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  • C12M25/00—Means for supporting, enclosing or fixing the microorganisms, e.g. immunocoatings
  • C12M25/16—Particles; Beads; Granular material; Encapsulation
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  • C08J7/00—Chemical treatment or coating of shaped articles made of macromolecular substances
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  • C12M33/00—Means for introduction, transport, positioning, extraction, harvesting, peeling or sampling of biological material in or from the apparatus
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  • C12M41/00—Means for regulation, monitoring, measurement or control, e.g. flow regulation
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  • C12M41/00—Means for regulation, monitoring, measurement or control, e.g. flow regulation
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  • C12M47/00—Means for after-treatment of the produced biomass or of the fermentation or metabolic products, e.g. storage of biomass
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  • C12N1/00—Microorganisms; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
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  • C12N11/00—Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
  • C12N11/02—Enzymes or microbial cells immobilised on or in an organic carrier
  • C12N11/08—Enzymes or microbial cells immobilised on or in an organic carrier the carrier being a synthetic polymer
  • C12N11/082—Enzymes or microbial cells immobilised on or in an organic carrier the carrier being a synthetic polymer obtained by reactions only involving carbon-to-carbon unsaturated bonds
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  • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
  • C12N5/0068—General culture methods using substrates
  • C12N5/0075—General culture methods using substrates using microcarriers
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  • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
  • C12N5/06—Animal cells or tissues; Human cells or tissues
  • C12N5/0602—Vertebrate cells
  • C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
  • C12N5/0662—Stem cells
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  • C08J2325/00—Characterised by the use of homopolymers or copolymers of compounds having one or more unsaturated aliphatic radicals, each having only one carbon-to-carbon double bond, and at least one being terminated by an aromatic carbocyclic ring; Derivatives of such polymers
  • C08J2325/02—Homopolymers or copolymers of hydrocarbons
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  • C08J2325/06—Polystyrene
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  • C08J2433/00—Characterised by the use of homopolymers or copolymers of compounds having one or more unsaturated aliphatic radicals, each having only one carbon-to-carbon double bond, and only one being terminated by only one carboxyl radical, or of salts, anhydrides, esters, amides, imides, or nitriles thereof; Derivatives of such polymers
  • C08J2433/04—Characterised by the use of homopolymers or copolymers of compounds having one or more unsaturated aliphatic radicals, each having only one carbon-to-carbon double bond, and only one being terminated by only one carboxyl radical, or of salts, anhydrides, esters, amides, imides, or nitriles thereof; Derivatives of such polymers esters
  • C08J2433/06—Characterised by the use of homopolymers or copolymers of compounds having one or more unsaturated aliphatic radicals, each having only one carbon-to-carbon double bond, and only one being terminated by only one carboxyl radical, or of salts, anhydrides, esters, amides, imides, or nitriles thereof; Derivatives of such polymers esters of esters containing only carbon, hydrogen, and oxygen, the oxygen atom being present only as part of the carboxyl radical
  • C08J2433/08—Homopolymers or copolymers of acrylic acid esters
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  • C08J2433/00—Characterised by the use of homopolymers or copolymers of compounds having one or more unsaturated aliphatic radicals, each having only one carbon-to-carbon double bond, and only one being terminated by only one carboxyl radical, or of salts, anhydrides, esters, amides, imides, or nitriles thereof; Derivatives of such polymers
  • C08J2433/24—Homopolymers or copolymers of amides or imides
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  • C12N2523/00—Culture process characterised by temperature
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  • C12N2531/00—Microcarriers
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  • C12N2533/00—Supports or coatings for cell culture, characterised by material
  • C12N2533/30—Synthetic polymers
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  • C12N2539/00—Supports and/or coatings for cell culture characterised by properties
  • C12N2539/10—Coating allowing for selective detachment of cells, e.g. thermoreactive coating

Definitions

• the present invention relates to a cell culture method using a temperature-responsive microcarrier, as well as temperature-responsive cell culture beads and a culture method thereof.
• Microcarriers which are bead-like particles made of synthetic polymers and natural polymers such as polysaccharides, have been developed. By introducing microcarriers into a container containing a cell suspension and culturing them, the surface area to which cells can adhere increases, making it possible to increase cell growth per unit volume of medium.
• the culture medium is stirred to suspend the cells and microcarriers, and the cells attached to the surface of the microcarriers grow.
• the culture method using microcarriers is characterized by being able to achieve a higher cell density per unit volume compared to the conventional culture method using dishes or flasks, because it is performed under stirring.
• the adherent cells need to be detached from the scaffold and collected.
• proteolytic enzymes such as trypsin are used to detach and collect cells that have adhered to the scaffold.
• proteolytic enzymes such as trypsin are generally used to detach and collect the cells from the microcarrier.
• proteolytic enzymes such as trypsin damage the cells because they degrade proteins on the cell surface, and that complicated operations are required to remove trypsin.
• Patent Document 1 discloses a method that uses a microcarrier coated with a temperature-responsive polymer that has a lower critical temperature. Cells adhere and grow at the culture temperature of 37°C, and by cooling to 20°C, the temperature-responsive polymer becomes hydrophilic, allowing cells to be recovered without using proteolytic enzymes. However, temperature-responsive microcarriers have the problem of low cell growth, and improvements were needed.
• Patent Documents 2 and 3 disclose a microcarrier coated with a polymer exhibiting a lower critical temperature. According to Patent Documents 2 and 3, the adhesion force of the microcarrier surface is weakened by causing a sol transition of the polymer exhibiting a lower critical temperature by cooling, and the cells can be detached from the microcarrier and recovered.
• a recovery method that combines pipetting operation as described in Patent Document 3 is usually used.
• pipetting operation is difficult to apply to mass culture, and the cell recovery methods described in Patent Documents 2 and 3 have not provided a mass culture technology, so a cell culture method more suitable for mass culture is required.
• the first object of this patent is to provide a cell culture method suitable for mass culture in a cell recovery method after cell culture on a microcarrier coated with a polymer that exhibits a lower critical temperature.
• the second object of the present invention is to provide a temperature-responsive microcarrier with high cell proliferation.
• the inventors have considered the problems with the culture method using microcarriers and conducted extensive research. As a result, they have discovered that in a cell culture method after cell culture on a microcarrier coated with a polymer that exhibits a lower critical temperature, a cooling step and a stirring step are included in the method, allowing cells to be recovered from the microcarrier with high efficiency and minimal invasiveness, and have completed the present invention.
• the present invention encompasses the following aspects.
• a method for culturing adherent cells in a culture vessel using a microcarrier coated with a polymer exhibiting a lower critical temperature comprising the steps of: (1) culturing the cells on the surface of the microcarriers in a culture medium at a temperature equal to or higher than the lower critical temperature; (2) after step (1), cooling the culture solution to a temperature equal to or lower than the lower critical temperature; (3) after step (2), stirring the culture solution in the culture vessel to detach cells from the surface of the microcarriers; (4) After step (3), recovering the cells detached from the microcarrier surface.
• ⁇ 1-2> 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.
• ⁇ 1-3> The method according to ⁇ 1-1> or ⁇ 1-2>, characterized in that the step (2) is carried out by replacing 10 (v/v) % to 90 (v/v) % of the culture medium used in the step (1) with a culture medium cooled to a temperature equal to or lower than the lower critical temperature.
• ⁇ 1-4> The method according to any one of ⁇ 1-1> to ⁇ 1-3>, wherein the step (4) is carried out by removing the microcarriers using a mesh.
• ⁇ 1-5> The method according to any one of ⁇ 1-1> to ⁇ 1-4>, wherein the particle size of the microcarrier is 50 ⁇ m to 1000 ⁇ m.
• ⁇ 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.
• ⁇ 1-7> The method according to any one of ⁇ 1-1> to ⁇ 1-6>, wherein the adherent cells are stem cells.
• the present inventors have conducted intensive research in view of the problem of low cell proliferation in temperature-responsive microcarriers, and have found that positively charged beads coated with a temperature-responsive polymer having a lower critical dissolution temperature of 0°C to 50°C can adhere cells well, and that cells can be detached and recovered by cooling after the end of culture, thereby completing the present invention. That is, the present invention also includes the following aspects. ⁇ 2-1> Temperature-responsive beads comprising a carrier and a polymer coating film made of a temperature-responsive polymer having a lower critical solution temperature of 0°C to 50°C, characterized in that the carrier surface has a positive charge and the film thickness of the temperature-responsive polymer is 10 nm to 1000 nm.
• ⁇ 2-2> The temperature-responsive bead according to ⁇ 2-1>, wherein the positively charged component 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> Temperature-responsive beads according to any one of ⁇ 2-1> to ⁇ 2-3>, wherein the carrier is made of polystyrene.
• ⁇ 2-5> Temperature-responsive beads according to any one of ⁇ 2-1> to ⁇ 2-4>, characterized in that the particle diameter of the carrier is 50 ⁇ m to 1000 ⁇ m.
• ⁇ 2-6> A cell culture method using the temperature-responsive bead according to any one of ⁇ 2-1> to ⁇ 2-5>.
• the method includes a cooling step and a stirring step, and allows cells to be recovered from the microcarrier with high efficiency and minimal invasiveness.
• temperature-responsive beads which are composed of a carrier and a polymer coating film made of a temperature-responsive polymer having a lower critical dissolution temperature of 0°C to 50°C, and are characterized in that the surface of the carrier has a positive charge, have high cell proliferation properties and can further enable cells to be recovered by cooling.
• 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 vessel using a microcarrier coated with a polymer exhibiting a lower critical temperature, the method comprising the following steps (1) to (4): (1) culturing cells on the surface of the microcarriers in a culture medium at a temperature equal to or higher than the lower critical temperature; (2) after step (1), cooling the culture solution to a temperature equal to or lower than the lower critical temperature; (3) after step (2), stirring the culture solution in the culture vessel to detach the cells from the surface of the microcarriers; (4) After step (3), recovering the cells detached from the microcarrier surface.
• LCST Lower Critical Solution Temperature
• LCST is the temperature below which a polymer dissolves in water to form a transparent solution, but above which it becomes insoluble and cloudy, or precipitates and undergoes phase separation.
• the LCST is preferably near the culture temperature, and as an example, it is in the range of 0°C to 50°C, preferably 20°C to 40°C, and more preferably 25°C to 35°C.
• the temperature at which the lower critical temperature appears varies depending on the concentration of the aqueous solution, but N-isopropylacrylamide is preferred because the expression of the lower critical temperature is less dependent on the concentration
• the repeating unit of the polymer exhibiting the lower critical temperature in the present invention may be of only one type, or may be a combination of two or more types. In addition to the repeating unit of the polymer exhibiting LCST, if it has LCST, it may contain a repeating unit of a polymer that does not exhibit LCST. In addition to the polymer exhibiting LCST, other polymer compounds may be introduced depending on the purpose, such as improving adhesion to cells or microcarriers. As an example, in the embodiment of the present invention, a polymer compound consisting of carboxystyrene and styrene is introduced to improve cell adhesion. As an example, the composition of N-isopropylacrylamide exhibiting LCST in the polymer is 10 mol% to 95 mol%, preferably 30 mol% to 80 mol%, and more preferably 65 mol% to 70 mol%.
• the material of the microcarrier is not particularly limited, but examples include polystyrene, polymethyl methacrylate, polyethylene terephthalate, polycarbonate, cellulose, cyclodextrin, acrylamide, alginate, dextran, gelatin, glass, or a mixture of two or more of these.
• the shape of the microcarrier is not particularly limited, but examples include spheres, ellipsoids, plates, and tubes. Furthermore, the microcarrier may or may not be porous. If it is porous, there is no limit to the pore size.
• the major axis of the microcarrier is preferably 50 ⁇ m to 1000 ⁇ m, more preferably 100 to 700 ⁇ m. If the major axis is smaller than the above range, separation from the cells becomes difficult, resulting in a lower recovery rate. Furthermore, if the major axis is larger than the above range, the culture area per volume becomes smaller.
• a temperature-responsive microcarrier refers to a microcarrier coated with a polymer that exhibits a lower critical temperature.
• the method for coating the microcarrier with a polymer that exhibits a lower critical temperature examples include a method in which the repeating units that exhibit a lower critical temperature are chemically coated by electron beam irradiation, and a method in which a surface treatment agent in which a polymer that exhibits a lower critical temperature is dissolved in a solvent is applied to the microcarrier to physically coat it.
• the surface of the microcarrier coated with the polymer exhibiting LCST may be further coated with an extracellular matrix.
• extracellular matrix There is no particular limitation on the type of extracellular matrix, and for example, collagen, atelocollagen, hyaluronic acid, elastin, proteoglycan, glycosaminoglycan, fibronectin, laminin, vitronectin, gelatin, or matrigel containing laminin, collagen IV, heparan sulfate proteoglycan, entactin/nidogen 1, 2, etc. as the main component may be used, and one of these may be used alone, or two or more types may be combined. It may also be a segment of these extracellular matrices.
• Adherent cells are cells that attach to the surface of cell culture substrates such as microcarriers. There are no particular limitations on the origin of the cells, but examples include humans, monkeys, dogs, cats, rabbits, rats, nude mice, mice, guinea pigs, pigs, sheep, Chinese hamsters, and cows.
• cells include 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 embryonic kidney-derived HEK293 cells, and human cervical cancer-derived HeLa cells, as well as epithelial cells and endothelial cells that constitute various tissues and organs in the body, skeletal muscle cells that exhibit contractility, smooth muscle cells, cardiac muscle cells, neuron cells, glial cells, and fibroblasts that constitute the nervous system, macrophages and dendritic cells that are involved in the immunity of the body, hepatic parenchymal cells, non-parenchymal liver cells, and adipocytes that are involved in the metabolism of the body, and cells having differentiation potential 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
• mesenchymal stem cells are preferably used.
• Mesenchymal stem cells refer to a population of stem cells and their precursor cells that can differentiate into all or some of the mesenchymal cells such as chondrocytes, osteoblasts, and adipocytes.
• mesenchymal stem cells can be derived from tissues such as bone marrow, fat, dental pulp, umbilical cord blood, placenta, and synovium, or from pluripotent stem cells such as ES cells and iPS cells.
• the temperature of the culture medium in the culture vessel is equal to or higher than the lower critical temperature, so that the polymer exhibiting the lower critical temperature gels, and the adhesive cells can adhere to the microcarrier and grow.
• the lower critical temperature When using human-derived cells, it is recommended to carry out the culture at around body temperature in order to obtain high culture efficiency.
• a temperature range of 30°C to 40°C is preferred, and a temperature range of 36°C to 38°C is even more preferred.
• the conditions other than the lower critical temperature and for example, either stationary culture or agitated culture may be used, but agitated culture is preferred, which allows for a larger culture area per unit volume.
• the cell culture density is not particularly limited as long as the cells adhere and grow, but in the case of human-derived mesenchymal stem cells, for example, the density is preferably 1.0 ⁇ 10 1 cells/cm 2 to 1.0 ⁇ 10 5 cells/cm 2 per surface area of the microcarrier, and more preferably 1.0 ⁇ 10 2 cells/cm 2 to 1.0 ⁇ 10 4 cells/cm 2.
• Other culture conditions are not particularly limited, and the culture may be performed under conditions normally used in this field.
• the temperature of the culture fluid during cooling in step (2) is preferably at least 1°C lower than the lower critical temperature.
• methods for lowering the temperature of the microcarriers include replacing the liquid in the culture vessel with a cooled liquid or storing in a cold place, but it is preferable to replace it with a cooled liquid in order to shorten the time required for cooling.
• There is no particular limit to the amount of liquid exchanged when replacing with a cooled liquid but as an example, 10% to 90% is preferable, and 50% to 90% is more preferable in order to increase the cooling rate.
• the liquid used for cooling and it can be selected according to the purpose, such as culture fluid, other medium solutions, or isotonic solutions.
• the cooling time is preferably 5 to 60 minutes.
• the stirring method in step (3) is not particularly limited, but an example is a method of stirring the culture solution by central stirring in which a stirring blade inserted vertically into the center of the culture vessel is rotated.
• the degree of stirring is not particularly limited, but stirring is preferably performed with a stirring Reynolds number, which is one of the indicators representing the degree of stirring, in the range of 50 to 2000, more preferably in the range of 100 to 1800, and most preferably in the range of 300 to 1500. If the stirring Reynolds number is below 50, the cells are not completely detached from the microcarriers and the recovery rate decreases. Also, if the stirring Reynolds number exceeds 2000, the cells are damaged and the viability rate decreases.
• the stirring Reynolds number is expressed by the following formula (1).
• the temperature of the liquid in the culture vessel in step (3) is preferably lower than the lower critical temperature by 1° C. or more to prevent the detached cells from re-adhering to the microcarriers.
• the liquid used for stirring can be selected according to the purpose, such as culture medium, other medium solution, isotonic liquid, etc.
• the stirring time is preferably 1 to 60 minutes, taking into consideration the recovery rate and damage of the cells.
• the method for separating the microcarriers and cells in step (4) is not particularly limited, and examples of the method include a method of separating based on differences in sedimentation velocity and a method of separating based on differences in particle size.
• An example of a method of separating based on differences in particle size is a method of separating the microcarriers and cell suspension using a mesh.
• the material of the mesh and one example is nylon.
• the mesh openings but one example is 20 ⁇ m to 100 ⁇ m, preferably 30 ⁇ m to 80 ⁇ m, and more preferably 40 ⁇ m to 60 ⁇ m. If the mesh openings are small, the cells will not pass through the mesh, and the cell recovery rate will decrease. Furthermore, if the mesh openings are large, the microcarriers will also pass through the mesh, making separation difficult.
• the cell culture vessel is not particularly limited as long as it is a vessel capable of stirring and suspending microcarriers.
• a 30 mL single-use bioreactor (Able Co., Ltd., product number: BWV-S03A) is used as an example.
• the composition of the culture medium used in the present invention is not particularly limited as long as the cells adhere and grow, and is composed of a basal culture medium and serum, and may contain antibiotics.
• the type of basal culture medium is not particularly limited, and for example, MEM, ⁇ MEM, DMEM, EMEM, GMEM, DMEM/Ham's F-12, Ham's F-12, Ham's F-10, Medium 199, RPMI 1640, 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, and human serum are used, but FBS is generally used because of its ease of availability.
• FBS fetal bovine serum
• the serum concentration in the culture medium is not particularly limited. For cost-effectiveness, it is generally used at a concentration of 20 vol% or less, but it may be at a concentration of more than 20 vol%.
• the medium may be a serum-free medium that does not contain untreated or unpurified serum, but contains purified blood-derived components or animal tissue-derived components (such as growth factors).
• the method for measuring the viability of cells there are no particular limitations on the method for measuring the viability of cells, but one known example is a method for determining whether cells are live or dead by trypan blue staining, which stains the cytoplasm of dead cells blue. There are no particular limitations on the method for determining whether cells are live or dead and for counting the number of cells, but there are methods in which an operator manually determines the viability using a hemocytometer, and methods in which an automatic cell counting device automatically determines the viability, but the method using an automatic cell counting device is preferred because it can be measured without relying on the level of skill.
• the present invention provides temperature-responsive beads for cell culture and a method for culturing the same.
• the temperature-responsive beads of the present invention are characterized in that a positively charged bead carrier is coated with a temperature-responsive polymer having a lower critical dissolution temperature of 0°C to 50°C.
• the structure of the positively charged bead carrier of the present invention is not particularly limited.
• a compound having a positive charge may be chemically or physically fixed to the carrier surface.
• Examples of a method of chemical fixation include a method of copolymerizing with a monomer containing a positive charge when manufacturing the carrier, and a method of chemically modifying the particle surface with a functional group that becomes positively charged.
• the functional group that becomes positively charged but examples include tertiary amino groups, quaternary ammonium groups, and alkaline earth metal salts.
• trimethylammonium groups, dimethylamino groups, diethylamino groups, and calcium phosphate can be exemplified because they are easy to modify.
• examples of a method of fixation by physical adsorption include a method of coating a carrier with a cationic polymer, and a method of coating the surface with an inorganic compound that is poorly soluble in water.
• the specific gravity is 1.0 to 1.1, and more preferably 1.01 to 1.06. If the specific gravity is less than 1.0, it will float in the medium and become difficult to culture, and if it is more than 1.1, it will become difficult to disperse in the medium.
• the material of the carrier includes synthetic polymers such as polyethylene, polypropylene, polystyrene, polyalkyl (meth)acrylate, polyalkyl (meth)acrylamide, polyester, polyurethane, polyvinyl chloride, polycarbonate, or resins of copolymers thereof, plant-derived polymers such as dextran and cellulose, or wood chips and ceramics.
• synthetic polymers such as polyethylene, polypropylene, polystyrene, polyalkyl (meth)acrylate, polyalkyl (meth)acrylamide, polyester, polyurethane, polyvinyl chloride, polycarbonate, or resins of copolymers thereof, plant-derived polymers such as dextran and cellulose, or wood chips and ceramics.
• the specific gravity of the beads can be designed to be close to the specific gravity of the culture solution and the settling of the beads can be suppressed during cell culture involving stirring, it is preferable to use a synthetic polymer bead carrier such as polystyrene, polyalkyl (meth)acrylate, polyalkyl (meth)acrylamide, polyester, or polyurethane, and more preferably a polystyrene bead carrier.
• a synthetic polymer bead carrier such as polystyrene, polyalkyl (meth)acrylate, polyalkyl (meth)acrylamide, polyester, or polyurethane, and more preferably a polystyrene bead carrier.
• the material constituting the bead carrier is crosslinked to prevent it from dissolving into the medium.
• the shape of the bead carrier is not particularly limited, but it may be plate-like or spherical, or may be porous.
• the particle size is preferably 50 ⁇ m to 1000 ⁇ m, more preferably 150 ⁇ m to 600 ⁇ m, and even more preferably 150 ⁇ m to 500 ⁇ m.
• the lower critical solution temperature is the temperature at which the solubility of a certain polymer in water changes.
• a polymer with an LCST dehydrates at temperatures higher than the LCST, and hydrophobic interactions become stronger, and hydrates, swells, or dissolves at temperatures lower than the LCST.
• the temperature-responsive polymer of the present invention refers to a polymer that contains, as a repeating unit, a monomer whose homopolymer has an LCST with respect to water between 0°C and 50°C.
• the structure of the temperature-responsive polymer of the present invention may include a repeating unit that exhibits an LCST, and may also include a repeating unit that does not exhibit an LCST as another structure. Since this shows good temperature responsiveness, it is preferable that the repeating unit that exhibits an LCST is a block structure.
• the temperature-responsive beads of the present invention are used for cell culture, the cells are generally cultured at around 37°C, so that 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.
• thermoresponsive polymer there are no particular limitations on the temperature-responsive polymer, but examples include the (N-isopropylacrylamide)-(n-butyl methacrylate) block copolymer and (N-isopropylacrylamide)-(n-butyl acrylate) block copolymer described in Japanese Patent No. 5,846,584, the (2-dimethylaminoethyl methacrylate)-(n-butyl methacrylate)-(N-isopropylacrylamide) block copolymer described in Japanese Patent No. 6,954,047, and the (N-isopropylacrylamide)-(n-butyl methacrylate)-(2-methoxyethyl acrylate) block copolymer described in Japanese Patent No. 7,127,330.
• the film thickness of the temperature-responsive polymer in the temperature-responsive beads is preferably 10 nm to 1000 nm, and more preferably 50 nm to 500 nm. If it is less than 10 nm, the effect of the positive charge will be strong, leading to poor temperature responsiveness, and if it exceeds 1000 nm, the effect of the positive charge will be weak.
• the aforementioned polymer may be chemically fixed or physically adsorbed.
• fixing by physical adsorption there is no particular limit to the method, and one example is a method in which a polymer solution is sprayed onto a bead carrier and then dried.
• drying method examples include air drying and drying under reduced pressure. If the carrier has pores, it is preferable to degas it by immersing it in a solvent for several hours before coating it with the temperature-responsive polymer.
• the temperature-responsive beads of the present invention can be used as microcarriers for cell culture.
• cells include 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 embryonic kidney-derived HEK293 cells, and human cervical cancer-derived HeLa cells, as well as epithelial cells and endothelial cells that constitute various tissues and organs in the body, skeletal muscle cells that exhibit contractility, smooth muscle cells, cardiac muscle cells, neuron cells, glial cells, and fibroblasts that constitute the nervous system, macrophages and dendritic cells that are involved in the immunity of the body, hepatic parenchymal cells, non-parenchymal liver cells, and adipocytes that are involved in the metabolism of the body, and cells having differentiation potential 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
• mesenchymal stem cells are preferably used.
• Mesenchymal stem cells refer to a population of stem cells and their precursor cells that can differentiate into all or some of the mesenchymal cells such as chondrocytes, osteoblasts, and adipocytes.
• mesenchymal stem cells can be derived from tissues such as bone marrow, fat, dental pulp, umbilical cord blood, placenta, and synovium, or from pluripotent stem cells such as ES cells and iPS cells.
• the composition of the culture medium used in cell culture using the temperature-responsive beads of the present invention is not particularly limited as long as the cells adhere and grow, and is composed of a basal culture medium and serum, and may contain antibiotics.
• the type of basal culture medium is not particularly limited, and for example, MEM, ⁇ MEM, DMEM, EMEM, GMEM, DMEM/Ham's F-12, Ham's F-12, Ham's F-10, Medium 199, RPMI 1640, 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, and human serum are used, but FBS is generally used because of its ease of availability.
• FBS fetal bovine serum
• the serum concentration in the culture medium is not particularly limited. For cost-effectiveness, it is generally used at a concentration of 20 vol% or less, but it may be at a concentration of more than 20 vol%.
• the medium may be a serum-free medium that does not contain untreated or unpurified serum, but contains purified blood-derived components or animal tissue-derived components (such as growth factors).
• the temperature-responsive beads of the present invention In cell culture using the temperature-responsive beads of the present invention, cells are cultured on the temperature-responsive beads at a temperature higher than the LCST of the temperature-responsive polymer, and after cell proliferation, the temperature is lowered to below the LCST of the temperature-responsive polymer to detach the proliferated cells from the beads.
• the temperature-responsive beads of the present invention are added to a cell culture vessel containing a medium, and after seeding the cells, the cells can be cultured by leaving them to stand, continuously stirring, or stirring or shaking for a certain period of time.
• the cells may be cooled in a cold place or the medium may be replaced with cooled medium.
• the culture substrate may be lightly tapped or shaken, the culture medium may be stirred, or pipetting may be used in combination.
• the seeding density of the cells is not particularly limited as long as the cells adhere and proliferate, but in the case of human-derived mesenchymal stem cells, for example, the seeding density per surface area of the microcarrier is preferably 1.0 ⁇ 10 1 cells/cm 2 to 1.0 ⁇ 10 5 cells/cm 2 , and more preferably 1.0 ⁇ 10 2 cells/cm 2 to 1.0 ⁇ 10 4 cells/cm 2.
• Other culture conditions are not particularly limited, and the culture may be performed under conditions normally used in this field.
• the reaction solution was purified by reprecipitation 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 collected using a separatory funnel.
• the collected chloroform phase was concentrated with an evaporator and purified by reprecipitation with heptane.
• the precipitate was collected by filtration and dried under reduced pressure to obtain 8.295 g of temperature-responsive polymer 1 poly(MEA-BA-IPAAm).
• the reaction solution was purified by reprecipitation 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 collected using a separatory funnel.
• the collected chloroform phase was concentrated with an evaporator and purified by reprecipitation with heptane.
• the precipitate was collected by filtration and dried under reduced pressure to obtain 7.591 g of temperature-responsive polymer 2 poly(BA-IPAAm).
• the mixture was purified in the same manner as in Example 1, and 0.92 g of polymer compound 1poly(CSt/St) was obtained.
• Reference Example 5 Preparation of Surface Treatment Agent 2 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 to stand overnight to dissolve. The mixture was then filtered through a 0.22 ⁇ m filter (manufactured by Millipore, hydrophilic filter) to obtain surface treatment agent 2.
• Reference Example 7 Preparation of Temperature-Responsive Microcarrier 1 5 g of untreated microcarrier (Corning, product number: 4625, particle size: 125 to 212 ⁇ m) and 10 g of surface treatment agent 1 were added to a 25 mL recovery flask and allowed to stand for 1 hour. The solvent was then removed by reducing the pressure with an evaporator to obtain temperature-responsive microcarrier 1.
• Reference Example 8 Preparation of temperature-responsive microcarrier 2 5 g of untreated microcarrier (Corning, product number: 4625, particle size: 125 to 212 ⁇ m) and 10 g of surface treatment agent 2 were added to a 25 mL recovery flask and allowed to stand for 1 hour. The solvent was then removed by reducing the pressure with an evaporator to obtain temperature-responsive microcarrier 2.
• Example 1 0.6 g of temperature-responsive microcarrier 1 was added to a 30 mL single-use bioreactor (Able Co., Ltd., product number: BWV-S03A), and 8.64 x 10 5 cells of bone marrow-derived human mesenchymal stem cells (Lonza Japan Co., Ltd., product number: PT-2501, Lot Number: 0000603525) were seeded and cultured at 37°C and 5% CO2 (resting for 59 minutes, then stirring at 55 rpm for 1 minute) x 10 times, followed by stirring culture 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.
• mesenchymal stem cell proliferation medium 2 PromoCell, product number: C-28009
• the obtained cell suspension was mixed with 0.4 w/v% trypan blue solution at a ratio of 1:1, and 10 ⁇ L was added to a cell count measurement slide (manufactured by Thermo Fisher Scientific Co., Ltd., product name: Countess Cell Counting Chamber Slide), and the cell number A and viability 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 viability was 95%.
• the microcarriers removed with the cell strainer were subjected to an enzyme treatment using a trypsin-EDTA solution, and the number of cells B that were not detached by cooling and stirring was also measured in the same manner as described above. Using these values, the cell recovery rate was calculated from formula (2). A/(A+B) ⁇ 100(%) Formula (2) As a result, the cell number B was 9.74 ⁇ 10 5 cells, and the cell recovery rate was 92%.
• Example 2 The same method as in Example 1 was used except for using the temperature-responsive microcarrier 2. As a result, the cell number A was 6.12 ⁇ 10 6 cells, the cell viability was 92%, the cell number B was 6.80 ⁇ 10 5 cells, and the cell recovery rate was 90%.
• Example 3 After culturing for 4 days, 24 mL of the culture medium was removed, 24 mL of culture medium cooled to 4°C was added, and the mixture was left to stand at room temperature (23°C) for 10 minutes, after which the culture medium was stirred at 15 rpm (stirring Reynolds number: 100) for 5 minutes to detach the cells from the microcarriers. The same procedure was followed as in Example 1. As a result, cell number A was 7.84 x 106 cells, cell viability was 98%, cell number B was 3.05 x 106 cells, and cell recovery rate was 72%.
• Example 4 After culturing for 4 days, 24 mL of the culture medium was removed, 24 mL of culture medium cooled to 4°C was added, and the mixture was left to stand at room temperature (23°C) for 10 minutes, after which the culture medium was stirred at 270 rpm (stirring Reynolds number: 1800) for 5 minutes to detach the cells from the microcarriers. The same procedure was followed as in Example 1. As a result, cell number A was 1.25 x 107 cells, cell viability was 89%, cell number B was 1.55 x 106 cells, and cell recovery rate was 98%.
• Example 5 The same procedure as in Example 1 was repeated, except that 0.4 g of temperature-responsive microcarrier 3 was added to a 30 mL single-use bioreactor, 8.64 x 10 5 cells of bone marrow-derived human mesenchymal stem cells were seeded, and the cells were cultured at 37°C and 5% CO2 (resting for 175 minutes, followed by stirring at 80 rpm for 5 minutes) x 8 times, followed by stirring culture at 80 rpm for 4 days. As a result, cell number A was 7.75 x 10 6 cells, cell viability was 96%, cell number B was 8.60 x 10 5 cells, and cell recovery rate was 90%.
• Comparative Example 1 The same procedure as in Example 1 was repeated, except that the culture medium cooled to 4°C was added, left to stand at room temperature (23°C) for 10 minutes, and then the culture medium was stirred at 5 rpm (Reynolds number of stirring: 33) for 5 minutes to detach the cells from the microcarriers.
• cell number A was 2.39 x 106 cells
• the viability was 88%
• cell number B was 8.47 x 106 cells
• the cell recovery rate was 22%, indicating a significant decrease in cell recovery rate.
• Comparative Example 2 The same procedure as in Example 1 was repeated, except that the culture medium cooled to 4°C was added, left to stand at room temperature (23°C) for 10 minutes, and then the culture medium was stirred at 350 rpm (Reynolds number of stirring: 2324) for 5 minutes to detach the cells from the microcarriers.
• cell number A was 1.29 x 107 cells
• the cell viability was 68%
• cell number B was 8.23 x 105 cells
• the cell recovery rate was 94%
• the cell viability was significantly reduced.
• Comparative Example 3 The same procedure as in Example 1 was repeated except that untreated microcarriers (Corning, product number: 4625, particle size: 125-212 ⁇ m) were used. As a result, cell number A was 4.98 ⁇ 10 5 cells, cell viability was 92%, cell number B was 9.47 ⁇ 10 6 cells, and cell recovery rate was 5%.
• Comparative Example 4 The same procedure as in Example 5 was repeated except that untreated microcarrier 2 (AmberChrom (registered trademark) 1x8 chloride form, 100-200 mesh (Sigma-Aldrich, product number: 217425, particle size: 54-154 ⁇ m)) was used. As a result, cell number A was 2.75 x 10 5 cells, cell viability was 85%, cell number B was 9.68 x 10 6 cells, and cell recovery rate was 3%.
• AmberChrom registered trademark
• 1x8 chloride form 100-200 mesh
• Temperature-responsive cell culture beads and culture method thereof> [Analysis of monomer addition rate and composition of temperature-responsive polymer]
• the temperature-responsive polymer was determined by 1H-NMR measurement using a Fourier transform nuclear magnetic resonance (NMR) method.
• the NMR device used was a JNM-ECZ400S/L1 (manufactured by JEOL Ltd.), and 10 mg of the temperature-responsive polymer was dissolved in 0.75 mL of deuterated chloroform for measurement.
• the measurements were performed under the following conditions: sample concentration 1 mg/mL, sample injection amount 0.1 mL, and eluent flow rate 0.6 mL/min.
• the calibration curve for molecular weight calculation was created by using polymethyl methacrylate (manufactured by PSS Polymer Standards Service GmbH) with a known molecular weight and performing measurements under the same conditions.
• the temperature-responsive beads were exposed to a ruthenium oxide vapor atmosphere for 2 hours to stain the polymer coating film.
• the stained temperature-responsive beads were embedded in room temperature curing epoxy resin and sliced with an ultramicrotome. The slices were observed with a transmission electron microscope (JEM-2100F, manufactured by JEOL Ltd.), and the film thickness was measured by image analysis.
• 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 95.1 mg (300 ⁇ mol) of cyanomethyl dodecyl trithiocarbonate, 4.8 mg (30 ⁇ mol) of azobisisobutyronitrile, and 30 mL of tert-butyl alcohol were further added, and the atmosphere was replaced with argon gas, and the reaction was carried out for 24 hours under the condition of 62° C. The monomer addition rate of the MEA after the reaction was 96%.
• MEA 2-methoxyethyl acrylate
• the entire reaction solution was dropped into a 3 L beaker containing 2 L of pure water, and the precipitated yellow viscous material was collected.
• This viscous material was immersed in 2 L of pure water for 12 hours, then heated to 40°C to collect the solid, which was then vacuum dried at 100°C for 12 hours.
• This solid was dissolved in 300 mL of chloroform, after which 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 then vacuum dried at 100°C for 12 hours, yielding 17.8 g of temperature-responsive polymer 4.
• the Mn and Mw/Mn of the temperature-responsive polymer 4 were such that the composition ratio of MEA/BA/IPAAm was 5/30/65 mol %.
• 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 recovery flask and allowed to stand for 2 hours. The solvent was then removed by reducing the pressure in an evaporator to obtain temperature-responsive beads 1. The thickness of the coating film 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 (Corning, product number 3471), human bone marrow-derived mesenchymal stem cells (Lonza, product number PT-2501, lot No.
• 21TL046615 were seeded at 1.0 x 105 cells/well, and cultured at 37°C and CO2 concentration of 5%.
• 5 mL of mesenchymal stem cell proliferation medium 2 (PromoCell, product number C-28009) was used as the medium.
• 4 mL of medium was removed, 4 mL of medium cooled to 4°C was added, and the plate was left to stand at room temperature for 30 minutes, and the cell suspension was passed through a cell strainer with a pore size of 100 ⁇ m to recover the cells.
• the strainer was washed twice with 4 mL of PBS (-), centrifuged with the cell suspension at 200 g for 5 minutes to remove the supernatant, and then suspended in culture medium to measure the number of cells recovered by cooling treatment. Furthermore, cells that were not detached by cooling treatment (remaining cells) were recovered by enzyme treatment using trypsin, and the number of cells was measured as the number of remaining cells.
• the cell proliferation rate was calculated from the total number of cells recovered by cooling treatment and the number of remaining cells, and the cell recovery rate was calculated from the total number of cells recovered by cooling treatment and the number of remaining cells and the number of cells recovered by cooling treatment.
• the cell proliferation rate was 590%, and the cell recovery rate by cooling treatment was 86%.
• Example 7 [Preparation of temperature-responsive beads 2] 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 4 were added to a 25 mL recovery flask and allowed to stand for 2 hours. The solvent was then removed by reducing the pressure in an evaporator to obtain temperature-responsive beads 2. The thickness of the temperature-responsive polymer coating was 62 nm. [Culture evaluation] The same procedure as in Example 6 was carried out except for using 200 mg of temperature-responsive beads 2. The cell proliferation rate was 710%, and the cell recovery rate was 89%.
• Example 8 [Preparation of temperature-responsive beads 3] 5 g of tertiary amine-modified polystyrene particles having fine pores (manufactured by Organo, product name Amberlyst A21, specific gravity 1.07) and 10 g of surface treatment agent 4 were added to a 25 mL recovery flask and allowed to stand for 2 hours. The solvent was then removed by reducing the pressure in an evaporator to obtain temperature-responsive beads 3. The thickness of the temperature-responsive polymer coating was 66 nm.
• Example 9 [Preparation of Surface Treatment Agent 5] 0.025 g of the temperature-responsive polymer 4 and 49.975 g of 1-methoxy-2-propanol were placed in a glass container and left to stand overnight to dissolve. The solution was then filtered through a 0.22 ⁇ m filter (manufactured by Millipore, hydrophilic filter) to prepare a surface treatment agent 5 with a polymer concentration of 0.05 wt %.
• 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 to stand overnight to dissolve. Then, the mixture was filtered through a 0.22 ⁇ m filter (manufactured by Millipore, hydrophilic filter) to prepare a surface treatment agent 6 with a polymer concentration of 1 wt %.
• 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 to stand overnight to dissolve. The solution was then filtered through a 0.22 ⁇ m filter (manufactured by Millipore, hydrophilic filter) to prepare a surface treatment agent 7 with a polymer concentration of 2 wt %.
• Example 12 Synthesis of temperature-responsive polymer 5
• n-butyl acrylate (BA) n-butyl acrylate
• 95.1 mg 300 ⁇ mol
• cyanomethyl dodecyl trithiocarbonate 95.1 mg
• azobisisobutyronitrile 95.1 mg
• 30 mL of tert-butyl alcohol 95.1 mg
• azobisisobutyronitrile 4.8 mg
• 30 tert-butyl alcohol tert-butyl alcohol
• the entire reaction solution was dropped into a 3 L beaker containing 2 L of pure water, and the precipitated yellow viscous material was collected.
• This viscous material was immersed in 2 L of pure water for 12 hours, then heated to 40°C to collect the solid, which was then vacuum dried at 100°C for 12 hours.
• This solid was dissolved in 300 mL of chloroform, after which 5 g of magnesium sulfate was added and the mixture was stirred at room temperature for 2 hours, 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 then vacuum dried at 100°C for 12 hours, yielding 21.2 g of temperature-responsive polymer 5.
• the Mn and Mw/Mn of the temperature-responsive polymer 5 were such that the composition ratio of BA/IPAAm was 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 to stand overnight to dissolve, then filtered through a 0.22 ⁇ m filter (manufactured by Millipore, hydrophilic filter) to obtain a surface treatment agent 8 with a polymer concentration of 0.2 wt %.
• Comparative Example 5 [Preparation of temperature-responsive beads 8] 5 g of polystyrene carrier (specific gravity 1.05) and 10 g of surface treatment agent 4 were added to a 25 mL recovery flask and allowed to stand for 2 hours. The solvent was then removed by reducing the pressure in an evaporator to obtain temperature-responsive beads 8. The thickness of the temperature-responsive polymer coating was 52 nm.
• Comparative Example 7 [Preparation of temperature-responsive beads 10] 5 g of sulfonic acid-modified polystyrene particles having fine pores (manufactured by Organo, product name Amberlite 200CT Na, specific gravity 1.04) and 10 g of surface treatment agent 4 were added to a 25 mL recovery flask and allowed to stand for 2 hours. The solvent was then removed by reducing the pressure in an evaporator to obtain temperature-responsive beads 10. The thickness of the temperature-responsive polymer coating was 55 nm. [Culture evaluation] The same procedure as in Example 6 was carried out except that 120 mg of temperature-responsive beads 10 were used. After the culture, the cells did not adhere to the beads, and the cell proliferation rate was 90%.
• Comparative Example 8 [Preparation of temperature-responsive beads 11] 5 g of polystyrene particles having 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 to a 25 mL recovery flask and allowed to stand for 2 hours. The solvent was then removed by reducing the pressure in an evaporator to obtain temperature-responsive beads 11. The thickness of the temperature-responsive polymer coating was 59 nm.
• Example 6 The same procedure as in Example 6 was carried out except that 140 mg of temperature-responsive beads 11 were used. After the culture, the cells did not adhere to the beads, and the cell proliferation rate was 90%. Comparative Example 9 [Culture evaluation] The same procedure as in Example 6 was repeated except that 60 mg of calcium phosphate-modified non-porous polystyrene particles were used as beads. The cell proliferation rate was 590%, and the cell recovery rate was 3%.
• Comparative Example 10 [Culture evaluation] 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 beads. The cell proliferation rate was 730%, and the cell recovery rate was 4%. Comparative Example 11 [Culture evaluation] 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 amines was used as beads. The cell proliferation rate was 460%, and the cell recovery rate was 2%.
• Example 13 [Preparation of temperature-responsive beads 12] 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 to a 25 mL recovery flask and allowed to stand for 2 hours. The solvent was then removed by reducing the pressure with an evaporator to obtain temperature-responsive beads 12. The thickness of the temperature-responsive polymer coating was 50 nm.
• Example 14 [Preparation of temperature-responsive beads 13] 5 g of polystyrene particles having pores modified with quaternary ammonium chloride (manufactured by Sigma-Aldrich, product name AmberChrom 1x8 100-200 mesh, specific gravity 1.09) and 10 g of surface treatment agent 5 were added to a 25 mL recovery flask and allowed to stand for 2 hours. The solvent was then removed by reducing the pressure with an evaporator to obtain temperature-responsive beads 13. The thickness of the temperature-responsive polymer coating was 12 nm.
• Example 15 [Preparation of temperature-responsive beads 14] 5 g of polystyrene particles having pores modified with quaternary ammonium chloride (manufactured by Sigma-Aldrich, product name AmberChrom 1x8 100-200 mesh, specific gravity 1.09) and 10 g of surface treatment agent 4 were added to a 25 mL recovery flask and allowed to stand for 2 hours. The solvent was then removed by reducing the pressure with an evaporator to obtain temperature-responsive beads 14. The thickness of the temperature-responsive polymer coating was 61 nm.
• Example 16 [Preparation of temperature-responsive beads 15] 5 g of polystyrene particles having pores modified with quaternary ammonium chloride (manufactured by Sigma-Aldrich, product name AmberChrom 1x8 50-100 mesh, specific gravity 1.09) and 10 g of surface treatment agent 4 were added to a 25 mL recovery flask and allowed to stand for 2 hours. The solvent was then removed by reducing the pressure with an evaporator to obtain temperature-responsive beads 15. The thickness of the temperature-responsive polymer coating was 44 nm.
• Comparative Example 12 [Preparation of temperature-responsive beads 16] 5 g of polystyrene particles having pores modified with quaternary ammonium chloride (manufactured by Sigma-Aldrich, product name AmberChrom 1x8 100-200 mesh, specific gravity 1.09) and 10 g of surface treatment agent 9 were added to a 25 mL recovery flask and allowed to stand for 2 hours. The solvent was then removed by reducing the pressure with an evaporator to obtain temperature-responsive beads 16. The temperature-responsive polymer coating had a thickness of 5 nm.

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