more preferably 150 μm to 500 μm.

[0041] In this specification, the lower critical solution temperature (LCST) is the temperature at which the solubility of certain polymers in water changes. Polymers having an LCST dehydrate on the high-temperature side of the LCST, and the hydrophobic interaction becomes stronger, and hydrate, swell, or dissolve on the low-temperature side of the LCST.

[0042] The temperature-responsive polymer of the present invention refers to a polymer containing, as a repeating unit, a monomer having an LCST of 0°C to 50°C in water for the homopolymer.

[0043] The structure of the temperature-responsive polymer of the present invention may include repeating units that exhibit an LCST, and may also include repeating units that do not exhibit an LCST as other structures. Since it exhibits good temperature responsiveness, the repeating unit that exhibits an LCST is preferably in a block structure. When the temperature-responsive beads of the present invention are used for cell culture, generally, since they are cultured at around 37°C, the LCST of the repeating unit having an LCST is preferably 0°C to 50°C, more preferably 10°C to 40°C, and even more preferably 20°C to 35°C. When the LCST is less than 0°C or exceeds 50°C, it is difficult to perform minimally invasive cell detachment because it damages the cells. As an example of a repeating unit having an LCST of 0°C to 50°C, repeating units generated 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), N-methyl-N-n-propylacrylamide (LCST = about 20°C) as monomers can be exemplified. The temperature-responsive polymer is not particularly limited, and as an example, the (N-isopropylacrylamide)-(n-butyl methacrylate) block copolymer described in Patent No. 5846584, the (N-isopropylacrylamide)-(n-butyl acrylate) block copolymer, the (2-dimethylaminoethyl methacrylate)-(n-butyl methacrylate)-(N-isopropylacrylamide) block copolymer described in Patent No. 6954047, and the (N-isopropylacrylamide)-(n-butyl methacrylate)-(2-methoxyethyl acrylate) block copolymer described in Patent No. 7127330 can be exemplified.

[0044] The film thickness of the temperature-responsive polymer of the temperature-responsive beads is not particularly limited, preferably 10 nm to 1000 nm, more preferably 50 nm to 500 nm. If it is less than 10 nm, the effect of the positive charge is strongly generated, leading to poor temperature responsiveness. If it exceeds 1000 nm, the effect of the positive charge weakens.

[0045] There is no particular limitation on the method of fixing the temperature-responsive polymer coating film to the carrier. The aforementioned polymer may be chemically fixed or physically adsorbed. When immobilizing by physical adsorption, there is no particular limitation on the method. As an example, a method of spraying a polymer solution onto a bead carrier and then drying it can be exemplified. There is no particular limitation on the drying method, and examples include air drying and drying under reduced pressure. When the carrier has pores, it is preferable to perform degassing by immersing it in a solvent for several hours before coating with the temperature-responsive polymer.

[0046] The temperature-responsive beads of the present invention can be used for cell culture as microcarriers. The type of cells is not particularly limited as long as they adhere to the temperature-responsive beads before cooling for cell detachment. Examples include cells derived from humans, monkeys, dogs, cats, rabbits, rats, nude mice, mice, guinea pigs, pigs, sheep, Chinese hamsters, cows, etc. Specific cells include, for example, various cultured cell lines such as Chinese hamster ovary-derived CHO cells, African green monkey kidney-derived Vero cells, mouse connective tissue L929 cells, human fetal kidney-derived HEK293 cells, and human cervical cancer-derived HeLa cells. In addition, for example, epithelial cells and endothelial cells that make up each tissue and organ in vivo, skeletal muscle cells, smooth muscle cells, cardiomyocytes that show contractility, neuron cells and glial cells that make up the nervous system, fibroblasts, macrophages and dendritic cells involved in the body's immunity, hepatocytes, non-parenchymal liver cells and adipocytes involved in the body's metabolism, and cells with differentiation ability such as induced pluripotent stem (iPS) cells, embryonic stem (ES) cells, embryonic germ (EG) cells, embryonic carcinoma (EC) cells, mesenchymal stem cells, hepatic stem cells, pancreatic stem cells, skin stem cells, muscle stem cells, germ stem cells, and other various stem cells, or progenitor cells of each tissue, and further cells differentiated therefrom, etc. are mentioned. In particular, mesenchymal stem cells are preferably used. Mesenchymal stem cells mean a population of stem cells capable of differentiating into all or some of mesenchymal cells such as chondrocytes, osteoblasts, and adipocytes, as well as their progenitor cells. The origin of mesenchymal stem cells is not particularly limited, and examples include tissues such as bone marrow, adipose tissue, dental pulp, umbilical cord blood, placenta, synovium, and those derived from pluripotent stem cells such as ES cells and iPS cells.

[0047] Regarding the composition of the culture medium used in cell culture using the temperature-responsive beads of the present invention, there is no particular limitation as long as the cells adhere and proliferate. It consists of a basal medium and serum, and may contain antibiotics. The type of basal medium is not particularly limited, and for example, MEM, αMEM, DMEM, EMEM, GMEM, DMEM / Ham’s F-12, Ham’s F-12, Ham’s F-10, Medium 199, RPMI1640, etc. can be used. The type of serum is not particularly limited, and for example, fetal bovine serum (FBS), calf serum, adult bovine serum, horse serum, sheep serum, goat serum, pig serum, chicken serum, rabbit serum, human serum are used, but FBS is generally often used due to its easy availability. The serum concentration in the culture medium is not particularly limited. Generally, it is often used at a concentration of 20 vol% or less from the cost-effectiveness, but a concentration exceeding 20 vol% may also be acceptable. Further, a serum-free medium containing no untreated or unpurified serum and containing purified blood-derived components or animal tissue-derived components (such as growth factors) may also be used.

[0048] In cell culture using the temperature-responsive beads of the present invention, the cells are cultured on the temperature-responsive beads at a temperature higher than the LCST of the temperature-responsive polymer, and after cell growth, the temperature is set below the LCST of the temperature-responsive polymer to detach the grown cells from the beads. There is no particular limitation on the method for culturing the cells. For example, the temperature-responsive beads of the present invention are added to a cell culture container containing a medium, and after seeding the cells, the cells can be cultured by standing, continuously stirring, or stirring or shaking for a certain period of time. There is no particular limitation on the cooling method when detaching the cells from the temperature-responsive beads of the present invention by cooling. It may be cooled in a cold place or the medium may be exchanged with a cooled medium. Further, in order to detach the cells efficiently, the culture substrate may be gently tapped, shaken, the culture solution may be stirred, or pipetting may be used in combination.

[0049] The seeding density of the cells is not particularly limited as long as the cells adhere and proliferate. In the case of human-derived mesenchymal stem cells, for example, 1.0×10 1 cells / cm 2 ~1.0×105 cells / cm 2 is preferred, and 1.0×10 2 cells / cm 2 ~1.0×10 4 cells / cm 2 is more preferred. Other culture conditions are not particularly limited, and culturing may be performed under conditions usually carried out in the art.

Examples

[0050] Examples of the present invention will be described below, but the present invention is not limited by these examples. Unless otherwise specified, commercially available reagents were used.

[0051] <Method for culturing adherent cells using microcarriers coated with a polymer showing a lower critical temperature in a culture vessel> Reference Example 1 Synthesis of Temperature-Responsive Polymer 1 0.650 g (5 mmol) of 2-methoxyethyl acrylate (MEA) was added to a 200 mL two-necked flask, and further 31.8 mg (100 μmol) of cyanomethyl dodecyl trithiocarbonate, 1.6 mg (10 μmol) of azobisisobutyronitrile, and 10 mL of tert-butyl alcohol were added. After purging with argon gas, the mixture was heated and stirred at 62°C for 24 hours.

[0052] 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-butyl alcohol were added. After purging with argon gas, the mixture was heated and stirred at 62°C for 24 hours.

[0053] After the second heating and stirring, 7.355 g (65 mmol) of N-isopropylacrylamide (IPAAm LCST = 32°C) was added to the above, and further 1.6 mg (10 μmol) of azobisisobutyronitrile and 85 mL of tert-butyl alcohol were added. After purging with argon gas, the mixture was heated and stirred at 62°C for 24 hours.

[0054] 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 1 poly(MEA-BA-IPAAm). The composition of the obtained temperature-responsive polymer 1 was MEA:BA:IPAAm = 5:30:65 (mol%), the lower critical temperature was 32 °C, and Mn was 11.8×10 4 , and Mw / Mn was 1.45.

[0055] Reference Example 2 Synthesis of Temperature-Responsive Polymer 2 3.845 g (30 mmol) of n-butyl acrylate (BA) was added to a 200 mL two-necked flask, and further 31.8 mg (100 μmol) of cyanomethyl dodecyl trithiocarbonate, 1.6 mg (10 μmol) of azobisisobutyronitrile, and 15 mL of tert-butyl alcohol were added. After purging with argon gas, the mixture was heated and stirred at 62 °C for 24 hours.

[0056] After heating and stirring, 7.355 g (65 mmol) of N-isopropylacrylamide (IPAAm LCST = 32 °C) was added thereto, and further 1.6 mg (10 μmol) of azobisisobutyronitrile and 85 mL of tert-butyl alcohol were added. After purging with argon gas, the mixture was heated and stirred at 62 °C for 24 hours.

[0057] 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 2 poly(BA-IPAAm). The composition of the obtained temperature-responsive polymer 2 was BA:IPAAm = 32:68 (mol%), the lower critical temperature was 32 °C, and Mn was 10.8×10 4 , and Mw / Mn was 1.35.

[0058] Synthesis method of Polymer Compound 1 (St / CSt), Reference Example 3 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 to a 100 mL two-necked flask. Further, 3.3 mg (20 μmol) of azobisisobutyronitrile and 20 mL of tert-butyl alcohol were added. After purging with nitrogen gas, the mixture was heated and stirred at 64 °C for 24 hours. By purifying in the same manner as in Example 1, 0.92 g of Polymer Compound 1 poly(CSt / St) was obtained. The composition of the obtained Polymer Compound 1 was CSt:St = 33:67 (mol%), the number average molecular weight Mn was 10.6×10 4 , and the molecular weight distribution Mw / Mn was 1.84.

[0059] Preparation of Surface Treatment Agent 1, Reference Example 4 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 placed in a glass container and left standing overnight to dissolve. Then, it was filtered through a 0.22 μm filter (manufactured by Millipore, hydrophilic filter) to obtain Surface Treatment Agent 1.

[0060] Preparation of Surface Treatment Agent 2, Reference Example 5 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 placed in a glass container and left standing overnight to dissolve. Then, it was filtered through a 0.22 μm filter (manufactured by Millipore, hydrophilic filter) to obtain Surface Treatment Agent 2.

[0061] Preparation of Surface Treatment Agent 3, Reference Example 6 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 placed in a glass container and left standing overnight to dissolve. Then, it was filtered through a 0.22 μm filter (manufactured by Millipore, hydrophilic filter) to obtain Surface Treatment Agent 3.

[0062] Preparation of Temperature-Responsive Microcarrier 1, Reference Example 7 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. Then, the solvent was distilled off by reducing the pressure with an evaporator to obtain temperature-responsive microcarrier 1.

[0063] Reference Example 8 Preparation of Temperature-Responsive Microcarrier 2 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. Then, the solvent was distilled off by reducing the pressure with an evaporator to obtain temperature-responsive microcarrier 2.

[0064] Reference Example 9 Preparation of Temperature-Responsive Microcarrier 3 5 g of AmberChrom (registered trademark) 1×8 chloride form, 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 flask and allowed to stand for 1 hour. Then, the solvent was distilled off by reducing the pressure with an evaporator to obtain temperature-responsive microcarrier 3.

[0065] Example 1 0.6 g of temperature-responsive microcarrier 1 was added to a 30 mL single-use bioreactor (ABLE Corporation, product number: BWV-S03A), and bone marrow-derived human mesenchymal stem cells (manufactured by Lonza Japan Co., Ltd., product number: PT-2501, Lot Number: 0000603525) were seeded at 8.64×10 5 cells. After (standing for 59 minutes → stirring at 55 rpm for 1 minute) × 10 times at 37 °C and a CO2 concentration of 5%, CO 2 At a concentration of 5% The cells were cultured with stirring at 55 rpm for 4 days. 30 mL of mesenchymal stem cell growth medium 2 (PromoCell, product number: C-28009) was used as the culture medium.

[0066] After culturing for 4 days, 24 mL of the culture medium was withdrawn, 24 mL of the culture medium cooled to 4 °C was added, and the mixture was allowed to stand at room temperature (23 °C) for 10 minutes. Then, the culture medium was stirred under the conditions of 200 rpm (stirring Reynolds number: 1330) × 5 minutes to detach the cells from the microcarriers. After stirring, the microcarriers were removed from the culture medium using a 100-μm mesh cell strainer to obtain a cell suspension. The collected cell suspension was centrifuged under the conditions of 160 rcf, 25 °C, and 5 minutes, the supernatant was removed, and PBS(-) was added and suspended. The obtained cell suspension was mixed with a 0.4 w / v% trypan blue solution at a ratio of 1:1, 10 μL was added to a cell counting slide (manufactured by Thermo Fisher Scientific Co., Ltd., product name: Countess Cell Counting Chamber Slid), and the cell number A and viable cell rate of the cell suspension were measured using an automatic cell counter (manufactured by Thermo Fisher Scientific Co., Ltd., product name: Countess II). As a result, the cell number A was 1.12×10 7 cells, and the viable cell rate was 95%. Also, for the microcarriers removed with the cell strainer, enzymatic treatment was performed using a trypsin-EDTA solution, and the cell number B that was not detached by the cooling and stirring operations was also measured in the same manner as described above. Using these values, the cell recovery rate was calculated from Equation (2). A / (A + B)×100 (%) ··· Equation (2) As a result, the cell number B was 9.74×10 5 cells, and the cell recovery rate was 92%.

[0067] Example 2 The same method as in Example 1 was performed 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%, the cell number B was 6.80×10 5 cells, and the cell recovery rate was 90%.

[0068] Example 3 After culturing for 4 days, 24 mL of the culture medium was removed, 24 mL of the culture medium cooled to 4 °C was added, and after standing at room temperature (23 °C) for 10 minutes, the culture medium was stirred under the conditions of 15 rpm (stirring Reynolds number: 100) × 5 minutes to detach the cells from the microcarriers, and the procedure was the same as in Example 1. As a result, the number of cells A was 7.84×10 6 cells, the viable cell rate was 98%, the number of cells B was 3.05×10 6 cells, and the cell recovery rate was 72%.

[0069] Example 4 After culturing for 4 days, 24 mL of the culture medium was removed, 24 mL of the culture medium cooled to 4 °C was added, and after standing at room temperature (23 °C) for 10 minutes, the culture medium was stirred under the conditions of 270 rpm (stirring Reynolds number: 1800) × 5 minutes to detach the cells from the microcarriers, and the procedure was the same as in Example 1. As a result, the number of cells A was 1.25×10 7 cells, the viable cell rate was 89%, the number of cells B was 1.55×10 6 cells, and the cell recovery rate was 98%.

[0070] Example 5 To a 30 mL single-use bioreactor, 0.4 g of temperature-responsive microcarrier 3 was added, and 8.64×10 5 cells of bone marrow-derived human mesenchymal stem cells were seeded. After (standing for 175 minutes → stirring at 80 rpm for 5 minutes) × 8 times at 37 °C and a CO2 concentration of 5%, CO 2 At a concentration of 5% The procedure was the same as in Example 1 except that stirring culture was carried out at 80 rpm for 4 days. As a result, the number of cells A was 7.75×10 6 cells, the viable cell rate was 96%, the number of cells B was 8.60×10 5 cells, and the cell recovery rate was 90%.

[0071] Comparative Example 1 The procedure was the same as in Example 1 except that the culture medium cooled to 4 °C was added, and after standing at room temperature (23 °C) for 10 minutes, the culture medium was stirred under the conditions of 5 rpm (stirring Reynolds number: 33) × 5 minutes to detach the cells from the microcarriers. As a result, the number of cells A was 2.39×10 6cells, the viable cell rate was 88%, and the cell count B was 8.47×10 6 cells, the cell recovery rate was 22%, and the cell recovery rate decreased significantly.

[0072] Comparative Example 2 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 under the conditions of 350 rpm (stirring Reynolds number: 2324) × 5 minutes to detach the cells from the microcarriers. The procedure was the same as in Example 1. As a result, the cell count A was 1.29×10 7 cells, the viable cell rate was 68%, and the cell count B was 8.23×10 5 cells, the cell recovery rate was 94%, and the viable cell rate decreased significantly.

[0073] Comparative Example 3 The procedure was the same as in Example 1 except that untreated microcarriers (Corning, product number: 4625, particle size: 125 - 212 μm) were used. As a result, the cell count A was 4.98×10 5 cells, the viable cell rate was 92%, and the cell count B was 9.47×10 6 cells, and the cell recovery rate was 5%.

[0074] Comparative Example 4 The procedure was the same as in Example 5 except that untreated microcarriers 2 (AmberChrom (registered trademark) 1×8 chloride form, 100 - 200 mesh (Sigma - Aldrich, product number: 217425, particle size: 54 - 154 μm)) were used. As a result, the cell count A was 2.75×10 5 cells, the viable cell rate was 85%, and the cell count B was 9.68×10 6 cells, and the cell recovery rate was 3%.

[0075]

Table 1

[0076] <Temperature - Responsive Beads for Cell Culture and Its Culture Method> [Analysis of Monomer Addition Rate and Composition of Temperature - Responsive Polymer] It was determined by 1H-NMR measurement using Fourier transform nuclear magnetic resonance (NMR) method. An NMR apparatus JNM-ECZ400S / L1 (manufactured by JEOL Ltd.) was used, and 10 mg of the temperature-responsive polymer was dissolved in 0.75 mL of deuterated chloroform for measurement. [Analysis of the molecular weight and molecular weight distribution of the temperature-responsive polymer] 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. A GPC apparatus HLC-8320GPC (manufactured by Tosoh Corporation) was used. Two TSKgel Super AWM-H (manufactured by Tosoh Corporation) columns were used, the column temperature was set at 40 °C, the concentration detector was a differential refractometer, and the eluent was a 10 mM sodium trifluoroacetate / 2,2,2-trifluoroethanol solution. 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. Also, a calibration curve for molecular weight calculation was prepared by measuring under the same conditions using polymethyl methacrylate (manufactured by PSS Polymer Standards Service GmbH) with a known molecular weight.

[0077] [Measurement of the polymer coat film thickness] The temperature-responsive beads were exposed to a vapor atmosphere of ruthenium oxide for 2 hours to stain the polymer coat film. After staining, the temperature-responsive beads were embedded in a room-temperature curable epoxy resin and sectioned with an ultramicrotome. The sections were observed with a transmission electron microscope (equipment name JEM-2100F, manufactured by JEOL Ltd.), and the film thickness was measured by image analysis.

[0078] Example 6 [Synthesis of temperature-responsive polymer 4] 1.952 g (15 mmol) of 2-methoxyethyl acrylate (MEA) was added to a 500 mL cylindrical flask (inner diameter 80 mm), and further 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. The atmosphere was replaced with argon gas, and the reaction was carried out at 62 °C for 24 hours. The monomer addition rate of MEA after the reaction was 96%.

[0079] After the first heating and stirring, 11.535 g (90 mmol) of n-butyl acrylate (BA) was added to the above reaction solution, and further 4.8 mg (30 μmol) of azobisisobutyronitrile and 10 mL of tert-butyl alcohol were added. The reaction solution was replaced with argon gas and reacted at 62 °C for 24 hours. The monomer addition rate of BA after the reaction was 95%.

[0080] After heating and stirring, 22.066 g (195 mmol) of N-isopropylacrylamide (IPAAm: LCST = 32 °C) was added to the above reaction solution, and further 4.8 mg (30 μmol) of azobisisobutyronitrile and 255 mL of tert-butyl alcohol were added. The reaction solution was replaced with argon gas and reacted at 62 °C for 24 hours. The monomer addition rate of IPAAm after the reaction was 99%.

[0081] The total amount of the reaction solution was dropped into a 3 L beaker containing 2 L of pure water, and the precipitated yellow viscous substance was collected. This viscous substance was immersed in 2 L of pure water for 12 hours, then heated to 40 °C to collect the solid, and vacuum dried at 100 °C for 12 hours. After dissolving this solid in 300 mL of chloroform, 5 g of magnesium sulfate was added and stirred at room temperature for 1 hour, and the filtrate was collected by filtration. The filtrate was dropped into a 3 L beaker containing 2 L of heptane, and the precipitated white solid was collected, and temperature-responsive polymer 4 (17.8 g) was obtained by drying under reduced pressure at 100 °C for 12 hours.

[0082] The Mn of temperature-responsive polymer 4, Mw / Mn, and the composition ratio were MEA / BA / IPAAm = 5 / 30 / 65 mol%. [Preparation of surface treatment agent 4] 0.1 g of temperature-responsive polymer 4 and 49.9 g of 1-methoxy-2-propanol were placed in a glass container and left standing overnight to dissolve. Then, it was filtered through a 0.22 μm filter (manufactured by Millipore, hydrophilic filter) to obtain surface treatment agent 4 with a polymer concentration of 0.2 wt%.

[0083] [Preparation of temperature-responsive beads 1] 5 g of non-porous polystyrene particles modified with calcium phosphate and 10 g of surface treatment agent 4 were added to a 25 mL eggplant flask and allowed to stand for 2 hours. Then, the solvent was distilled off by reducing the pressure with an evaporator to obtain temperature-responsive beads 1. The coat film thickness of the temperature-responsive polymer was approximately 58 nm. [Culture evaluation] 60 mg of temperature-responsive beads 1 washed with PBS(-) were added to a Costar(R) ultra-low adhesion surface 6-well plate (manufactured by Corning, product number 3471), and human bone marrow-derived mesenchymal stem cells (manufactured by Lonza, product number PT-2501, lot No. 21TL046615) were seeded at 1.0×10 5 Cells / well and cultured at 37 °C with a CO2 concentration of 5%. 5 mL of mesenchymal stem cell growth medium 2 (manufactured by PromoCell, product number C-28009) was used as the culture medium. After culturing for 4 days, 4 mL of the culture medium was withdrawn, 4 mL of the culture medium cooled to 4 °C was added, and the mixture was allowed to stand at room temperature for 30 minutes. The cells were recovered by passing the cell suspension through a cell strainer with a pore size of 100 μm. The strainer was washed twice with 4 mL of PBS(-), and after centrifuging the cell suspension under the conditions of 200 g × 5 minutes to remove the supernatant, the cells were suspended in the culture medium and the number of cells recovered by the cooling treatment was measured. Furthermore, the cells that did not detach by the cooling treatment (remaining cells) were recovered by enzymatic treatment using trypsin, and the number of cells was measured as the number of remaining cells. The cell growth rate was calculated from the sum of the number of cells recovered by the cooling treatment and the number of remaining cells, and the cell recovery rate was calculated from the sum of the number of cells recovered by the cooling treatment and the number of remaining cells and the number of cells recovered by the cooling treatment. The cell growth rate was 590%, and the cell recovery rate by the cooling treatment was 86%.

[0084] Example 7 [Preparation of temperature-responsive beads 2] 5 g of polystyrene particles with pores modified with quaternary ammonium chloride (manufactured by Organo, product name Amberlite IRA900J Cl, specific gravity 1.06) and 10 g of surface treatment agent 4 were added to a 25 mL eggplant flask and allowed to stand for 2 hours. Then, the solvent was distilled off by reducing the pressure with an evaporator to obtain temperature-responsive beads 2. The coat film thickness of the temperature-responsive polymer was 62 nm. [Culture evaluation] The procedure was the same as in Example 6, except that 200 mg of the temperature-responsive beads 2 were used. The cell growth rate was 710% and the cell recovery rate was 89%.

[0085] Example 8 [Preparation of Temperature-Responsive Beads 3] To a 25 mL eggplant flask, 5 g of polystyrene particles having pores modified with a tertiary amine (manufactured by Organo, product name Amberlyst A21, specific gravity 1.07) and 10 g of surface treatment agent 4 were added, and the mixture was allowed to stand for 2 hours. Then, the solvent was distilled off by reducing the pressure with an evaporator to obtain temperature-responsive beads 3. The thickness of the temperature-responsive polymer coating film was 66 nm.

[0086] [Culture Evaluation] The procedure was the same as in Example 6, except that 90 mg of the temperature-responsive beads 3 were used. The cell growth rate was 430% and the cell recovery rate was 91%.

[0087] Example 9 [Preparation of Surface Treatment Agent 5] 0.025 g of temperature-responsive polymer 4 and 49.975 g of 1-methoxy-2-propanol were placed in a glass container and allowed to stand overnight for dissolution. Then, the mixture was filtered through a 0.22 μm filter (manufactured by Millipore, hydrophilic filter) to prepare a surface treatment agent 5 with a polymer concentration of 0.05 wt%. [Preparation of Temperature-Responsive Beads 4] To a 25 mL eggplant flask, 5 g of polystyrene particles having pores modified with quaternary ammonium chloride (manufactured by Organo, product name Amberlite IRA900J Cl, specific gravity 1.06) and 10 g of surface treatment agent 5 were added, and the mixture was allowed to stand for 2 hours. Then, the solvent was distilled off by reducing the pressure with an evaporator to obtain temperature-responsive beads 4. The thickness of the temperature-responsive polymer coating film was 16 nm.

[0088] [Culture Evaluation] The procedure was the same as in Example 6, except that 200 mg of the temperature-responsive beads 4 were used. The cell growth rate was 750% and the cell recovery rate was 83%.

[0089] Example 10 [Preparation of Surface Treatment Agent 6] 0.5 g of the temperature-responsive polymer 4 and 49.5 g of 1-methoxy-2-propanol were placed in a glass container and left standing overnight to dissolve. Then, it was filtered through a 0.22-μm filter (Millipore, hydrophilic filter) to prepare a surface treatment agent 6 with a polymer concentration of 1 wt%. [Preparation of Temperature-Responsive Beads 5] 5 g of polystyrene particles (manufactured by Organo, product name Amberlite IRA900J Cl, specific gravity 1.06) having pores modified with 5 g of quaternary ammonium chloride and 10 g of the surface treatment agent 6 were added to a 25-mL eggplant flask and left standing for 2 hours. Then, the solvent was distilled off by reducing the pressure with an evaporator to obtain temperature-responsive beads 5. The temperature-responsive polymer coat film thickness was 253 nm.

[0090] [Culture Evaluation] It was carried out in the same manner as in Example 6 except that 200 mg of the temperature-responsive beads 5 were used. The cell growth rate was 720% and the cell recovery rate was 90%.

[0091] Example 11 [Preparation of Surface Treatment Agent 7] 1 g of the temperature-responsive polymer 4 and 49 g of 1-methoxy-2-propanol were placed in a glass container and left standing overnight to dissolve. Then, it was filtered through a 0.22-μm filter (Millipore, hydrophilic filter) to prepare a surface treatment agent 7 with a polymer concentration of 2 wt%. [Preparation of Temperature-Responsive Beads 6] 5 g of polystyrene particles (manufactured by Organo, product name Amberlite IRA900J Cl, specific gravity 1.06) having pores modified with 5 g of quaternary ammonium chloride and 10 g of the surface treatment agent 7 were added to a 25-mL eggplant flask and left standing for 2 hours. Then, the solvent was distilled off by reducing the pressure with an evaporator to obtain temperature-responsive beads 6. The temperature-responsive polymer coat film thickness was 698 nm.

[0092] [Culture Evaluation] Example 6 was repeated except that 200 mg of temperature-responsive beads 6 were used. The cell growth rate was 680%, and the cell recovery rate was 92%.

[0093] Example 12 [Synthesis of Temperature-Responsive Polymer 5] To 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. The mixture was purged with argon gas and reacted at 62 °C for 24 hours. The monomer addition rate of BA after the reaction was 97%.

[0094] After heating and stirring, 27.158 g (240 mmol) of N-isopropylacrylamide (IPAAm: LCST = 32 °C) was added to the above reaction solution. Further, 4.8 mg (30 μmol) of azobisisobutyronitrile and 255 mL of tert-butyl alcohol were added. The mixture was purged with argon gas and reacted at 62 °C for 24 hours. The monomer addition rate after the reaction was 98%.

[0095] The total amount of the reaction solution was dropped into a 3 L beaker containing 2 L of pure water, and the precipitated yellow viscous substance was recovered. This viscous substance was immersed in 2 L of pure water for 12 hours, then heated to 40 °C to recover the solid, and vacuum dried at 100 °C for 12 hours. After dissolving this solid in 300 mL of chloroform, 5 g of magnesium sulfate was added and stirred at room temperature for 2 hours, and the filtrate was recovered by filtration. The filtrate was dropped into a 3 L beaker containing 2 L of heptane, and the precipitated white solid was recovered and dried under reduced pressure at 100 °C for 12 hours to obtain 21.2 g of temperature-responsive polymer 5.

[0096] The Mn of the temperature-responsive polymer 5, Mw / Mn, and the composition ratio were BA / IPAAm = 25 / 75 mol%. [Preparation of Surface Treatment Agent 8] 0.1 g of the temperature-responsive polymer 5 and 49.9 g of 1-methoxy-2-propanol were placed in a glass container and left standing overnight to dissolve. Thereafter, it was filtered through a 0.22 μm filter (Millipore, hydrophilic filter) to obtain a surface treatment agent 8 with a polymer concentration of 0.2 wt%.

[0097] [Preparation of Temperature-Responsive Beads 7] Into a 25 mL eggplant flask, 5 g of polystyrene particles (manufactured by Organo, product name Amberlite IRA900J Cl, specific gravity 1.06) having pores modified with 5 g of quaternary ammonium chloride and 10 g of the surface treatment agent 8 were added and left standing for 2 hours. Thereafter, the solvent was distilled off by reducing the pressure with an evaporator to obtain temperature-responsive beads 7. The temperature-responsive polymer coat film thickness was 52 nm.

[0098] [Culture Evaluation] It was carried out in the same manner as in Example 6 except that 200 mg of the temperature-responsive beads 7 were used. The cell growth rate was 720% and the cell recovery rate was 71%.

[0099] Comparative Example 5 [Preparation of Temperature-Responsive Beads 8] Into a 25 mL eggplant flask, 5 g of a polystyrene carrier (specific gravity 1.05) and 10 g of the surface treatment agent 4 were added and left standing for 2 hours. Thereafter, the solvent was distilled off by reducing the pressure with an evaporator to obtain temperature-responsive beads 8. The temperature-responsive polymer coat film thickness was 52 nm.

[0100] [Culture Evaluation] It was carried out in the same manner as in Example 6 except that 60 mg of the temperature-responsive beads 8 were used. The cell growth rate was 250% and the cell recovery rate was 80%.

[0101] Comparative Example 6 [Preparation of Surface Treatment Agent 9] 0.05 g of the temperature-responsive polymer 1 and 499.95 g of 1-methoxy-2-propanol were placed in a glass container and left standing overnight to dissolve. Then, it was filtered through a 0.22-μm filter (Millipore, hydrophilic filter) to obtain a surface treatment agent 9 with a polymer concentration of 0.01 wt%.

[0102] [Preparation of temperature-responsive beads 9] Into a 25-mL eggplant flask, 5 g of polystyrene particles (manufactured by Organo, product name Amberlite IRA900J Cl, specific gravity 1.06) having pores modified with quaternary ammonium chloride and 10 g of the surface treatment agent 9 were added and left standing for 2 hours. Then, the solvent was distilled off by reducing the pressure with an evaporator to obtain temperature-responsive beads 9. The temperature-responsive polymer coat film thickness was 5 nm.

[0103] [Culture evaluation] The procedure was the same as in Example 6 except that 200 mg of the temperature-responsive beads 9 were used. The cell growth rate was 770% and the cell recovery rate was 4%.

[0104] Comparative Example 7 [Preparation of temperature-responsive beads 10] Into a 25-mL eggplant flask, 5 g of polystyrene particles (manufactured by Organo, product name Amberlite 200CT Na, specific gravity 1.04) having pores modified with sulfonic acid and 10 g of the surface treatment agent 4 were added and left standing for 2 hours. Then, the solvent was distilled off by reducing the pressure with an evaporator to obtain temperature-responsive beads 10. The temperature-responsive polymer coat film thickness was 55 nm. [Culture evaluation] The procedure was the same as in Example 6 except that 120 mg of the temperature-responsive beads 10 were used. After culturing, the cells did not adhere to the beads and the cell growth rate was 90%.

[0105] Comparative Example 8 [Preparation of temperature-responsive beads 11] In a 25 mL eggplant flask, 5 g of polystyrene particles with pores modified with carboxylic acid (manufactured by Organo, product name Amberlite IRC76, specific gravity 1.04) and 10 g of surface treatment agent 4 were added, and the mixture was allowed to stand for 2 hours. Then, the solvent was distilled off by reducing the pressure with an evaporator to obtain temperature-responsive beads 11. The temperature-responsive polymer coat thickness was 59 nm.

[0106] [Culture evaluation] The procedure was the same as in Example 6 except that 140 mg of temperature-responsive beads 11 were used. After culturing, the cells did not adhere to the beads, and the cell growth rate was 90%. Comparative Example 9 [Culture evaluation] The procedure was the same as in Example 6 except that 60 mg of non-porous polystyrene particles modified with calcium phosphate were used as beads. The cell growth rate was 590%, and the cell recovery rate was 3%.

[0107] Comparative Example 10 [Culture evaluation] The procedure was the same as in Example 6 except that 200 mg of Amberlite IRA900J Cl (manufactured by Organo, specific gravity 1.06) with pores modified with quaternary ammonium chloride were used as beads. The cell growth rate was 730%, and the cell recovery rate was 4%. Comparative Example 11 [Culture evaluation] The procedure was the same as in Example 6 except that 90 mg of Amberlyst A21 (manufactured by Organo, specific gravity 1.07) with pores modified with tertiary amine were used as beads. The cell growth rate was 460%, and the cell recovery rate was 2%.

[0108] Example 13 [Preparation of temperature-responsive beads 12] In a 25 mL eggplant flask, 5 g of polystyrene particles having pores modified with quaternary ammonium chloride (manufactured by Sigma-Aldrich, product name AmberChrom 1×8 200 - 400 mesh, specific gravity 1.09) and 10 g of surface treatment agent 4 were added, and the mixture was allowed to stand for 2 hours. Then, the solvent was distilled off by reducing the pressure with an evaporator to obtain temperature-responsive beads 12. The thickness of the temperature-responsive polymer coat was 50 nm.

[0109] [Culture evaluation] The procedure was the same as in Example 6 except that 20 mg of temperature-responsive beads 12 were used. The cell growth rate was 790% and the cell recovery rate was 88%.

[0110] Example 14 [Preparation of temperature-responsive beads 13] In a 25 mL eggplant flask, 5 g of polystyrene particles having pores modified with quaternary ammonium chloride (manufactured by Sigma-Aldrich, product name AmberChrom 1×8 100 - 200 mesh, specific gravity 1.09) and 10 g of surface treatment agent 5 were added, and the mixture was allowed to stand for 2 hours. Then, the solvent was distilled off by reducing the pressure with an evaporator to obtain temperature-responsive beads 13. The thickness of the temperature-responsive polymer coat was 12 nm.

[0111] [Culture evaluation] The procedure was the same as in Example 6 except that 35 mg of temperature-responsive beads 13 were used. The cell growth rate was 830% and the cell recovery rate was 80%.

[0112] Example 15 [Preparation of temperature-responsive beads 14] In a 25 mL eggplant flask, 5 g of polystyrene particles having pores modified with quaternary ammonium chloride (manufactured by Sigma-Aldrich, product name AmberChrom 1×8 100 - 200 mesh, specific gravity 1.09) and 10 g of surface treatment agent 4 were added, and the mixture was allowed to stand for 2 hours. Then, the solvent was distilled off by reducing the pressure with an evaporator to obtain temperature-responsive beads 14. The thickness of the temperature-responsive polymer coat was 61 nm.

[0113] [Culture evaluation] The procedure was the same as in Example 6, except that 35 mg of temperature-responsive beads 14 were used. The cell growth rate was 750% and the cell recovery rate was 86%.

[0114] Example 16 [Preparation of Temperature-Responsive Beads 15] To a 25 mL eggplant flask, 5 g of polystyrene particles having pores modified with quaternary ammonium chloride (manufactured by Sigma-Aldrich, product name AmberChrom 1×8 50-100 mesh, specific gravity 1.09) and 10 g of surface treatment agent 4 were added, and the mixture was allowed to stand for 2 hours. Then, the solvent was distilled off by reducing the pressure with an evaporator to obtain temperature-responsive beads 15. The thickness of the temperature-responsive polymer coat was 44 nm.

[0115] [Culture Evaluation] The procedure was the same as in Example 6, except that 100 mg of temperature-responsive beads 15 were used. The cell growth rate was 790% and the cell recovery rate was 85%.

[0116] Comparative Example 12 [Preparation of Temperature-Responsive Beads 16] To a 25 mL eggplant flask, 5 g of polystyrene particles having pores modified with quaternary ammonium chloride (manufactured by Sigma-Aldrich, product name AmberChrom 1×8 100-200 mesh, specific gravity 1.09) and 10 g of surface treatment agent 9 were added, and the mixture was allowed to stand for 2 hours. Then, the solvent was distilled off by reducing the pressure with an evaporator to obtain temperature-responsive beads 16. The thickness of the temperature-responsive polymer coat was 5 nm.

[0117] [Culture Evaluation] The procedure was the same as in Example 6, except that 35 mg of temperature-responsive beads 16 were used. The cell growth rate was 750% and the cell recovery rate was 6%.

[0118] [Table 2]

[0119] [Table 3]

Claims

1. A method for culturing adherent cells in a culture vessel using a microcarrier coated with a polymer that exhibits a lower critical temperature, the method comprising the following steps (1) to (4): (1) culturing the cells on the surface of the microcarrier in a culture medium at a temperature equal to or higher than the lower critical temperature; (2) after step (1), cooling the culture medium to a temperature equal to or lower than the lower critical temperature; (3) after step (2), stirring the culture medium in the culture vessel to detach the cells from the surface of the microcarrier; and (4) after step (3), recovering the cells detached from the surface of the microcarrier.

2. The method according to claim 1, wherein in step (3), the stirring is carried out at a stirring Reynolds number in the range of 50 to 2000.

3. The method according to claim 2, wherein step (2) is carried out by replacing 10 (v / v) % to 90 (v / v) % of the culture medium used in step (1) with a culture medium cooled to a temperature not higher than the lower critical temperature.

4. The method according to claim 3, wherein step (4) is carried out by removing the microcarriers using a mesh.

5. The method according to claim 4, wherein the particle size of the microcarriers is 50 μm to 1000 μm.

6. The method according to claim 5, wherein the lower critical temperature is 0°C to 50°C.

7. The method according to claim 6, wherein the adherent cells are stem cells.

8. Temperature-responsive beads comprising a carrier and a polymer coating film made of a temperature-responsive polymer having a lower critical solution temperature between 0°C and 50°C, characterized in that the carrier surface has a positive charge and the film thickness of the temperature-responsive polymer is between 10 nm and 1000 nm.

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

10. Beads according to claim 9, characterized in that the carrier has a specific gravity of 1.0 to 1.

1.

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

12. Temperature-responsive beads 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 beads described in claim 12.

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