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WO2024253069A1 - Microsupport thermosensible - Google Patents

Microsupport thermosensible Download PDF

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Publication number
WO2024253069A1
WO2024253069A1 PCT/JP2024/020256 JP2024020256W WO2024253069A1 WO 2024253069 A1 WO2024253069 A1 WO 2024253069A1 JP 2024020256 W JP2024020256 W JP 2024020256W WO 2024253069 A1 WO2024253069 A1 WO 2024253069A1
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WO
WIPO (PCT)
Prior art keywords
cells
temperature
cooling
microcarrier
polymer
Prior art date
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Pending
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PCT/JP2024/020256
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English (en)
Japanese (ja)
Inventor
伸哉 今富
聖也 平床
大空 池谷
栄鎮 金
政浩 林
聡文 最上
博之 伊藤
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Tosoh Corp
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Tosoh Corp
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Publication date
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Priority to JP2025526105A priority Critical patent/JPWO2024253069A1/ja
Publication of WO2024253069A1 publication Critical patent/WO2024253069A1/fr
Anticipated expiration legal-status Critical
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    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08F—MACROMOLECULAR COMPOUNDS OBTAINED BY REACTIONS ONLY INVOLVING CARBON-TO-CARBON UNSATURATED BONDS
    • C08F293/00—Macromolecular compounds obtained by polymerisation on to a macromolecule having groups capable of inducing the formation of new polymer chains bound exclusively at one or both ends of the starting macromolecule
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08J—WORKING-UP; GENERAL PROCESSES OF COMPOUNDING; AFTER-TREATMENT NOT COVERED BY SUBCLASSES C08B, C08C, C08F, C08G or C08H
    • C08J3/00—Processes of treating or compounding macromolecular substances
    • C08J3/12—Powdering or granulating
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M3/00—Tissue, human, animal or plant cell, or virus culture apparatus
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • 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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • 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

Definitions

  • the present invention relates to a temperature-responsive microcarrier consisting of beads coated with a polymer coating film that contains a temperature-responsive polymer and a negatively charged polymer.
  • Patent Document 1 describes a microcarrier made of beads made from synthetic polymers and natural polymers such as polysaccharides. By placing the microcarrier in a container containing a cell suspension and culturing it, the cells grow on the surface of the microcarrier, and cell growth per unit volume of medium can be increased.
  • the temperature-responsive microcarrier described in Patent Document 2 when using the temperature-responsive microcarrier described in Patent Document 2, cells can be detached from the microcarrier surface by cooling without the need for proteolytic enzymes, simplifying the process.
  • the temperature-responsive microcarrier described in Patent Document 2 does not always detach cells sufficiently, and there is a demand for a temperature-responsive microcarrier that exhibits more suitable temperature responsiveness.
  • the present inventors have conducted extensive research and found that a suitable temperature responsiveness can be achieved by using a microcarrier made of beads coated with a polymer coating film containing a temperature-responsive polymer having a lower critical solution temperature of 0° C. to 50° C. and a negatively charged polymer, and have thus completed the present invention. That is, the present invention includes the following aspects. ⁇ 1> A microcarrier comprising beads coated with a polymer coating film containing a temperature-responsive polymer having a lower critical solution temperature of 0°C to 50°C and a negatively charged polymer. ⁇ 2> The microcarrier according to ⁇ 1>, wherein the proportion of the negatively charged polymer is 0.1 to 20 wt %.
  • ⁇ 3> The microcarrier according to ⁇ 1> or ⁇ 2>, wherein the negatively charged polymer contains a carboxy group.
  • ⁇ 4> The microcarrier according to any one of ⁇ 1> to ⁇ 3>, wherein the ratio of carboxyl groups in the negatively charged polymer is 10 to 50 wt %.
  • ⁇ 5> The microcarrier according to any one of ⁇ 1> to ⁇ 4>, wherein the bead surface is cationic.
  • ⁇ 6> The microcarrier according to any one of ⁇ 1> to ⁇ 5>, wherein the beads are modified with a functional group having a positive charge.
  • ⁇ 7> The microcarrier according to any one of ⁇ 1> to ⁇ 6>, wherein the beads are trimethylammonium chloride-modified polystyrene.
  • ⁇ 8> The microcarrier according to any one of ⁇ 1> to ⁇ 7>, wherein the particle size of the beads is 20 ⁇ m to 1000 ⁇ m.
  • ⁇ 9> The microcarrier according to any one of ⁇ 1> to ⁇ 8>, wherein the temperature-responsive polymer is a block copolymer containing the following blocks (A), (B), and (C): (A) A polymer block having an HLB value (Griffin method) in the range of 7 or more and 20 or less.
  • HLB value Griffin method
  • (B) A polymer block having an HLB value (Griffin method) in the range of 0 or more and less than 7.
  • C A temperature-responsive polymer block having a lower critical solution temperature (LCST) in water in the range of 0°C to 50°C.
  • LCST lower critical solution temperature
  • ⁇ 10> A method for producing a microcarrier according to any one of ⁇ 1> to ⁇ 9>.
  • ⁇ 11> A method for producing a microcarrier according to any one of ⁇ 1> to ⁇ 9>, comprising a step of coating the beads with a polymer coating film containing a temperature-responsive polymer having a lower critical solution temperature of 0°C to 50°C and a polymer having a negative charge.
  • ⁇ 12> A cell culture method using the microcarrier according to any one of ⁇ 1> to ⁇ 9>.
  • a cell culture method comprising the step of culturing cells in a medium containing the microcarrier according to any one of ⁇ 1> to ⁇ 9>.
  • a microcarrier consisting of beads coated with a polymer coating film containing a temperature-responsive polymer with a lower critical dissolution temperature between 0°C and 50°C and a negatively charged polymer can exhibit suitable temperature responsiveness.
  • the present invention is a microcarrier consisting of beads coated with a polymer coating film that contains a temperature-responsive polymer with a lower critical solution temperature between 0°C and 50°C and a negatively charged polymer.
  • the temperature-responsive polymer in the present invention is not particularly limited as long as the compatibility changes when the lower critical solution temperature is from 0° C. to 50° C. However, in order to significantly exhibit the change in compatibility, it is preferable that the temperature-responsive polymer is a block copolymer.
  • An example of the block copolymer is a block copolymer containing the following blocks (A), (B), and (C).
  • B A polymer block having an HLB value (Griffin method) in the range of 0 or more and less than 7.
  • C A temperature-responsive polymer block having a lower critical solution temperature (LCST) in water in the range of 0°C to 50°C.
  • the HLB value (Hydrophile-Lipophile Balance: HLB) is a value expressing the degree of affinity for water and oil, as described in W. C. Griffin, Journal of the Society of Cosmetic Chemists, 1, 311 (1949). It takes a value from 0 to 20, and the closer to 0, the higher the hydrophobicity, and the closer to 20, the higher the hydrophilicity.
  • Methods for calculating the HLB value by a formula include the Atlas method, the Griffin method, the Davis method, and the Kawakami method.
  • the value calculated by the Griffin method is used, and the value is calculated by the following formula based on the formula weight of the hydrophilic portion in the repeating unit of each block constituting the block copolymer of the present invention and the total formula weight of the repeating units.
  • HLB value 20 x (formula weight of hydrophilic portion in repeating unit) ⁇ (total formula weight of repeating units)
  • hydrophilic moieties in the repeating units of each block include a sulfone moiety (-SO 3 -), a phosphono group (-PO 3 -), a carboxyl group (-COOH), an ester group (-COO-), an amide group (-CONH-), an imide group (-CON-), an aldehyde group (-CHO), a carbonyl group (-CO-), a hydroxyl group (-OH), an amino group (-NH 2 ), an acetyl group (-COCH 3 ), an ethyleneamine group (-CH 2 CH 2 N-), an ethyleneoxy group (-CH 2 CH 2 O-), an alkali metal ion, an alkaline earth metal ion, an ammonium ion, a halide ion, and an acetate ion.
  • the atoms constituting the hydrophilic portion must not overlap with the atoms constituting other hydrophilic portions.
  • An example of calculating the HLB value in a repeating unit is shown below.
  • the hydrophilic portion is one part ester portion, one part phosphono group portion, and one part ethyleneamine portion, and the molecular weight of the hydrophilic portion is 181.04, so the HLB value is 12.3.
  • the hydrophilic portion is one part ester portion and one part ethyleneamine portion, and the molecular weight of the hydrophilic portion is 86.07, so the HLB value is 11.0.
  • the hydrophilic portion is one part ester portion, and the molecular weight of the hydrophilic portion is 44.01, so the HLB value is 8.8.
  • the hydrophilic portion is one part ester, and the molecular weight of the hydrophilic portion is 44.01, so the HLB value is 6.2.
  • the lower critical solution temperature is the temperature below which a polymer dissolves in water to form a transparent solution, but above which it becomes insoluble and either becomes cloudy or precipitates, resulting in phase separation.
  • temperature-responsive polymers include homopolymers composed only of component (C), copolymers composed of components (A) and (C), and copolymers composed of components (B) and (C).
  • the copolymers may be random or block copolymers.
  • the number average molecular weight of the temperature-responsive polymer is not particularly limited, but an example is 1,000 to 1,000,000, preferably 5,000 to 500,000, and more preferably 10,000 to 200,000.
  • a negatively charged polymer is a polymer having a functional group that ionizes in water and exhibits anionic properties.
  • the functional group that exhibits anionic properties examples include hydroxyl groups, carboxy groups, and sulfo groups. Among these, carboxy groups are preferred because of the ease of controlling the acidity.
  • the repeating units of the negatively charged polymer examples include vinylphenol, acrylic acid, methacrylic acid, and styrene sulfonic acid.
  • the negatively charged polymer may also contain repeating units of a polymer that does not have a negative charge, and an example is a copolymer of styrene sulfonic acid and styrene.
  • the copolymer may also be a random copolymer or a block copolymer.
  • the functional group of the negatively charged polymer is a carboxy group
  • the proportion of carboxy groups in the negatively charged polymer is 10 to 50 wt%, preferably 15 to 40 wt%, and more preferably 20 to 30 wt%.
  • the number average molecular weight of the negatively charged polymer is not particularly limited, but an example is 1,000 to 1,000,000, preferably 5,000 to 500,000, and more preferably 10,000 to 200,000.
  • beads are powder particles.
  • the particle size of the beads there is no particular limitation on the particle size of the beads, and examples thereof are 1 ⁇ m to 10 mm, 5 ⁇ m to 5 mm, and 20 ⁇ m to 1000 ⁇ m.
  • the shape of the beads there is no particular limitation on the shape of the beads, and examples thereof include a sphere, a rectangular parallelepiped, a cylindrical column, a cylindrical shape, and a star shape.
  • the true density of the beads but the higher the density of the polymer solution used in producing the temperature-responsive microcarrier, the more preferable it is, and an example thereof is 0.9 to 20.0 g/cm 2.
  • the beads may or may not have pores.
  • the specific surface area per dry weight of the beads there is no particular limitation on the specific surface area per dry weight of the beads, and an example thereof is 10 to 100,000 cm 2 /g.
  • the specific surface area per dry weight can be measured by the BET (Brunauer Emmett Teller) method, but in this application, it is calculated from the average particle size and particle specific gravity obtained from SEM images.
  • the material of the beads there is no particular limit to the material of the beads, and various materials can be used, such as metals, metal oxides, inorganic salts, synthetic polymers, biopolymers, or composites of these.
  • the surface of the beads it is preferable that the surface is cationic or modified with a positively charged functional group in order to enhance cell proliferation.
  • positively charged functional groups include ammonium groups and ammonium group derivatives.
  • Specific examples of beads include polystyrene, and trimethylammonium chloride-modified polystyrene is preferred.
  • the method for producing the temperature-responsive microcarrier is not particularly limited, but examples include a general method of physically coating the beads by adsorbing a polymer, or a chemical coating method by fixing a polymer to the beads by covalent bonding.
  • the coating method is not particularly limited, and examples include the impregnation method, immersion method, and Henschel mixer method. In the impregnation method and immersion method, the beads are immersed or sprayed with a polymer solution, and the solvent is removed by reducing the pressure to obtain a temperature-responsive microcarrier.
  • the beads are stirred with an impeller in a reaction vessel equipped with an impeller, and a polymer coating solution is added while blowing a drying gas into the reaction vessel, thereby obtaining a temperature-responsive microcarrier in a short time.
  • the Henschel mixer method and the impregnation method have the advantage that the entire amount of the polymer solution used can be adsorbed, making it easy to adjust the composition ratio and coating amount of the temperature-responsive polymer and the negatively charged polymer.
  • the polymer is fixed to the beads by a chemical covalent bond, there is no particular limitation on the type of chemical covalent bond, examples of which include ester bonds, amide bonds, ether bonds, and thioether bonds.
  • Temperature-responsive microcarriers can be obtained by removing unreacted compounds by washing after the reaction.
  • the drying gas there is no particular limitation on the drying gas, and any gas that is not reactive with the beads and polymer coating solution, such as nitrogen, air, argon, and neon, can be used.
  • the method for producing a temperature-responsive microcarrier may be, for example, a method characterized by comprising a step of coating beads with a polymer coating film containing a temperature-responsive polymer having a lower critical solution temperature of 0°C to 50°C and a polymer having a negative charge.
  • the proportion of the negatively charged polymer in the temperature-responsive microcarrier is not particularly limited, but is, for example, 0.1 to 20 wt %, and in order to exhibit suitable temperature responsiveness, is preferably 0.3 to 10 wt %, more preferably 0.5 to 5 wt %, even more preferably 0.5 to 4 wt %, and particularly preferably 0.5 to 3 wt %.
  • the temperature-responsive microcarriers can be used for cell culture, particularly for cell culture of adherent cells.
  • Adherent cells are cells that grow while adhering to the surface of a cell culture substrate such as a microcarrier.
  • a cell culture substrate such as a microcarrier.
  • adherent cells There are no particular limitations on the origin of the adherent cells to be cultured, but examples include mammals such as humans, monkeys, dogs, cats, rabbits, rats, nude mice, mice, guinea pigs, pigs, sheep, Chinese hamsters, and cows, and birds such as chickens and ducks.
  • the type of cells (adherent cells) to be cultured is not particularly limited, and examples thereof include human or animal-derived mesenchymal stem cells, CHO cells derived from Chinese hamster ovaries, VERO cells derived from African green monkey kidney epithelium, MDCK cells derived from dog kidneys, MRC-5 cells derived from normal human fetal lung tissue, CEF cells derived from chicken fetuses, BHK cells derived from hamster kidneys, L929 cells derived from mouse connective tissue, HEK293 cells derived from human fetal kidneys, MDBK cells derived from bovine kidneys, CRFK cells derived from cat kidneys, CPK cells derived from guinea pig kidneys, HeLa cells derived from human cervical cancer, and further, epithelial cells and endothelial cells constituting each tissue and organ in the body, skeletal muscle cells that exhibit contractility, smooth muscle cells, cardiac muscle cells, neuron cells that constitute the nervous system, glial cells, fibros,
  • cell lines in which the above-mentioned cell types have been immortalized.
  • Other examples include cells contained in blood, lymph, cerebrospinal fluid, sputum, urine, or feces, and microorganisms, viruses, protozoa, etc. present in the body or in the environment.
  • the type of medium used it may be a serum medium or a serum-free medium, and can be selected appropriately depending on the type of cell.
  • the type of basal medium used in the culture of the present invention is not particularly limited, and examples of the medium that can be used include MEM, ⁇ MEM, DMEM, EMEM, GMEM, DMEM/Ham's F-12, Ham's F-12, Ham's F-10, Medium 199, and RPMI 1640.
  • the medium may be a serum medium or a serum-free medium, and the serum used is not particularly limited, and examples of the serum that can be used include fetal bovine serum (FBS), calf serum, adult bovine serum, horse serum, sheep serum, goat serum, pig serum, chicken serum, rabbit serum, and human serum.
  • FBS fetal bovine serum
  • the complete medium may or may not contain antibiotics, and can be selected appropriately depending on the type of cells.
  • the cells may be cultured under stationary conditions or under agitated conditions.
  • the agitation speed there is no particular limitation on the agitation speed, and one example is 1 to 1000 rpm.
  • the culture temperature during culture but it is preferable to perform the culture at or above the LCST, which can provide an environment in which the surface of the temperature-responsive polymer-coated microcarrier becomes hydrophobic and cells can easily adhere. After culture, the cells on the microcarrier can be recovered by cooling to a temperature below the LCST.
  • the cells may be directly cooled using a cooler, or the medium may be replaced with a cooling liquid.
  • the cooling liquid may be a basal medium, a complete medium containing growth factors and serum, or a phosphate buffer, but by selecting a phosphate buffer, the cells are easily detached from the microcarrier. If a chelating agent such as EDTA or EGTA is introduced into the phosphate buffer for cooling, the cells are more easily detached from the microcarrier as single cells.
  • single cells mean that the cells have left the state in which they were connected to each other and have been separated into individual cells.
  • the concentration of the chelating agent in the phosphate buffer is not particularly limited and is between 1 ⁇ M and 1 mM.
  • the cooling liquid it is preferable for the cooling liquid to have a flow, as this can promote cooling of the temperature-responsive microcarriers. For example, a flow can be obtained by stirring or pipetting.
  • the cell culture method using the temperature-responsive microcarrier of the present invention may be, for example, a method characterized by including a step of culturing cells in a medium containing the microcarrier of the present invention.
  • the cell culture method using the temperature-responsive microcarrier of the present invention may further include a cooling cell detachment step of detaching the cells from the microcarrier using a cooling liquid containing a chelating agent.
  • the culture performance of the temperature-responsive microcarrier of the present invention is evaluated by the total cell number and the cooling detachment rate. If the total cell number is equal to or lower than the total cell number cultured in a current general-purpose microcarrier with the same culture area, it is judged to have poor culture performance.
  • the current general-purpose microcarrier is Cytodex1 with a cationic surface from Cytiva and/or Cellbind with an anionic surface from Corning.
  • the method for evaluating the temperature-responsive cooling detachment rate there is no particular limitation on the method for evaluating the temperature-responsive cooling detachment rate, but as an example, (1) the number of cells recovered by cooling is measured, and (2) the number of cells that were not detached by cooling is measured using a protease, and the number of cells in (1) is divided by the sum of the number of cells in (1) and (2), and the result is multiplied by 100 to evaluate the cooling detachment rate.
  • the method for measuring the number of cells but it can be evaluated using a hemocytometer or the like.
  • the GPC device used was a Tosoh Corporation HLC-8320GPC, and two Tosoh Corporation TSKgel SuperAWM-H columns were used. The column temperature was set to 40°C, and the eluent was 2,2,2-trifluoroethanol containing 10 mM sodium trifluoroacetate.
  • the measurement sample was prepared at 1.0 mg/mL and measured.
  • polymethylmethacrylate Sigma-Aldrich
  • 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 in an evaporator and purified by reprecipitation with hexane.
  • the precipitate was collected by filtration and dried under reduced pressure to obtain 5.805 g of polymer MBI.
  • ⁇ Polymer CStS having negative charge> 1.156 g (8 mmol) of p-carboxystyrene (CSt) and 1.271 g (12 mmol) of styrene (St) were added to a 100 mL two-neck flask, and 3.3 mg (20 ⁇ mol) of azobisisobutyronitrile and 20 mL of tert-butyl alcohol were added. After argon gas replacement, the mixture was heated and stirred at 64° C. for 24 hours. The reaction solution was purified by reprecipitation with n-heptane and dried under reduced pressure to obtain 0.92 g of polymer CStS. The composition ratio of polymer CStS was CSt/St 33/67 (mol%), the number average molecular weight Mn was 106,000, and the molecular weight distribution Mw/Mn was 1.84.
  • the rotary evaporator was a Tokyo Rika Kikai N-1300
  • the small cooling water circulation device was a Tokyo Rika Kikai CCA-1112A
  • the diaphragm type vacuum pump was a Tokyo Rika Kikai NVP-2100V
  • the water bath was a Tokyo Rika Kikai OSB-2200.
  • Water was used as the fluid for the small cooling water circulation device, and the temperature was set to 3°C. The temperature of the water bath was set to 50°C.
  • Example 1 10 g of 1-methoxy-2-propanol solution of 0.050 wt% temperature-responsive block copolymer MBI and 10 g of 1-methoxy-2-propanol solution of 0.0025 wt% polymer CStS were added to a 200 mL flask. 10 g of dried polystyrene beads (surface: cationic, average specific gravity: 1.08) with a diameter of 130 ⁇ m were further added and allowed to stand for 1 minute. The flask was placed in an evaporator, the stirring speed was set to 60 rpm, and the mixture was subjected to constant pressure treatment at 12,000 Pa for 1 minute.
  • the mixture was further subjected to constant pressure treatment at 10,000 Pa for 1 minute, constant pressure treatment at 8,000 Pa for 1 minute, constant pressure treatment at 6,000 Pa for 2 minutes, and constant pressure treatment at 4,000 Pa for 3 minutes.
  • the stirring speed was set to 120 rpm, and the mixture was finally subjected to vacuum impregnation treatment by reducing the pressure to 1,500 Pa by treating for 30 minutes with the vacuum pump at its maximum output.
  • the flask was removed from the evaporator, 150 mL of ion-exchanged water was added, ultrasonic waves were applied, and the flask was left to stand for 10 minutes.
  • the beads collected by filtration were transferred to a 200 mL flask, and the stirring speed in the evaporator was set to 120 rpm.
  • the vacuum pump was set to maximum output and the flask was left to stand for 30 minutes to obtain temperature-responsive microcarrier 1, which contains 4.76 wt% of negatively charged polymer.
  • ⁇ Human bone marrow-derived mesenchymal stem cell culture> To a 30 mL single-use bioreactor manufactured by Able, 204 mg (culture area 87 cm2 ) of temperature-responsive microcarrier 1 and 30 mL of mesenchymal stem cell proliferation medium 2 manufactured by PromoCell were added. Furthermore, 5 x 105 cells of human bone marrow-derived mesenchymal stem cells (passage number 2) manufactured by Lonza were added and cultured for 6 days under stirring conditions of 100 rpm in an incubator with a 5 vol% CO2 atmosphere at 37°C.
  • the 30 mL single-use bioreactor was transferred to a safety cabinet at room temperature, left to settle the microcarriers, 22.5 mL of the medium was removed, and 22.5 mL of phosphate buffered saline (PBS(-)) containing 1 mM EDTA at 4 ° C. without calcium and magnesium was added and left to stand for 10 minutes. After standing, the mixture was stirred at 300 rpm for 3 minutes at room temperature.
  • the cell suspension was passed through a 50 mL tube with a cell strainer with a 70 ⁇ m opening, and 5 mL of PBS(-) was added twice on the cell strainer, the cells were collected in the tube, concentrated by centrifugation, and the cell number was counted.
  • the number of cells collected by cooling was 70.8 ⁇ 10 5 cells.
  • the microcarriers on the cell strainer were then transferred to a 15 mL tube, 5 mL of TrypLE Express Enzyme (manufactured by Thermo Fisher Scientific) was added, and the tube was left to stand for 5 minutes in a 5 vol% CO2 atmosphere at 37 °C.
  • the cell suspension was passed through a 50 mL tube equipped with a cell strainer with a 70 ⁇ m opening, and 5 mL of PBS (-) was added twice onto the cell strainer to collect the cells in the tube, which were then concentrated by centrifugation to count the number of cells.
  • the number of cells that could not be collected by cooling was 22.1 ⁇ 105.
  • the cooling detachment rate was 76%.
  • Example 2 10 g of 1-methoxy-2-propanol solution of 0.050 wt% temperature-responsive block copolymer MBI and 10 g of 1-methoxy-2-propanol solution of 0.0015 wt% polymer CStS were added to a 200 mL flask. 10 g of dried polystyrene beads (surface: cationic, average specific gravity: 1.08) with a diameter of 130 ⁇ m were added and left to stand for 1 minute. The flask was placed in an evaporator, the stirring speed was set to 60 rpm, and the mixture was treated at a constant pressure of 12,000 Pa for 1 minute.
  • the mixture was further treated at a constant pressure of 10,000 Pa for 1 minute, at 8,000 Pa for 1 minute, at 6,000 Pa for 2 minutes, and at 4,000 Pa for 3 minutes.
  • the stirring speed was set to 120 rpm, and the mixture was finally treated for 30 minutes with the vacuum pump at its maximum output, reducing the pressure to 1,500 Pa and performing vacuum impregnation.
  • the flask was removed from the evaporator, 150 mL of ion-exchanged water was added, and the flask was left to stand for 10 minutes while being exposed to ultrasonic waves.
  • the beads collected by filtration were transferred to a 200 mL flask, and the stirring speed in the evaporator was set to 120 rpm.
  • the vacuum pump was set to maximum output and the flask was left to stand for 30 minutes to obtain temperature-responsive microcarrier 2, which contains 2.91 wt% of negatively charged polymer.
  • Human bone marrow-derived mesenchymal stem cells were cultured and the number of cells was counted in the same manner as in Example 1 ⁇ Human bone marrow-derived mesenchymal stem cell culture>, except that temperature-responsive microcarrier 2 was used.
  • the number of cells recovered by cooling was 78.2 x 105
  • the number of cells that could not be recovered by cooling was 18.2 x 105.
  • the cooling detachment rate was 81%.
  • Example 3 10 g of 1-methoxy-2-propanol solution of 0.050 wt% temperature-responsive block copolymer MBI and 10 g of 1-methoxy-2-propanol solution of 0.0005 wt% polymer CStS were added to a 200 mL flask. 10 g of dried polystyrene beads (surface: cationic, average specific gravity: 1.08) with a diameter of 130 ⁇ m were further added and allowed to stand for 1 minute. The flask was placed in an evaporator, the stirring speed was set to 60 rpm, and the mixture was subjected to constant pressure treatment at 12000 Pa for 1 minute.
  • the mixture was further subjected to constant pressure treatment at 10000 Pa for 1 minute, constant pressure treatment at 8000 Pa for 1 minute, constant pressure treatment at 6000 Pa for 2 minutes, and constant pressure treatment at 4000 Pa for 3 minutes.
  • the stirring speed was set to 120 rpm, and the vacuum pump was finally set to maximum output for 30 minutes to reduce the pressure to 1500 Pa, and vacuum impregnation treatment was performed.
  • the flask was removed from the evaporator, 150 mL of ion-exchanged water was added, ultrasonic waves were applied, and the flask was left to stand for 10 minutes.
  • the beads collected by filtration were transferred to a 200 mL flask, and the stirring speed in the evaporator was set to 120 rpm.
  • the vacuum pump was set to maximum output and the flask was left to stand for 30 minutes to obtain temperature-responsive microcarrier 3, which contains 0.99 wt% of negatively charged polymer.
  • Human bone marrow-derived mesenchymal stem cells were cultured and the number of cells was counted in the same manner as in Example 1 ⁇ Human bone marrow-derived mesenchymal stem cell culture>, except for using temperature-responsive microcarrier 3.
  • the number of cells recovered by cooling was 96.4 x 105
  • the number of cells that could not be recovered by cooling was 19.1 x 105.
  • the cooling detachment rate was 83%.
  • Example 4 10 g of 1-methoxy-2-propanol solution of 0.050 wt% temperature-responsive block copolymer MBI and 10 g of 1-methoxy-2-propanol solution of 0.00038 wt% polymer CStS were added to a 200 mL flask. 10 g of dried polystyrene beads (surface: cationic, average specific gravity: 1.08) with a diameter of 130 ⁇ m were further added and allowed to stand for 1 minute. The flask was placed in an evaporator, the stirring speed was set to 60 rpm, and the mixture was subjected to constant pressure treatment at 12000 Pa for 1 minute. The mixture was further subjected to constant pressure treatment at 10000 Pa for 1 minute, constant pressure treatment at 8000 Pa for 1 minute, constant pressure treatment at 6000 Pa for 2 minutes, and constant pressure treatment at 4000 Pa for 3 minutes, for vacuum impregnation treatment.
  • the flask was removed from the evaporator, 150 mL of ion-exchanged water was added, ultrasonic waves were applied, and the flask was left to stand for 10 minutes.
  • the beads collected by filtration were transferred to a 200 mL flask, and the stirring speed in the evaporator was set to 120 rpm.
  • the vacuum pump was set to maximum output and the flask was left to stand for 30 minutes to obtain temperature-responsive microcarrier 4, which contains 0.74 wt% of negatively charged polymer.
  • Human bone marrow-derived mesenchymal stem cells were cultured and the number of cells was counted in the same manner as in Example 1 ⁇ Human bone marrow-derived mesenchymal stem cell culture>, except that temperature-responsive microcarrier 4 was used.
  • the number of cells recovered by cooling was 93.4 x 105
  • the number of cells that could not be recovered by cooling was 3.6 x 105.
  • the cooling detachment rate was 96%.
  • Example 5 10 g of 1-methoxy-2-propanol solution of 0.050 wt% temperature-responsive block copolymer MBI and 10 g of 1-methoxy-2-propanol solution of 0.00025 wt% polymer CStS were added to a 200 mL flask. 10 g of dried polystyrene beads (surface: cationic, average specific gravity: 1.08) with a diameter of 130 ⁇ m were further added and allowed to stand for 1 minute. The flask was placed in an evaporator, the stirring speed was set to 60 rpm, and the mixture was subjected to constant pressure treatment at 12,000 Pa for 1 minute.
  • the mixture was further subjected to constant pressure treatment at 10,000 Pa for 1 minute, constant pressure treatment at 8,000 Pa for 1 minute, constant pressure treatment at 6,000 Pa for 2 minutes, and constant pressure treatment at 4,000 Pa for 3 minutes.
  • the stirring speed was set to 120 rpm, and the vacuum pump was finally set to maximum output for 30 minutes to reduce the pressure to 1,500 Pa, and vacuum impregnation treatment was performed.
  • the flask was removed from the evaporator, 150 mL of ion-exchanged water was added, ultrasonic waves were applied, and the flask was left to stand for 10 minutes.
  • the beads collected by filtration were transferred to a 200 mL flask, and the stirring speed in the evaporator was set to 120 rpm.
  • the vacuum pump was set to maximum output and the flask was left to stand for 30 minutes to obtain temperature-responsive microcarrier 5, which contains 0.50 wt% of negatively charged polymer.
  • Human bone marrow-derived mesenchymal stem cells were cultured and the number of cells was counted in the same manner as in Example 1 ⁇ Human bone marrow-derived mesenchymal stem cell culture>, except that temperature-responsive microcarrier 5 was used.
  • the number of cells recovered by cooling was 73.2 x 105
  • the number of cells that could not be recovered by cooling was 29.3 x 105.
  • the cooling detachment rate was 71%.
  • Example 6 12 g of 1-methoxy-2-propanol solution of 0.050 wt% temperature-responsive block copolymer MBI and 12 g of 1-methoxy-2-propanol solution of 0.00038 wt% polymer CStS were added to a 200 mL flask. 10 g of dried polystyrene beads (surface: cationic, average specific gravity: 1.08) with a diameter of 130 ⁇ m were added and allowed to stand for 1 minute. The flask was placed in an evaporator, the stirring speed was set to 60 rpm, and the mixture was subjected to constant pressure treatment at 12,000 Pa for 1 minute.
  • the mixture was further subjected to constant pressure treatment at 10,000 Pa for 1 minute, constant pressure treatment at 8,000 Pa for 1 minute, constant pressure treatment at 6,000 Pa for 2 minutes, and constant pressure treatment at 4,000 Pa for 3 minutes.
  • the stirring speed was set to 120 rpm, and the vacuum pump was set to maximum output for 30 minutes to reduce the pressure to 1,500 Pa, and vacuum impregnation treatment was performed.
  • the flask was removed from the evaporator, 150 mL of ion-exchanged water was added, and the mixture was exposed to ultrasound and left to stand for 10 minutes.
  • the beads collected by filtration were transferred to a 200 mL flask, and the stirring speed in the evaporator was set to 120 rpm.
  • the vacuum pump was set to maximum output and the mixture was treated for 30 minutes to obtain temperature-responsive microcarriers 6 with a negatively charged polymer ratio of 0.74 wt%.
  • Human bone marrow-derived mesenchymal stem cells were cultured and the number of cells was counted in the same manner as in Example 1 ⁇ Human bone marrow-derived mesenchymal stem cell culture>, except that temperature-responsive microcarriers 6 were used.
  • the number of cells recovered by cooling was 109.0 x 105
  • the number of cells that could not be recovered by cooling was 1.6 x 105.
  • the cooling detachment rate was 98%.
  • the mixture was further subjected to constant pressure treatment at 10,000 Pa for 1 minute, constant pressure treatment at 8,000 Pa for 1 minute, constant pressure treatment at 6,000 Pa for 2 minutes, and constant pressure treatment at 4,000 Pa for 3 minutes.
  • the stirring speed was set to 120 rpm, and the vacuum pump was set to maximum output for 30 minutes to reduce the pressure to 1,500 Pa, and vacuum impregnation treatment was performed.
  • the flask was removed from the evaporator, 150 mL of ion-exchanged water was added, ultrasonic waves were applied, and the flask was left to stand for 10 minutes.
  • the beads collected by filtration were transferred to a 200 mL flask, and the stirring speed in the evaporator was set to 120 rpm.
  • the vacuum pump was set to maximum output and the flask was left to stand for 30 minutes to obtain temperature-responsive microcarrier 7, which contains 0.74 wt% of negatively charged polymer.
  • Human bone marrow-derived mesenchymal stem cells were cultured and the number of cells was counted in the same manner as in Example 1 ⁇ Human bone marrow-derived mesenchymal stem cell culture>, except for using temperature-responsive microcarriers 7.
  • the number of cells recovered by cooling was 94.7 x 105 , and the number of cells that could not be recovered by cooling was 20.5 x 105.
  • the cooling detachment rate was 82%.
  • Example 8 In a 200 mL flask, 6 g of a 1-methoxy-2-propanol solution of 0.050 wt% temperature-responsive block copolymer MBI and 6 g of a 1-methoxy-2-propanol solution of 0.00038 wt% polymer CStS were added. Furthermore, 10 g of dried polystyrene beads (surface: cationic, average specific gravity: 1.08) having a diameter of 130 ⁇ m were added and left to stand for 1 minute. The flask was placed in an evaporator, the stirring speed was set to 60 rpm, and the mixture was subjected to constant pressure treatment at 12000 Pa for 1 minute.
  • a 1-methoxy-2-propanol solution of 0.050 wt% temperature-responsive block copolymer MBI and 6 g of a 1-methoxy-2-propanol solution of 0.00038 wt% polymer CStS were added. Furthermore, 10 g of dried polystyrene
  • constant pressure treatment was performed at 10000 Pa for 1 minute, constant pressure treatment at 8000 Pa for 1 minute, constant pressure treatment at 6000 Pa for 2 minutes, and constant pressure treatment at 4000 Pa for 3 minutes.
  • the stirring speed was set to 120 rpm, and finally, the vacuum pump was set to maximum output and treated for 30 minutes to reduce the pressure to 1500 Pa, and vacuum impregnation treatment was performed.
  • Human bone marrow-derived mesenchymal stem cells were cultured and the number of cells was counted in the same manner as in Example 1 ⁇ Human bone marrow-derived mesenchymal stem cell culture>, except that temperature-responsive microcarriers 8 were used.
  • the number of cells recovered by cooling was 84.7 x 105
  • the number of cells that could not be recovered by cooling was 28.6 x 105.
  • the cooling detachment rate was 75%.
  • Example 9 ⁇ VERO cell culture> 356 mg (culture area 150 cm 2 ) of temperature-responsive microcarrier 2 and 30 mL of DMEM (Dulbecco's Modified Eagle Medium, Wako Pure Chemical Industries) containing 10% fetal bovine serum (Biowest Co., Ltd.) and 1% antibiotic-antimycotic solution (Wako Pure Chemical Industries, Ltd.) were added to a 30 mL single-use bioreactor manufactured by Able Co., Ltd. Furthermore, 1 x 10 6 cells of VERO cells (passage number 16) manufactured by JCRB Cell Bank were added and cultured for 8 days under stirring conditions of 120 rpm in an incubator with a 5 vol% CO 2 atmosphere at 37°C.
  • DMEM Dynamic Eagle Medium
  • fetal bovine serum Biowest Co., Ltd.
  • antibiotic-antimycotic solution (Wako Pure Chemical Industries, Ltd.)
  • the 30 mL single-use bioreactor was transferred to a safety cabinet at room temperature, the cell suspension was passed through a 50 mL tube with a cell strainer with a mesh size of 70 ⁇ m, the floating cells were removed, and only the microcarriers to which the cells on the cell strainer were attached were collected, and phosphate buffered saline (PBS (-)) free of calcium and magnesium at 4 ° C was added and allowed to stand for 10 minutes. After standing, pipetting was performed 60 times, and the detached cells were collected by passing them through a 50 mL tube with a cell strainer with a mesh size of 70 ⁇ m, and the cells were concentrated by centrifugation and counted.
  • PBS (-) phosphate buffered saline
  • Example 10 ⁇ VERO cell culture> VERO cells were cultured and the number of cells was counted in the same manner as in ⁇ VERO cell culture> of Example 9, except that temperature-responsive microcarrier 3 was used.
  • the number of cells recovered by cooling was 11.0 x 106
  • the number of cells that could not be recovered by cooling was 1.8 x 106.
  • the cooling detachment rate was 86%.
  • Example 11 ⁇ VERO cell culture> VERO cells were cultured and the number of cells was counted in the same manner as in ⁇ VERO cell culture> of Example 9, except that temperature-responsive microcarrier 4 was used.
  • the number of cells recovered by cooling was 12.8 x 106
  • the number of cells that could not be recovered by cooling was 0.8 x 106.
  • the cooling detachment rate was 94%.
  • Comparative Example 1 10 g of 1-methoxy-2-propanol solution of 0.050 wt% temperature-responsive block copolymer MBI and 10 g of 1-methoxy-2-propanol solution of 0.050 wt% polymer CStS were added to a 200 mL flask. 10 g of dried polystyrene beads (surface: cationic, average specific gravity: 1.08) with a diameter of 130 ⁇ m were further added and allowed to stand for 1 minute. The flask was placed in an evaporator, the stirring speed was set to 60 rpm, and the mixture was subjected to constant pressure treatment at 12,000 Pa for 1 minute.
  • Human bone marrow-derived mesenchymal stem cells were cultured and the number of cells was counted in the same manner as in Example 1 ⁇ Human bone marrow-derived mesenchymal stem cell culture>, except that temperature-responsive microcarrier 9 was used.
  • the number of cells recovered by cooling was 22.3 x 105
  • the number of cells that could not be recovered by cooling was 89.0 x 105.
  • the cooling detachment rate was 20%.
  • Comparative Example 2 10 g of a 1-methoxy-2-propanol solution of 0.050 wt% temperature-responsive block copolymer MBI was added to a 200 mL flask. 10 g of dried polystyrene beads (surface: cationic, average specific gravity: 1.08) with a diameter of 130 ⁇ m was further added and allowed to stand for 1 minute. The flask was placed in an evaporator, the stirring speed was set to 60 rpm, and the mixture was subjected to constant pressure treatment at 12,000 Pa for 1 minute.
  • the mixture was further subjected to constant pressure treatment at 10,000 Pa for 1 minute, constant pressure treatment at 8,000 Pa for 1 minute, constant pressure treatment at 6,000 Pa for 2 minutes, and constant pressure treatment at 4,000 Pa for 3 minutes.
  • the stirring speed was set to 120 rpm, and the vacuum pump was set to maximum output for 30 minutes to reduce the pressure to 1,500 Pa, and vacuum impregnation treatment was performed.
  • the flask was removed from the evaporator, 150 mL of ion-exchanged water was added, and the flask was left to stand for 10 minutes while being exposed to ultrasonic waves.
  • the beads collected by filtration were transferred to a 200 mL flask, and the stirring speed in the evaporator was set to 120 rpm.
  • the vacuum pump was set to maximum output and the flask was left to stand for 30 minutes to obtain temperature-responsive microcarriers 10 with a negatively charged polymer ratio of 0.00 wt%.
  • Human bone marrow-derived mesenchymal stem cells were cultured and the number of cells was counted in the same manner as in Example 1 ⁇ Human bone marrow-derived mesenchymal stem cell culture>, except that the temperature-responsive microcarrier 10 was used.
  • the number of cells recovered by cooling was 39.3 x 105
  • the number of cells that could not be recovered by cooling was 10.7 x 105.
  • the cooling detachment rate was 79%, but the total number of cells was lower than the total number of cells cultured using the current general-purpose microcarrier described in Reference Example 1.
  • Comparative Example 3 10 g of 1-methoxy-2-propanol solution of 0.050 wt% polymer CStS was added to a 200 mL flask. 10 g of dried polystyrene beads (surface: cationic, average specific gravity: 1.08) with a diameter of 130 ⁇ m was added and allowed to stand for 1 minute. The flask was placed in an evaporator, the stirring speed was set to 60 rpm, and the mixture was subjected to constant pressure treatment at 12,000 Pa for 1 minute. The mixture was then subjected to constant pressure treatment at 10,000 Pa for 1 minute, constant pressure treatment at 8,000 Pa for 1 minute, constant pressure treatment at 6,000 Pa for 2 minutes, and constant pressure treatment at 4,000 Pa for 3 minutes. The stirring speed was set to 120 rpm, and the vacuum pump was set to maximum output for 30 minutes to reduce the pressure to 1,500 Pa, and vacuum impregnation treatment was performed.
  • the flask was removed from the evaporator, 150 mL of ion-exchanged water was added, ultrasonic waves were applied, and the flask was left to stand for 10 minutes.
  • the beads collected by filtration were transferred to a 200 mL flask, and the stirring speed in the evaporator was set to 120 rpm.
  • the vacuum pump was set to maximum output and the flask was left to stand for 30 minutes to obtain microcarriers 11 with a negatively charged polymer ratio of 100.00 wt%.
  • Human bone marrow-derived mesenchymal stem cells were cultured and the number of cells was counted in the same manner as in Example 1 ⁇ Human bone marrow-derived mesenchymal stem cell culture>, except for using the temperature-responsive microcarrier 11.
  • the number of cells recovered by cooling was 2.3 x 105
  • the number of cells that could not be recovered by cooling was 98.5 x 105.
  • the cooling detachment rate was 2%.
  • VERO cells were cultured and the number of cells was counted in the same manner as in ⁇ VERO cell culture> of Example 9, except that temperature-responsive microcarrier 11 was used.
  • the number of cells recovered by cooling was 1.4 x 106
  • the number of cells that could not be recovered by cooling was 11.1 x 106.
  • the cooling detachment rate was 85%.
  • Reference Example 1 Human bone marrow-derived mesenchymal stem cell culture> 20 mg (culture area 87 cm2 ) of autoclave-sterilized Cytodex1 (surface: cationic, average specific gravity: 1.03) was added to a 30 mL single-use bioreactor manufactured by Able Co., Ltd. Furthermore, 5 x 105 cells of human bone marrow-derived mesenchymal stem cells (passage number 2) manufactured by Lonza were added and cultured for 6 days under stirring conditions of 60 rpm in an incubator with a 5 vol% CO2 atmosphere at 37°C.
  • the 30 mL single-use bioreactor was transferred to a safety cabinet at room temperature, left to settle Cytodex1, 27 mL of medium was removed, and 27 mL of PBS(-) was added. Cytodex1 was left to settle, 27 mL of liquid was removed, 5 mL of TrypLE Express Enzyme (manufactured by Thermo Fisher Scientific) was added, and the mixture was left to stand for 5 minutes in a 5 vol% CO2 atmosphere at 37 ° C.
  • the cell suspension was passed through a 50 mL tube with a cell strainer with a 70 ⁇ m opening, and 5 mL of PBS(-) was added twice on the cell strainer, the cells were collected in the tube, concentrated by centrifugation, and the cell number was measured. The cell number was 51.7 ⁇ 10 5 cells.
  • Reference Example 2 ⁇ VERO cell culture> VERO cells were cultured and the number of cells was counted in the same manner as in ⁇ VERO cell culture> in Example 9, except that 34.1 mg (culture area 150 cm2 ) of the microcarrier of Reference Example 1 (Cytodex 1, surface: cationic, average specific gravity: 1.03) was used and the stirring speed was set to 60 rpm.
  • the number of cells recovered by cooling was 1.4 x 106
  • the number of cells that could not be recovered by cooling was 8.7 x 106.
  • the cooling detachment rate was 14%.
  • Reference Example 3 ⁇ VERO cell culture> 416 mg (culture area 150 cm 2 ) of autoclave-sterilized Cellbind (surface: anionic, average specific gravity: 1.026) was added to a 30 mL single-use bioreactor manufactured by Able. 30 mL of DMEM (Dulbecco's Modified Eagle Medium, Wako Pure Chemical Industries) containing 10% fetal bovine serum (Biowest) and 1% antibiotic-antimycotic solution (Wako Pure Chemical Industries) was then added. 1 ⁇ 10 6 cells of VERO cells (passage number 16) manufactured by JCRB Cell Bank were then added and cultured for 8 days at 37° C. in an incubator with a 5 vol% CO 2 atmosphere at 120 rpm.
  • DMEM Disulbecco's Modified Eagle Medium, Wako Pure Chemical Industries
  • VERO cells were cultured and the number of cells was counted in the same manner as in ⁇ VERO cell culture> in Example 9, except that Cellbind microcarriers were used.
  • the number of cells recovered by cooling was 1.4 x 106
  • the number of cells that could not be recovered by cooling was 14.1 x 106.
  • the cooling detachment rate was 9%.
  • Example 12 12 g of 1-methoxy-2-propanol solution of 0.050 wt% temperature-responsive block copolymer MBI and 12 g of 1-methoxy-2-propanol solution of 0.00038 wt% polymer CStS were added to a 200 mL flask. 10 g of dried polystyrene beads (surface: cationic, average specific gravity: 1.03) with a diameter of 130 ⁇ m were added and allowed to stand for 1 minute. The flask was placed in an evaporator, the stirring speed was set to 60 rpm, and the mixture was subjected to constant pressure treatment at 12,000 Pa for 1 minute.
  • the mixture was further subjected to constant pressure treatment at 10,000 Pa for 1 minute, constant pressure treatment at 8,000 Pa for 1 minute, constant pressure treatment at 6,000 Pa for 2 minutes, and constant pressure treatment at 4,000 Pa for 3 minutes.
  • the stirring speed was set to 120 rpm, and the vacuum pump was set to maximum output for 30 minutes to reduce the pressure to 1,500 Pa, and vacuum impregnation treatment was performed.
  • the flask was removed from the evaporator, 150 mL of ion-exchanged water was added, ultrasonic waves were applied, and the flask was left to stand for 10 minutes.
  • the beads collected by filtration were transferred to a 200 mL flask, and the stirring speed in the evaporator was set to 120 rpm.
  • the vacuum pump was set to maximum output and the flask was left to stand for 30 minutes to obtain temperature-responsive microcarriers 12 with a negatively charged polymer ratio of 0.74 wt%.
  • Human bone marrow-derived mesenchymal stem cells were cultured and the number of cells was counted in the same manner as in Example 1 ⁇ Human bone marrow-derived mesenchymal stem cell culture>, except that 195 mg (culture area 87 cm2 ) of temperature-responsive microcarriers 12 were used and the stirring speed was set to 60 rpm.
  • the number of cells recovered by cooling was 61.3 x 105 , and the number of cells that could not be recovered by cooling was 1.4 x 105.
  • the cooling detachment rate was 98%.
  • Example 13 A 500 mL separable flask was set and equipped with a PTFE (poly(1,1,2,2-tetrafluoroethylene)) stirring blade. 40 g of dried polystyrene beads (surface: cationic, average specific gravity: 1.08) with a diameter of 130 ⁇ m was added and purged with nitrogen gas in the flask while stirring at 100 rpm.
  • PTFE poly(1,1,2,2-tetrafluoroethylene)
  • Example 4 Human bone marrow-derived mesenchymal stem cell culture> Human bone marrow-derived mesenchymal stem cells were cultured and the number of cells was counted in the same manner as in Example 1 ⁇ Human bone marrow-derived mesenchymal stem cell culture>, except that 238 mg (culture area 87 cm2 ) of Cellbind (surface: anionic, average specific gravity: 1.026) microcarriers sterilized by autoclave were used and the stirring speed was set to 60 rpm. The number of cells recovered by cooling was 20.0 x 105 , and the number of cells that could not be recovered by cooling was 28.2 x 105. The cooling detachment rate was 59%.
  • Example 5 Human bone marrow-derived mesenchymal stem cell culture> Human bone marrow-derived mesenchymal stem cells were cultured and the number of cells was counted in the same manner as in Example 1 ⁇ Human bone marrow-derived mesenchymal stem cell culture>, except that 20 mg (culture area 87 cm2 ) of autoclave-sterilized Cytodex1 microcarriers (surface: cationic, average specific gravity: 1.03) was used and the stirring speed was set to 60 rpm. The number of cells recovered by cooling was 21.1 x 105 , and the number of cells that could not be recovered by cooling was 28.9 x 105. The cooling detachment rate was 42%.
  • Example 14 ⁇ VERO cell culture> VERO cells were cultured and the number of cells was counted in the same manner as in ⁇ VERO cell culture> of Example 9, except that 340 mg (culture area 150 cm2 ) of temperature-responsive microcarriers 13 were used and the stirring speed was set to 60 rpm. The number of cells recovered by cooling was 122.0 x 105 , and the number of cells that could not be recovered by cooling was 12.0 x 105. The cooling detachment rate was 91%.
  • Example 15 Serum-free culture of VERO cells 356 mg (culture area 150 cm 2 ) of temperature-responsive microcarrier 13 and 1 L of VP-SFM (1X) (Thermo Fisher Scientific, product number: 11681020) were added to a 30 mL single-use bioreactor manufactured by Able, and 20 mL of 200 mM L-Glutamine Solution (1X) (ATCC, product number: 30-2214) were added to the VP-SFM (1X) and 30 mL of a final medium with a final concentration of 20 mM were added. Furthermore, 1 x 10 6 cells of Vero cells (American Type Culture Collection (ATCC), product number: CCL-81) were added and cultured for 8 days at 37 ° C. in an incubator with a 5 vol % CO 2 atmosphere at 100 rpm stirring condition.
  • ATCC American Type Culture Collection
  • the 30 mL single-use bioreactor was transferred to a safety cabinet at room temperature, the cell suspension was passed through a 50 mL tube with a cell strainer with a mesh size of 70 ⁇ m, the floating cells were removed, and only the microcarriers to which the cells on the cell strainer were attached were collected, and phosphate buffered saline (PBS (-)) free of calcium and magnesium at 4 ° C was added and allowed to stand for 10 minutes. After standing, pipetting was performed 60 times, and the detached cells were collected by passing them through a 50 mL tube with a cell strainer with a mesh size of 70 ⁇ m, and the cells were concentrated by centrifugation and counted.
  • PBS (-) phosphate buffered saline
  • the number of cells collected by cooling was 46.5 ⁇ 10 5 cells. Furthermore, the microcarriers on the cell strainer were transferred to a 15 mL tube, 5 mL of CTS TrypLE Select Enzyme (manufactured by Thermo Fisher Scientific) was added, and the mixture was allowed to stand for 5 minutes in a 5 vol% CO 2 atmosphere at 37 ° C. The cell suspension was passed through a 50 mL tube equipped with a cell strainer with a mesh size of 70 ⁇ m, the cells were collected, concentrated by centrifugation, and the cell number was counted. The number of cells that could not be collected by cooling was 4.2 ⁇ 10 5 cells. The cooling detachment rate was 92%.
  • Example 16 ⁇ BHK-21 cell culture> 340 mg (culture area 150 cm 2 ) of temperature-responsive microcarrier 12 and 30 mL of complete medium of DMEM (Dulbecco's Modified Eagle Medium, Wako Pure Chemical Industries) containing 10% fetal bovine serum (FBS, Biowest Co., Ltd.) and 1% antibiotic-antimycotic solution (Wako Pure Chemical Industries, Ltd.) were added to a 30 mL single-use bioreactor manufactured by Able Co., Ltd. Furthermore, 1 x 10 6 cells of BHK-21 cells (ATCC, product number: CCL-10) were added and cultured for 7 days at 60 rpm stirring in an incubator with a 5 vol% CO 2 atmosphere at 37°C.
  • DMEM Dynamic fetal bovine serum
  • the 30 mL single-use bioreactor was transferred to a safety cabinet at room temperature, the cell suspension was passed through a 50 mL tube equipped with a cell strainer with a mesh size of 70 ⁇ m, the floating cells were removed, and only the microcarriers to which the cells on the cell strainer were attached were collected, and phosphate buffered saline (PBS (-)) containing no calcium or magnesium at 4 ° C was added and allowed to stand for 10 minutes. After standing, pipetting was performed 60 times, and the detached cells were collected by passing them through a 50 mL tube equipped with a cell strainer with a mesh size of 70 ⁇ m, and the cells were concentrated by centrifugation and counted.
  • PBS (-) phosphate buffered saline
  • the number of cells collected by cooling was 148.0 ⁇ 10 5 cells.
  • the microcarriers on the cell strainer were then transferred to a 15 mL tube, 5 mL of CTS TrypLE Select Enzyme (manufactured by Thermo Fisher Scientific) was added, and the mixture was allowed to stand for 5 minutes in a 5 vol% CO2 atmosphere at 37 °C.
  • the cell suspension was passed through a 50 mL tube equipped with a cell strainer with a 70 ⁇ m opening, the cells were collected, and the cells were concentrated by centrifugation to count the number of cells.
  • the number of cells that could not be collected by cooling was 19.0 ⁇ 105.
  • the cooling detachment rate was 89%.
  • Example 17 ⁇ CRFK cell culture> 356 mg (culture area 150 cm 2 ) of temperature-responsive microcarrier 4 and 30 mL of complete medium of EMEM (ATCC-formulated Eagle's Minimum Essential Medium, ATCC, product number: 30-2003) containing 10% horse serum (HS, Sigma-Aldrich Japan, LLC) were added to a 30 mL single-use bioreactor manufactured by Able Co., Ltd. Furthermore, 0.8 x 10 6 cells of CRFK cells (ATCC, product number: CCL-94) were added and cultured for 7 days at 100 rpm stirring in an incubator with a 5 vol% CO 2 atmosphere at 37°C.
  • EMEM ATCC-formulated Eagle's Minimum Essential Medium, ATCC, product number: 30-2003
  • horse serum HS, Sigma-Aldrich Japan, LLC
  • the 30 mL single-use bioreactor was transferred to a safety cabinet at room temperature, the cell suspension was passed through a 50 mL tube equipped with a cell strainer with a mesh size of 70 ⁇ m, the floating cells were removed, and only the microcarriers to which the cells on the cell strainer were attached were collected, and phosphate buffered saline (PBS (-)) containing no calcium or magnesium at 4 ° C was added and allowed to stand for 10 minutes. After standing, pipetting was performed 60 times, and the detached cells were collected by passing them through a 50 mL tube equipped with a cell strainer with a mesh size of 70 ⁇ m, and the cells were concentrated by centrifugation and counted.
  • PBS (-) phosphate buffered saline
  • the number of cells collected by cooling was 26.8.0 ⁇ 10 5 cells.
  • the microcarriers on the cell strainer were then transferred to a 15 mL tube, 5 mL of CTS TrypLE Select Enzyme (manufactured by Thermo Fisher Scientific) was added, and the mixture was allowed to stand for 5 minutes in a 5 vol% CO2 atmosphere at 37 °C.
  • the cell suspension was passed through a 50 mL tube equipped with a cell strainer with a 70 ⁇ m opening, the cells were collected, and the cells were concentrated by centrifugation to count the number of cells.
  • the number of cells that could not be collected by cooling was 3.1 ⁇ 10 5 cells.
  • the cooling detachment rate was 90%.
  • Example 18 CRFK cell culture> CRFK cells were cultured and the number of cells was counted in the same manner as in ⁇ CRFK cell culture> in Example 17, except that 340 mg of microcarriers 12 (culture area 150 cm 2 ) was used and the stirring speed was set to 60 rpm. The number of cells recovered by cooling was 13.8 ⁇ 10 5 , and the number of cells that could not be recovered by cooling was 3.8 ⁇ 10 5 . The cooling detachment rate was 78%.
  • Example 19 CRFK cell culture> CRFK cells were cultured and the number of cells was counted in the same manner as in ⁇ CRFK cell culture> in Example 17, except that 356 mg of microcarrier 13 (culture area 150 cm 2 ) was used. The number of cells recovered by cooling was 21.2 ⁇ 10 5 , and the number of cells that could not be recovered by cooling was 4.6 ⁇ 10 5 . The cooling detachment rate was 82%.
  • Example 20 ⁇ MDCK cell culture> 356 mg (culture area 150 cm 2 ) of temperature-responsive microcarrier 4 and 30 mL of complete medium of DMEM (Dulbecco's Modified Eagle Medium, Wako Pure Chemical Industries) containing 10% fetal bovine serum (FBS, Biowest Co., Ltd.) and 1% antibiotic-antimycotic solution (Wako Pure Chemical Industries, Ltd.) were added to a 30 mL single-use bioreactor manufactured by Able Co., Ltd. Furthermore, 0.6 x 10 6 cells of MDCK cells (manufactured by Japanese Collection of Research Bioresources (JCRB), product number: JCRB9029) were added and cultured for 7 days at 37 ° C. in an incubator with 5 vol % CO 2 atmosphere at 100 rpm stirring conditions.
  • DMEM Dynamic Eagle Medium
  • FBS fetal bovine serum
  • antibiotic-antimycotic solution Wako Pure Chemical Industries, Ltd.
  • the 30 mL single-use bioreactor was transferred to a safety cabinet at room temperature, the cell suspension was passed through a 50 mL tube equipped with a cell strainer with a mesh size of 70 ⁇ m, the floating cells were removed, and only the microcarriers to which the cells on the cell strainer were attached were collected, and phosphate buffered saline (PBS (-)) containing no calcium or magnesium at 4 ° C was added and allowed to stand for 10 minutes. After standing, pipetting was performed 60 times, and the detached cells were collected by passing them through a 50 mL tube equipped with a cell strainer with a mesh size of 70 ⁇ m, and the cells were concentrated by centrifugation and counted.
  • PBS (-) phosphate buffered saline
  • the number of cells collected by cooling was 137.0 ⁇ 10 5 cells.
  • the microcarriers on the cell strainer were then transferred to a 15 mL tube, 5 mL of CTS TrypLE Select Enzyme (manufactured by Thermo Fisher Scientific) was added, and the mixture was allowed to stand for 5 minutes in a 5 vol% CO2 atmosphere at 37 °C.
  • the cell suspension was passed through a 50 mL tube equipped with a cell strainer with a 70 ⁇ m opening, the cells were collected, and the cells were concentrated by centrifugation to count the number of cells.
  • the number of cells that could not be collected by cooling was 37.0 ⁇ 10 5 cells.
  • the cooling detachment rate was 79%.
  • Example 21 ⁇ MDCK cell culture> MDCK cells were cultured and the number of cells was counted in the same manner as in ⁇ MDCK cell culture> of Example 19, except that 340 mg of microcarriers 12 (culture area 150 cm 2 ) was used and the stirring speed was set to 60 rpm. The number of cells recovered by cooling was 112.0 ⁇ 10 5 , and the number of cells that could not be recovered by cooling was 32.0 ⁇ 10 5 . The cooling detachment rate was 78%.
  • Example 11 MDCK cells were cultured and the number of cells was counted in the same manner as in ⁇ MDCK cell culture> of Example 20, except that 416 mg (culture area 150 cm2 ) of Cellbind (surface: anionic, average specific gravity: 1.026) microcarriers sterilized by autoclave were used and the stirring speed was set to 60 rpm.
  • the number of cells recovered by cooling was 2.3 x 105
  • the number of cells that could not be recovered by cooling was 68.8 x 105.
  • the cooling detachment rate was 3%.
  • Example 12 MDCK cell culture> MDCK cells were cultured and the number of cells was counted in the same manner as in ⁇ MDCK cell culture> of Example 20, except that 34.1 mg (culture area 150 cm2 ) of autoclave-sterilized Cytodex1 (surface: cationic, average specific gravity: 1.03) microcarriers were used and the stirring speed was set to 60 rpm.
  • the number of cells recovered by cooling was 16.7 x 105
  • the number of cells that could not be recovered by cooling was 157.4 x 105.
  • the cooling detachment rate was 10%.
  • Example 22 ⁇ MRC-5 cell culture> 204 mg (culture area 87 cm 2 ) of temperature-responsive microcarrier 4 and 30 mL of complete medium of DMEM (Dulbecco's Modified Eagle Medium, Wako Pure Chemical Industries) containing 10% fetal bovine serum (FBS, Biowest Co., Ltd.) and 1% antibiotic-antimycotic solution (Wako Pure Chemical Industries, Ltd.) were added to a 30 mL single-use bioreactor manufactured by Able Co., Ltd. Furthermore, 0.5 x 10 6 cells of MRC-5 cells (manufactured by Japanese Collection of Research Bioresources (JCRB), product number: JCRB9008) were added and cultured for 6 days at 37 ° C. in an incubator with 5 vol % CO 2 atmosphere at 100 rpm stirring conditions.
  • DMEM Dynamic Eagle Medium
  • FBS fetal bovine serum
  • antibiotic-antimycotic solution Wako Pure Chemical Industries, Ltd.
  • the 30 mL single-use bioreactor was transferred to a safety cabinet at room temperature, the cell suspension was passed through a 50 mL tube with a cell strainer with a mesh size of 70 ⁇ m, the floating cells were removed, and only the microcarriers to which the cells on the cell strainer were attached were collected, and phosphate buffered saline (PBS (-)) free of calcium and magnesium at 4 ° C was added and allowed to stand for 10 minutes. After standing, pipetting was performed 60 times, and the detached cells were collected by passing them through a 50 mL tube with a cell strainer with a mesh size of 70 ⁇ m, and the cells were concentrated by centrifugation and counted.
  • PBS (-) phosphate buffered saline
  • the number of cells collected by cooling was 9.8 ⁇ 10 5 cells. Furthermore, the microcarriers on the cell strainer were transferred to a 15 mL tube, 5 mL of CTS TrypLE Select Enzyme (manufactured by Thermo Fisher Scientific) was added, and the mixture was allowed to stand for 5 minutes in a 5 vol% CO 2 atmosphere at 37 ° C. The cell suspension was passed through a 50 mL tube equipped with a cell strainer with a mesh size of 70 ⁇ m, the cells were collected, concentrated by centrifugation, and the number of cells was counted. The number of cells that could not be collected by cooling was 4.1 ⁇ 10 5 cells. The cooling detachment rate was 71%.
  • Example 23 ⁇ MRC-5 cell culture> MRC-5 cells were cultured and the number of cells was counted in the same manner as in ⁇ MRC-5 cell culture> in Example 22, except that 195 mg of microcarriers 12 (culture area 87 cm 2 ) were used and the stirring speed was 60 rpm.
  • the number of cells recovered by cooling was 18.8 ⁇ 10 5
  • the number of cells that could not be recovered by cooling was 1.0 ⁇ 10 5 .
  • the cooling detachment rate was 95%.
  • MRC-5 cells were cultured and the number of cells was counted in the same manner as in ⁇ MRC-5 cell culture> in Example 22, except that 238 mg (culture area 87 cm 2 ) of Cellbind (surface: anionic, average specific gravity: 1.026) microcarriers sterilized by autoclave were used and the stirring speed was set to 60 rpm.
  • the number of cells recovered by cooling was 3.4 ⁇ 10 5
  • the number of cells that could not be recovered by cooling was 8.8 ⁇ 10 5 .
  • the cooling detachment rate was 28%.
  • MRC-5 cells were cultured and the number of cells was counted in the same manner as in ⁇ MRC-5 cell culture> in Example 22, except that 20 mg (culture area 87 cm 2 ) of autoclave-sterilized Cytodex 1 (surface: cationic, average specific gravity: 1.03) microcarriers were used and the stirring speed was set to 60 rpm.
  • the number of cells recovered by cooling was 4.2 ⁇ 10 5
  • the number of cells that could not be recovered by cooling was 9.2 ⁇ 10 5 .
  • the cooling detachment rate was 31%.

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Abstract

La présente invention concerne un microsupport contenant des billes enduites d'un film de revêtement polymère qui comprend un polymère thermosensible qui présente une température de solution critique inférieure comprise entre 0 °C et 50 °C et un polymère présentant une charge négative.
PCT/JP2024/020256 2023-06-06 2024-06-03 Microsupport thermosensible Pending WO2024253069A1 (fr)

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JP2010508851A (ja) * 2006-11-13 2010-03-25 エシコン・インコーポレイテッド マイクロキャリアを使用した、産褥由来の細胞の生体外での拡大
JP2011514169A (ja) * 2008-03-17 2011-05-06 エージェンシー フォー サイエンス，テクノロジー アンド リサーチ 幹細胞培養のためのマイクロキャリア
WO2015093393A1 (fr) * 2013-12-20 2015-06-25 Dic株式会社 Substrat de culture cellulaire réactif à la température et son procédé de production
WO2015111686A1 (fr) * 2014-01-23 2015-07-30 日産化学工業株式会社 Composition de milieu de culture
JP2016501026A (ja) * 2012-12-14 2016-01-18 デピュイ・シンセス・プロダクツ・インコーポレイテッド ｈＵＴＣ増殖用栄養強化培地
JP2016036274A (ja) * 2014-08-06 2016-03-22 株式会社Ihi 細胞剥離方法、細胞剥離装置、及び、細胞培養システム
JP2019094421A (ja) * 2017-11-22 2019-06-20 東ソー株式会社 ブロック共重合体コートビーズおよびその製造方法
WO2020054755A1 (fr) * 2018-09-11 2020-03-19 日産化学株式会社 Dispositif de séparation et procédé de séparation de matériau à séparer l'utilisant
WO2022202911A1 (fr) * 2021-03-26 2022-09-29 東ソー株式会社 Agent de traitement de surface polymère thermosensible

Patent Citations (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2010508851A (ja) * 2006-11-13 2010-03-25 エシコン・インコーポレイテッド マイクロキャリアを使用した、産褥由来の細胞の生体外での拡大
JP2011514169A (ja) * 2008-03-17 2011-05-06 エージェンシー フォー サイエンス，テクノロジー アンド リサーチ 幹細胞培養のためのマイクロキャリア
JP2016501026A (ja) * 2012-12-14 2016-01-18 デピュイ・シンセス・プロダクツ・インコーポレイテッド ｈＵＴＣ増殖用栄養強化培地
WO2015093393A1 (fr) * 2013-12-20 2015-06-25 Dic株式会社 Substrat de culture cellulaire réactif à la température et son procédé de production
WO2015111686A1 (fr) * 2014-01-23 2015-07-30 日産化学工業株式会社 Composition de milieu de culture
JP2016036274A (ja) * 2014-08-06 2016-03-22 株式会社Ihi 細胞剥離方法、細胞剥離装置、及び、細胞培養システム
JP2019094421A (ja) * 2017-11-22 2019-06-20 東ソー株式会社 ブロック共重合体コートビーズおよびその製造方法
WO2020054755A1 (fr) * 2018-09-11 2020-03-19 日産化学株式会社 Dispositif de séparation et procédé de séparation de matériau à séparer l'utilisant
WO2022202911A1 (fr) * 2021-03-26 2022-09-29 東ソー株式会社 Agent de traitement de surface polymère thermosensible

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