WO2024253070A1 - Tissu non tissé sensible à la température - Google Patents

Tissu non tissé sensible à la température Download PDF

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Publication number
WO2024253070A1
WO2024253070A1 PCT/JP2024/020258 JP2024020258W WO2024253070A1 WO 2024253070 A1 WO2024253070 A1 WO 2024253070A1 JP 2024020258 W JP2024020258 W JP 2024020258W WO 2024253070 A1 WO2024253070 A1 WO 2024253070A1
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Prior art keywords
nonwoven fabric
cells
temperature
less
responsive
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PCT/JP2024/020258
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English (en)
Japanese (ja)
Inventor
栄鎮 金
伸哉 今富
聡文 最上
博之 伊藤
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Tosoh Corp
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Tosoh Corp
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Priority to JP2025526106A priority Critical patent/JPWO2024253070A1/ja
Publication of WO2024253070A1 publication Critical patent/WO2024253070A1/fr
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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
    • 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
    • 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
    • D—TEXTILES; PAPER
    • D06—TREATMENT OF TEXTILES OR THE LIKE; LAUNDERING; FLEXIBLE MATERIALS NOT OTHERWISE PROVIDED FOR
    • D06M—TREATMENT, NOT PROVIDED FOR ELSEWHERE IN CLASS D06, OF FIBRES, THREADS, YARNS, FABRICS, FEATHERS OR FIBROUS GOODS MADE FROM SUCH MATERIALS
    • D06M15/00—Treating fibres, threads, yarns, fabrics, or fibrous goods made from such materials, with macromolecular compounds; Such treatment combined with mechanical treatment
    • D06M15/19—Treating fibres, threads, yarns, fabrics, or fibrous goods made from such materials, with macromolecular compounds; Such treatment combined with mechanical treatment with synthetic macromolecular compounds
    • D06M15/21—Macromolecular compounds obtained by reactions only involving carbon-to-carbon unsaturated bonds
    • D06M15/263—Macromolecular compounds obtained by reactions only involving carbon-to-carbon unsaturated bonds of unsaturated carboxylic acids; Salts or esters thereof
    • D—TEXTILES; PAPER
    • D06—TREATMENT OF TEXTILES OR THE LIKE; LAUNDERING; FLEXIBLE MATERIALS NOT OTHERWISE PROVIDED FOR
    • D06M—TREATMENT, NOT PROVIDED FOR ELSEWHERE IN CLASS D06, OF FIBRES, THREADS, YARNS, FABRICS, FEATHERS OR FIBROUS GOODS MADE FROM SUCH MATERIALS
    • D06M15/00—Treating fibres, threads, yarns, fabrics, or fibrous goods made from such materials, with macromolecular compounds; Such treatment combined with mechanical treatment
    • D06M15/19—Treating fibres, threads, yarns, fabrics, or fibrous goods made from such materials, with macromolecular compounds; Such treatment combined with mechanical treatment with synthetic macromolecular compounds
    • D06M15/21—Macromolecular compounds obtained by reactions only involving carbon-to-carbon unsaturated bonds
    • D06M15/285—Macromolecular compounds obtained by reactions only involving carbon-to-carbon unsaturated bonds of unsaturated carboxylic acid amides or imides
    • D—TEXTILES; PAPER
    • D06—TREATMENT OF TEXTILES OR THE LIKE; LAUNDERING; FLEXIBLE MATERIALS NOT OTHERWISE PROVIDED FOR
    • D06M—TREATMENT, NOT PROVIDED FOR ELSEWHERE IN CLASS D06, OF FIBRES, THREADS, YARNS, FABRICS, FEATHERS OR FIBROUS GOODS MADE FROM SUCH MATERIALS
    • D06M2101/00—Chemical constitution of the fibres, threads, yarns, fabrics or fibrous goods made from such materials, to be treated
    • D06M2101/16—Synthetic fibres, other than mineral fibres
    • D06M2101/30—Synthetic polymers consisting of macromolecular compounds obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds
    • D06M2101/32—Polyesters

Definitions

  • the purpose of this patent is to provide a culture substrate that has high cell productivity per unit medium and enables the recovery of a cell suspension that does not contain fibrous impurities.
  • the present inventors have conducted extensive research and found that a nonwoven fabric made of plastic with a true density of 1.0 to 1.5 g/ cm3 and a water absorption rate of less than 0.3%, the surface of which is coated with a block copolymer containing the following blocks (A), (B), and (C), has high cell productivity per unit medium and enables the recovery of a cell suspension free of fibrous impurities, thereby completing the present invention. That is, the present invention encompasses the following aspects.
  • C A temperature-responsive polymer block having a lower critical solution temperature (LCST) in water in the range of 0°C to 50°C.
  • ⁇ 2> The nonwoven fabric according to ⁇ 1>, characterized in that the water absorption rate is 0.2% or less.
  • ⁇ 3> The nonwoven fabric according to ⁇ 1> or ⁇ 2>, characterized in that the plastic is polyethylene terephthalate.
  • ⁇ 4> The nonwoven fabric according to any one of ⁇ 1> to ⁇ 3>, wherein the coating amount of the block copolymer is 1.0 to 100.0 ⁇ g/ cm2 .
  • ⁇ 5> The nonwoven fabric according to any one of ⁇ 1> to ⁇ 4>, wherein the fibers constituting the nonwoven fabric have a thickness of 1 to 1000 ⁇ m.
  • ⁇ 6> The nonwoven fabric according to any one of ⁇ 1> to ⁇ 5>, wherein the proportion of the block (C) in the block copolymer is 30 to 90 mol %.
  • ⁇ 7> The nonwoven fabric according to any one of ⁇ 1> to ⁇ 6>, wherein the block copolymer has a number average molecular weight of 1,000 to 1,000,000.
  • ⁇ 8> A method for producing the nonwoven fabric according to any one of ⁇ 1> to ⁇ 7>.
  • a method for producing a nonwoven fabric according to any one of ⁇ 1> to ⁇ 7> comprising a step of coating a surface of a nonwoven fabric made of a plastic having a true density of 1.0 to 1.5 g/ cm3 and a water absorption rate of less than 0.3% with a block copolymer containing the following blocks (A), (B), and (C): (A) A polymer block having an HLB value (Griffin method) in the range of 7 or more and 20 or less. (B) A polymer block having an HLB value (Griffin method) in the range of 0 or more and less than 7.
  • (C) A temperature-responsive polymer block having a lower critical solution temperature (LCST) in water in the range of 0°C to 50°C.
  • LCST lower critical solution temperature
  • ⁇ 10> The method according to ⁇ 9>, wherein the water absorption rate is 0.2% or less.
  • ⁇ 11> A cell culture method using the nonwoven fabric according to any one of ⁇ 1> to ⁇ 7>.
  • ⁇ 12> A cell culture method comprising a step of culturing cells in a medium containing the nonwoven fabric according to any one of ⁇ 1> to ⁇ 7>.
  • ⁇ 13> The cell culture method according to ⁇ 11> or ⁇ 12>, further comprising a cooling cell detachment step of detaching cells from microcarriers using a cooling liquid containing a chelating agent.
  • a nonwoven fabric made of plastic having a true density of 1.0 to 1.5 g/ cm3 and a water absorption rate of less than 0.3%, the surface of which is coated with a block copolymer containing the following blocks (A), (B), and (C), has high cell productivity per unit medium and can recover a cell suspension that does not contain fibrous impurities.
  • 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 present invention relates to a nonwoven fabric made of plastic having a true density of 1.0 to 1.5 g/ cm3 and a water absorption rate of less than 0.3%, the surface of which is coated with a block copolymer containing blocks (A), (B), and (C).
  • nonwoven fabric is fabric made by bonding or intertwining fibers through heat, mechanical or chemical action.
  • nonwoven fabric is fabric made by forming fibers into a cloth and bonding the fibers together.
  • sheeting method used to form the nonwoven fabric typically examples include the dry method in which the fabric is formed in air, the wet method in which the fabric is formed in water, and the spunbond method.
  • bonding method used to form bonds between the fibers typically examples include thermal bonding, chemical bonding, and mechanical bonding. As long as it is within the scope of the present invention, it can be selected appropriately depending on the ease of processing.
  • the true density of the fibers constituting the nonwoven fabric is a density calculated from the volume excluding the gaps (pores) between the fibers constituting the nonwoven fabric, and is 1.0 to 1.5 g/cm 3 in order to settle in water, and is preferably 1.0 to 1.3 g/cm 3 , and more preferably 1.0 to 1.2 g/cm 3 in order to easily disperse under stirring.
  • the water absorption rate of the fibers constituting the nonwoven fabric is less than 0.3% in order to maintain strength without decomposing in water, preferably 0.2% or less, more preferably 0.15% or less, and particularly preferably 0.1% or less.
  • the method for evaluating the water absorption rate refers to JIS K 7209:2000, "Plastics - Determination of water absorption," which is a translation of ISO 62:1999, Plastics - Determination of water absorption.
  • the material of the fibers constituting the nonwoven fabric is not particularly limited, but examples of materials having true densities and water absorption rates within the ranges of the present invention include polyamideimide, polyoxymethylene, polycarbonate, polyethylene terephthalate, polybutylene terephthalate, polyvinyl chloride, polyether ether ketone, polyphenylene sulfide, polystyrene, polypropylene, etc., and polyethylene terephthalate is preferred because of its ease of processing.
  • the material may be made of a copolymer or may be a multilayer structure yarn as long as it is within the true density and water absorption rate ranges of the present invention.
  • organic and/or inorganic substances and/or metals may be added to the nonwoven fabric as long as it is within the true density and water absorption rate ranges of the present invention.
  • the thickness of the fibers that make up the nonwoven fabric is preferably 1 to 1000 ⁇ m, and more preferably 10 to 300 ⁇ m, but it is preferable to select an appropriate thickness taking into consideration the type and characteristics of the cells used.
  • the structure of the fibers that make up the nonwoven fabric is not particularly limited, and an example of a cross-sectional structure is a circle, but it may also be flower-shaped or flattened and multi-lobed to increase the surface area, or it may have a hollow fiber structure to allow the cell culture medium to reach the entire nonwoven fabric. It is preferable to select an appropriate structure taking into account the type and characteristics of the cells used.
  • the thickness of the nonwoven fabric is not particularly limited, but as an example, it is 0.01 to 5 mm, and preferably 0.1 to 3 mm, but it is preferable to select an appropriate thickness taking into consideration the type and characteristics of the cells used.
  • the mesh size of the nonwoven fabric is 1 ⁇ m to 1000 ⁇ m.
  • the block copolymer with which the nonwoven fabric of the present invention is coated contains 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.
  • LCST lower critical solution temperature
  • 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 proportion of block (C) is preferably 30 to 90 mol%, and more preferably 50 to 80 mol%.
  • the method for producing the nonwoven fabric of the present invention is not particularly limited, and examples include the impregnation method and the immersion method, in which the nonwoven fabric is immersed or sprayed with a polymer solution, and then dried to remove the solvent.
  • the nonwoven fabric of the present invention may be coated with a polymer other than the block copolymer containing blocks (A), (B), and (C), and as an example, coating with a polymer having a positive or negative charge also facilitates cell adhesion.
  • the method for producing a nonwoven fabric of the present invention may be, for example, a method characterized by including a step of coating a surface of a nonwoven fabric made of a plastic having a true density of 1.0 to 1.5 g/ cm3 and a water absorption rate of less than 0.3%, with a block copolymer containing the following blocks (A), (B), and (C):
  • a polymer block having an HLB value (Griffin method) in the range of 7 or more and 20 or less.
  • B A polymer block having an HLB value (Griffin method) in the range of 0 or more and less than 7.
  • C A temperature-responsive polymer block having a lower critical solution temperature (LCST) in water in the range of 0°C to 50°C.
  • the nonwoven fabric of the present invention can be used for cell culture, particularly for culturing 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 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, 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, epithelial cells and endothelial cells constituting each tissue and organ in the body, skeletal muscle cells exhibiting contractility, smooth muscle cells, cardiac muscle cells, neuron cells constituting the nervous system, glial cells, fibroblasts, hepatic parenchymal cells involved in the metabolism of the body, non-parenchymal liver cells and adipocytes, and cells having differentiation potential, such as stem cells present in various tissues
  • examples include cells contained in blood, lymph, cerebrospinal fluid, sputum, urine or stool, 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 cells.
  • the type of basal medium used in the culture of the present invention 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.
  • it may be a serum medium or a serum-free medium, but the serum used 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 can be used.
  • the complete medium may or may not contain antibiotics, and can be selected appropriately depending on the type of cells.
  • the cell culture method using the nonwoven fabric of the present invention is not particularly limited, and may be cultured under static conditions, shaking conditions, stirring conditions, or a combination of the above methods under intermittent shaking and/or intermittent stirring conditions.
  • culturing under shaking conditions there is no particular limit to the shaking speed, and there is no particular limit to the shaking direction.
  • stirring speed there is no particular limit to the stirring speed, and one example is 1 to 1000 rpm.
  • the culture temperature during culture but it is preferable to carry out the culture at a temperature above the LCST, which can provide an environment in which the temperature-responsive polymer becomes hydrophobic and cells can easily adhere. After culture, the cells on the nonwoven fabric can be recovered by cooling to a temperature below the LCST.
  • the cooling method there is no particular limit to the cooling method, and the cells may be directly cooled using a cooler, or the culture medium may be replaced with a cooling liquid.
  • the cooling liquid can be a culture medium, and the culture medium may be a complete medium containing growth factors, serum, etc., or a basal medium not containing growth factors, serum, etc.
  • the cooling liquid may be a phosphate buffer or a cell detachment liquid containing a protease, but by selecting a cell detachment liquid containing a protease, the number of cells recovered is likely to be high.
  • the protease is trypsin, and it may be recombinant trypsin that does not contain animal-derived components.
  • the cells are more likely to be detached from the nonwoven fabric as single cells.
  • single cells mean that the cells have escaped from a state in which they were connected to each other and are separated into individual cells.
  • the concentration of the chelating agent in the phosphate buffer is not particularly limited and is 1 ⁇ M to 1 mM.
  • the cooling liquid has a flow, since this can promote the cooling of the nonwoven fabric. For example, the flow can be obtained by stirring or pipetting.
  • the cell culture method using the nonwoven fabric of the present invention may be, for example, a method characterized by including a step of culturing cells in a medium containing the nonwoven fabric of the present invention.
  • the cell culture method using the nonwoven fabric of the present invention may further include a cooling cell detachment step of detaching cells from microcarriers using a cooling liquid containing a chelating agent.
  • the culture performance of the nonwoven fabric of the present invention is evaluated based on the number of cells recovered using a cell detachment solution containing a chilled protease and/or a chilled detachment solution not containing a protease and/or a cell detachment solution containing a chelating agent, and the presence or absence of nonwoven fabric fragments in the recovered cell suspension.
  • a cell detachment solution containing a chilled protease and/or a chilled detachment solution not containing a protease and/or a cell detachment solution containing a chelating agent and the presence or absence of nonwoven fabric fragments in the recovered cell suspension.
  • the protease but an example is TrypLE Express Enzyme (manufactured by Thermo Fisher Scientific).
  • the chilled detachment solution not containing a protease but an example is phosphate buffered saline (PBS(-)) not containing calcium and magnesium.
  • the cell detachment solution containing a chelating agent there is no particular limitation on the cell detachment solution containing a chelating agent, but an example is PBS(-) containing 1 mM EDTA.
  • the method for measuring the number of cells there is no particular limitation on the method for measuring the number of cells, but the evaluation can be performed 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.
  • Example 1 10 g of a 1.00 wt% 1-methoxy-2-propanol solution of polymer MBI and 10 g of a 0.03 wt% 1-methoxy-2-propanol solution of polymer CStS were added to a 50 mL beaker. 5 g of BioNOCII (manufactured by CESCO BIOENGINEERING, specific surface area 2400 cm 2 /g, water absorption rate 0.1%), which is a polyethylene terephthalate nonwoven fabric, was immersed in the solution in the beaker, and after leaving it to stand for 5 minutes, the nonwoven fabric was removed from the solution and dried at room temperature for 2 hours.
  • BioNOCII manufactured by CESCO BIOENGINEERING, specific surface area 2400 cm 2 /g, water absorption rate 0.15%
  • the nonwoven fabric was dried under reduced pressure at 50°C to obtain a temperature-responsive nonwoven fabric 1.
  • 1 g of the block copolymer coating film of the temperature-responsive nonwoven fabric 1 was extracted with 2,2,2-trifluoroethanol, and the coating amount (fixed amount) of the block copolymer was evaluated from the peak intensity of GPC, which was 5.0 ⁇ g/cm 2 .
  • 0.1 g of the temperature-responsive nonwoven fabric 1 and 10 mL of mesenchymal stem cell proliferation medium 2 (PromoCell) were added to a 100 mm untreated dish (Corning).
  • 5 x 10 cells of human bone marrow-derived mesenchymal stem cells (passage number 2) (Lonza) were further added and cultured for 6 days in an incubator at 37°C with a 5 vol% CO2 atmosphere.
  • the dish was transferred to a safety cabinet at room temperature, the medium was removed, 10 mL of calcium- and magnesium-free phosphate buffered saline (PBS(-)) at 37 ° C was added and left to stand for 1 minute, and the PBS(-) was removed. Furthermore, 5 mL of TrypLE Express Enzyme (manufactured by Thermo Fisher Scientific) at 4 ° C was added and left to stand for 30 minutes.
  • PBS(-) calcium- and magnesium-free phosphate buffered saline
  • mesenchymal stem cell growth medium 2 5 mL of mesenchymal stem cell growth medium 2 was added, and after pipetting 30 times with a 1 mL pipette, the cell suspension was passed through a 15 mL tube with a cell strainer with a mesh size of 70 ⁇ m set, and 2 mL of PBS(-) was added on the cell strainer, the cell suspension was collected in a tube, concentrated by centrifugation, and the cell number was counted. The cell number was 38.0 ⁇ 10 5 cells, and no fibrous impurities were observed.
  • Example 2 A temperature-responsive nonwoven fabric 2 was obtained in the same manner as in Example 1, except that 10 g of a 4.00 wt % 1-methoxy-2-propanol solution of polymer MBI and 10 g of a 0.12 wt % 1-methoxy-2-propanol solution of polymer CStS were added to a 50 mL beaker. The coating amount (fixed amount) of the block copolymer of the temperature-responsive nonwoven fabric 2 was evaluated and found to be 35.0 ⁇ g/ cm2 .
  • Human bone marrow-derived mesenchymal stem cells were cultured in the same manner as in Example 1, except that temperature-responsive nonwoven fabric 2 was used.
  • the cell suspension was collected in the same manner as in Example 1 except that temperature-responsive nonwoven fabric 2 was used, concentrated by centrifugation, and the cell number was counted.
  • the cell number was 38.8 x 10 5 cells, and no fibrous impurities were found.
  • Example 3 Temperature-responsive nonwoven fabric 3 was obtained in the same manner as in Example 1, except that 10 g of a 0.50 wt % 1-methoxy-2-propanol solution of polymer MBI and 10 g of a 0.015 wt % 1-methoxy-2-propanol solution of polymer CStS were added to a 50 mL beaker. The coating amount (fixed amount) of the block copolymer of temperature-responsive nonwoven fabric 3 was evaluated and found to be 1.7 ⁇ g/ cm2 .
  • Human bone marrow-derived mesenchymal stem cells were cultured in the same manner as in Example 1, except that temperature-responsive nonwoven fabric 3 was used.
  • the cell suspension was collected in the same manner as in Example 1 except that temperature-responsive nonwoven fabric 3 was used, concentrated by centrifugation, and the cell number was counted.
  • the cell number was 33.3 x 10 5 cells, and no fibrous impurities were found.
  • Comparative Example 1 Human bone marrow-derived mesenchymal stem cells were cultured in the same manner as in Example 1, except that 0.1 g of untreated BioNOCII (defined as non-temperature responsive nonwoven fabric A) was used in a 100 mm untreated dish (manufactured by Corning).
  • BioNOCII defined as non-temperature responsive nonwoven fabric A
  • Example 1 A cell suspension was collected in the same manner as in Example 1 except that non-temperature responsive nonwoven fabric A was used, concentrated by centrifugation, and the cell count was measured. No fibrous impurities were found, but the cell count was 10.2 ⁇ 10 5 cells, which was only about one-third of the number of cells collected in Examples 1 to 3.
  • Example 2 A temperature-responsive nonwoven fabric A was obtained in the same manner as in Example 1, except that a nylon nonwoven fabric (specific surface area: 3,500 cm2 /g, water absorption rate: 1.1%) was used. The coating amount (fixed amount) of the block copolymer on the temperature-responsive nonwoven fabric A was evaluated to be 6.0 ⁇ g/ cm2 .
  • Human bone marrow-derived mesenchymal stem cells were cultured in the same manner as in Example 1, except that temperature-responsive nonwoven fabric A was used.
  • the cell suspension was collected in the same manner as in Example 1 except that temperature-responsive nonwoven fabric A was used, concentrated by centrifugation, and the cell number was counted.
  • the cell number was 32.8 ⁇ 10 5 cells, but fibrous impurities were found.
  • Comparative Example 3 Human bone marrow-derived mesenchymal stem cells were cultured in the same manner as in Comparative Example 1, except that a nylon nonwoven fabric (specific surface area: 3500 cm 2 /g, water absorption rate: 1.1%) (defined as non-temperature responsive nonwoven fabric B) was used.
  • a cell suspension was collected in the same manner as in Example 1 except that non-temperature responsive nonwoven fabric B was used, concentrated by centrifugation, and the cell count was measured.
  • the cell count was 10.0 ⁇ 10 5 cells, which was only about one-third of the number of cells collected in Examples 1 to 3, and fibrous impurities were also found.
  • Example 4 Temperature-responsive nonwoven fabric 4 was obtained in the same manner as in Example 1, except that 10 g of a 6.00 wt % 1-methoxy-2-propanol solution of polymer MBI and 10 g of a 0.09 wt % 1-methoxy-2-propanol solution of polymer CStS were added to a 50 mL beaker. The coating amount (fixed amount) of the block copolymer of temperature-responsive nonwoven fabric 4 was evaluated to be 41.6 ⁇ g/ cm2 .
  • the 24-well plate was transferred to a safety cabinet at room temperature, the medium was removed, 2 mL of 37°C PBS(-) was added, and the plate was left to stand for 1 minute for washing, and the PBS(-) was removed. Furthermore, 2 mL of 4°C PBS(-) was added and left to stand for 10 minutes. Then, the plate was pipetted 20 times with a 1 mL pipette for cooling treatment.
  • the cell suspension obtained after the cooling treatment was passed through a 50 mL tube equipped with a cell strainer (manufactured by ASONEO, product number: VCS-70) with a mesh size of 70 ⁇ m, and 2 mL of fresh 4°C PBS(-) was added on the cell strainer for washing, and the cell suspension was collected in a tube and concentrated by centrifugation to count the number of cells.
  • the cell number was 2.81 ⁇ 10 5 cells/sheet, and no fibrous impurities were observed.
  • Example 5 Temperature responsive nonwoven fabric 5 was obtained in the same manner as in Example 1, except that 10 g of a 1-methoxy-2-propanol solution of 8.00 wt % polymer MBI and 10 g of a 1-methoxy-2-propanol solution of 0.16 wt % polymer CStS were added to a 50 mL beaker. The coating amount (fixed amount) of the block copolymer of temperature responsive nonwoven fabric 5 was evaluated to be 52.5 ⁇ g/ cm2 .
  • VERO cells were cultured in the same manner as in Example 4, except that temperature-responsive nonwoven fabric 5 was used.
  • the cell suspension was recovered in the same manner as in Example 4 except that temperature responsive nonwoven fabric 5 was used, concentrated by centrifugation, and the cell number was counted.
  • the cell number was 2.48 ⁇ 10 5 cells/sheet, and no fibrous impurities were found.
  • Comparative Example 4 VERO cells were cultured in the same manner as in Example 4, except that non-temperature responsive nonwoven fabric A was used.
  • Example 4 A cell suspension was collected in the same manner as in Example 4 except that non-temperature responsive nonwoven fabric A was used, concentrated by centrifugation, and the cell count was counted. No fibrous impurities were found, but the cell count was 1.22 ⁇ 10 5 cells/sheet, which was about half the number of cells collected compared to Examples 4 and 5.
  • Example 6 Two sheets of temperature-responsive nonwoven fabric 4 and 2 mL of DMEM (Dulbecco's Modified Eagle Medium, Wako Pure Chemical Industries, Ltd.) containing 10% fetal bovine serum (FBS, Biowest Co., Ltd.) and 1% antibiotic-antimycotic solution (Wako Pure Chemical Industries, Ltd.) were added to an ultra-low attachment surface 24-well plate (Corning, product number: 3473). Furthermore, BHK-21 cells (ATCC, product number: CCL-10) were added at a seeding concentration of 0.25 x 10 5 cells/sheet and cultured for 6 days in an incubator with a 5 vol% CO 2 atmosphere at 37°C.
  • DMEM Dynamic Eagle Medium
  • FBS fetal bovine serum
  • antibiotic-antimycotic solution Wako Pure Chemical Industries, Ltd.
  • the 24-well plate was transferred to a safety cabinet at room temperature, the medium was removed, 2 mL of 37°C PBS(-) was added, and the plate was left to stand for 1 minute for washing, and the PBS(-) was removed. Furthermore, 2 mL of 4°C PBS(-) was added and left to stand for 10 minutes. Then, the plate was pipetted 20 times with a 1 mL pipette for cooling treatment.
  • the cell suspension obtained after the cooling treatment was passed through a 50 mL tube equipped with a cell strainer (manufactured by ASONEO, product number: VCS-70) with a mesh size of 70 ⁇ m, and 2 mL of fresh 4°C PBS(-) was added on the cell strainer for washing, and the cell suspension was collected in a tube and concentrated by centrifugation to count the number of cells.
  • the cell number was 2.80 ⁇ 10 5 cells/sheet, and no fibrous impurities were observed.
  • Example 7 BHK-21 cells were cultured in the same manner as in Example 6, except that temperature-responsive nonwoven fabric 5 was used.
  • the cell suspension was collected in the same manner as in Example 6 except that temperature-responsive nonwoven fabric 5 was used, concentrated by centrifugation, and the cell count was measured. The cell count was 4.22 ⁇ 10 5 cells/sheet, and no fibrous impurities were observed.
  • Example 6 A cell suspension was collected in the same manner as in Example 6 except that non-temperature responsive nonwoven fabric A was used, concentrated by centrifugation, and the cell count was measured. No fibrous impurities were found, but the cell count was 1.41 ⁇ 10 5 cells/sheet, which was only about one-third of the number of cells collected in Examples 6 and 7.
  • Example 8 BHK-21 cells were cultured in the same manner as in Example 6, except that temperature-responsive nonwoven fabric 4 was used.
  • the 24-well plate was transferred to a safety cabinet at room temperature, the medium was removed, 2 mL of 37°C PBS(-) was added, washed for 1 minute, and the PBS(-) was removed. Furthermore, 2 mL of PBS(-) containing 1 mM EDTA at 4°C was added and left to stand for 10 minutes. Then, the plate was pipetted 20 times with a 1 mL pipette and cooled.
  • the cell suspension obtained after the cooling treatment was passed through a 50 mL tube with a cell strainer (manufactured by ASONEO, product number: VCS-70) with a mesh size of 70 ⁇ m, washed by adding 2 mL of fresh 4°C PBS(-), the cell suspension was collected in a tube, concentrated by centrifugation, and the number of cells was counted.
  • the number of cells was 3.09 ⁇ 10 5 cells/sheet, which was an increase in the number of cells collected compared to Example 6. In addition, no fibrous impurities were observed.
  • Example 9 BHK-21 cells were cultured in the same manner as in Example 6, except that temperature-responsive nonwoven fabric 5 was used.
  • Example 8 A cell suspension was collected in the same manner as in Example 8 except that temperature-responsive nonwoven fabric 5 was used, concentrated by centrifugation, and the number of cells was counted, resulting in a cell count of 4.46 ⁇ 10 5 cells/sheet, which was an increase in the number of cells collected compared to Example 7. In addition, no fibrous impurities were observed.
  • Comparative Example 6 BHK-21 cells were cultured in the same manner as in Example 6, except that non-temperature responsive nonwoven fabric A was used.
  • Example 8 A cell suspension was collected in the same manner as in Example 8, except that non-temperature responsive nonwoven fabric A was used, concentrated by centrifugation, and the cell count was counted. No fibrous impurities were found, but the cell count was 1.66 ⁇ 10 5 cells/sheet, which was about one-third of the number of cells collected in Examples 8 and 9.

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Abstract

La présente invention concerne un tissu non tissé dans lequel un copolymère séquencé comprenant des blocs (A), (B) et (C) est appliqué sur une surface du tissu non tissé, qui comprend un plastique ayant une densité réelle de 1,0 à 1,5 g/cm3 et un taux d'absorption d'eau inférieur à 0,3 %. (A) Un bloc polymère ayant une valeur HLB (méthode de Griffin) dans la plage de 7 à 20 inclus. (B) Un bloc polymère ayant une valeur HLB (méthode de Griffin) dans la plage d'au moins 0 et inférieure à 7. (C) Un bloc polymère sensible à la température ayant une température de solution critique inférieure (LCST) dans l'eau dans la plage de 0 à 50 °C.
PCT/JP2024/020258 2023-06-06 2024-06-03 Tissu non tissé sensible à la température Ceased WO2024253070A1 (fr)

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Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH1156353A (ja) * 1997-11-20 1999-03-02 Nippon Bio Serapii Kk Ｐｅｔ（ポリエチレンテレフタレート）不織布を用いた動物組織等付着性細胞の培養方法
WO2004058936A1 (fr) * 2002-12-25 2004-07-15 National Institute Of Advanced Industrial Science And Technology Appareil de separation et de collecte de cellules et procede de separation et de collecte de cellules
JP2018174919A (ja) * 2017-04-12 2018-11-15 東ソー株式会社 ブロック共重合体およびそれを用いた表面処理剤
JP2020110140A (ja) * 2019-01-11 2020-07-27 東ソー株式会社 細胞培養方法
JP2022073687A (ja) * 2020-11-02 2022-05-17 東ソー株式会社 細胞数計測に適した冷却回収方法

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH1156353A (ja) * 1997-11-20 1999-03-02 Nippon Bio Serapii Kk Ｐｅｔ（ポリエチレンテレフタレート）不織布を用いた動物組織等付着性細胞の培養方法
WO2004058936A1 (fr) * 2002-12-25 2004-07-15 National Institute Of Advanced Industrial Science And Technology Appareil de separation et de collecte de cellules et procede de separation et de collecte de cellules
JP2018174919A (ja) * 2017-04-12 2018-11-15 東ソー株式会社 ブロック共重合体およびそれを用いた表面処理剤
JP2020110140A (ja) * 2019-01-11 2020-07-27 東ソー株式会社 細胞培養方法
JP2022073687A (ja) * 2020-11-02 2022-05-17 東ソー株式会社 細胞数計測に適した冷却回収方法

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