WO2021181819A1 - 細胞懸濁液の製造方法、及び、接着細胞の製造方法 - Google Patents

細胞懸濁液の製造方法、及び、接着細胞の製造方法 Download PDF

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Publication number
WO2021181819A1
WO2021181819A1 PCT/JP2020/047981 JP2020047981W WO2021181819A1 WO 2021181819 A1 WO2021181819 A1 WO 2021181819A1 JP 2020047981 W JP2020047981 W JP 2020047981W WO 2021181819 A1 WO2021181819 A1 WO 2021181819A1
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Prior art keywords
cell suspension
adherent cells
cells
microcarriers
volume
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PCT/JP2020/047981
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English (en)
French (fr)
Japanese (ja)
Inventor
尚武 陳
史子 中川
靖彦 多田
雅弘 岡野定
優至 佐藤
悠午 鈴木
克彦 中島
亮介 高橋
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Resonac Corp
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Showa Denko Materials Co Ltd
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Application filed by Showa Denko Materials Co Ltd filed Critical Showa Denko Materials Co Ltd
Priority to JP2021538009A priority Critical patent/JP7767147B2/ja
Priority to CN202080098424.XA priority patent/CN115485363A/zh
Priority to US17/419,985 priority patent/US20220315898A1/en
Priority to EP20904261.3A priority patent/EP3907278B8/en
Publication of WO2021181819A1 publication Critical patent/WO2021181819A1/ja
Anticipated expiration legal-status Critical
Priority to JP2023113824A priority patent/JP2023134611A/ja
Ceased legal-status Critical Current

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    • 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
    • C12N5/0068—General culture methods using substrates
    • C12N5/0075—General culture methods using substrates using microcarriers
    • 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
    • C12N5/0602—Vertebrate cells
    • C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
    • C12N5/0662—Stem cells
    • C12N5/0663—Bone marrow mesenchymal stem cells (BM-MSC)
    • 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
    • C12N2511/00—Cells for large scale production
    • 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
    • C12N2531/00—Microcarriers

Definitions

  • the present disclosure relates to a method for producing a cell suspension, a method for producing adherent cells, a method for producing a useful substance-containing liquid, and a method for producing a useful substance.
  • Adherent cells are cells that require a scaffold for proliferation.
  • a culture carrier that serves as a scaffold is used for culturing adherent cells.
  • adherent cells When such adherent cells are used for cell preparation, production of useful substances, etc., it is necessary to increase them in large quantities.
  • Biochemical Engineering Journal, 120 (2017), pp. 49-62 a method of culturing a large amount of human mesenchymal stromal cells using a microcarrier as a culture carrier is being investigated.
  • WO 2009/105044 discusses methods for producing particles, including isolating particles containing at least one biological property of mesenchymal stem cells from a mesenchymal stem cell conditioned medium (MSC-CM). Has been done.
  • Stem cells have proliferative and differentiating abilities, and are expected to be clinically applied to the field of regenerative medicine. A very large number of stem cells are required for clinical application. In addition, since exosomes isolated from stem cells contain various physiologically active substances, they are expected to be used in therapeutic and diagnostic methods for diseases. A large number of stem cells is still needed to obtain a sufficient amount of exosomes. However, the current situation is that there is room for improvement in the technique for culturing stem cells on a large scale.
  • the present disclosure provides a method for efficiently producing a large amount of cell suspension.
  • the present disclosure also provides a method for efficiently producing a large amount of adherent cells.
  • the present disclosure provides a method for efficiently producing a useful substance-containing liquid in a large amount, and a method for efficiently producing a useful substance in a large amount.
  • the disclosure includes various embodiments of the present invention. Examples of embodiments are given below.
  • One embodiment relates to a method for producing a cell suspension, which comprises the following (A), (B), and (C).
  • A Culturing the adherent cells in a cell suspension containing adherent cells, microcarriers, and a medium and having a volume of 0.3 L or more
  • B Adhesion obtained through the above (A). Culturing the adherent cells in a cell suspension containing cells, microcarriers, and medium and having a volume of 5 L or more, and (C) Culturing the adherent cells in a cell suspension containing the adherent cells, microcarriers, and medium obtained through the above (B) and having a volume of 10 L or more.
  • the cells are obtained by obtaining a cell suspension containing adherent cells, fresh microcarriers, and a medium, and intermittently stirring the cell suspension, and after undergoing the intermittent stirring, the cells.
  • the present invention relates to a method for producing a cell suspension, which comprises continuously stirring the suspension.
  • Yet another embodiment is the production of adherent cells, which comprises preparing a cell suspension obtained by the production method according to any of the above embodiments, and obtaining adherent cells from the cell suspension. Regarding the method.
  • Yet another embodiment is a useful substance, which comprises preparing a cell suspension obtained by the production method according to any one of the above embodiments, and obtaining a useful substance-containing liquid from the cell suspension.
  • the present invention relates to a method for producing a containing liquid.
  • Yet another embodiment is to prepare a cell suspension obtained by the production method according to any one of the above embodiments, or a useful substance-containing liquid obtained by the production method according to the above embodiment, and the above-mentioned.
  • the present invention relates to a method for producing a useful substance, which comprises obtaining a useful substance from a cell suspension or the liquid containing the useful substance.
  • the present disclosure provides a method for efficiently producing a large amount of cell suspension.
  • the present disclosure also provides a method for efficiently producing a large amount of adherent cells. Further, the present disclosure provides a method for efficiently producing a useful substance-containing liquid in a large amount, and a method for efficiently producing a useful substance in a large amount.
  • FIG. 1 is a conceptual diagram showing a method for producing a cell suspension in an example.
  • the method for producing a cell suspension includes the following (A), (B), and (C).
  • the above (A), (B) and (C) may be referred to as a process (A), a process (B) and a process (C), respectively.
  • the "process” is not limited to an independent process, and even if it cannot be clearly distinguished from other processes, it is clearly distinguished from other processes as long as the operation specified in the "process” is performed. Indistinguishable steps are also included.
  • adherent cells can be easily mass-cultured, and after mass-culturing, the survival rate is good.
  • a homogeneous cell population is obtained.
  • a method of exfoliating the adherent cells from a culture carrier that is a scaffold, subculturing them, and culturing them may be used.
  • the subculture operation involving the separation of the adherent cells and the microcarriers by peeling or the like may cause contamination or damage of the adherent cells.
  • cell suspensions for culture containing fresh microcarriers are adhered by a simple method of sequentially performing steps (A), (B) and (C) while controlling a specific volume. It promotes the movement of cells between microcarriers, enables efficient subculture of adherent cells, and realizes mass culture of adherent cells.
  • the cell suspension means a liquid containing cells, and the cells here may be in a state of being attached to a microcarrier or a state in which they are not attached.
  • the adherent cells are not particularly limited as long as they are known to exhibit adhesion to the selected substrate, and examples thereof include somatic cells and stem cells.
  • somatic cells for example, endothelial cells, epidermal cells, epithelial cells, myocardial cells, myoblasts, nerve cells, bone cells, osteoblasts, fibroblasts, fat cells, hepatocytes, renal cells, pancreatic cells, adrenal cells , Dental cells, gingival cells, bone membrane cells, skin cells, dendritic cells, macrophages and the like.
  • the adherent cells are preferably animal-derived cells, and more preferably mammalian-derived cells. Mammals include, for example, humans, monkeys, chimpanzees, cows, pigs, horses, sheep, goats, rabbits, rats, mice, guinea pigs, dogs, cats and the like.
  • Adhesive cells may be, for example, cells derived from tissues such as skin, liver, kidney, muscle, bone, blood vessel, blood, and nervous tissue. Usually, one type of cell is used for culturing alone, but two or more types may be used for culturing in combination.
  • the cell may be a primary cell from a tissue or a cell line established by immortalization. Further, the cell may be an artificially established cell.
  • the adherent cells may be stem cells.
  • stem cells include somatic stem cells such as mesenchymal stem cells, hematopoietic stem cells, neural stem cells, bone marrow stem cells, and germline stem cells, and can be mesenchymal stem cells or bone marrow mesenchymal stem cells. ..
  • Mesenchymal stem cells broadly mean somatic stem cells that are present in various tissues of the human body and can be differentiated into all or some of mesenchymal cells such as osteoblasts, chondrocytes and adipocytes. do.
  • the stem cells may further include induced pluripotent stem cells (iPS cells) and embryonic stem cells (ES cells).
  • iPS cells induced pluripotent stem cells
  • ES cells embryonic stem cells
  • the method for producing a cell suspension according to the present disclosure is a method suitable for mass production of mesenchymal stem cells.
  • Microcarriers are carriers that provide a scaffold for cell proliferation in the culture of adherent cells. Microcarriers known as carriers for cell culture can be used.
  • the material of the microcarrier may be an organic substance, an inorganic substance, or a composite material thereof, and may be soluble or insoluble.
  • organic substances for example, synthetic polymers such as polystyrene, polyester, polyurethane, polyethylene, polypropylene, polyvinyl alcohol, (meth) acrylic polymer, (meth) acrylamide polymer, silicone polymer, epoxy resin, urethane resin; cellulose, dextran , Collagen, polygalacturonic acid, polyargicic acid, natural polymers such as gelatin and the like.
  • the inorganic substance examples include glass, ceramics, metals, alloys, metal oxides and the like.
  • the material of the microcarrier preferably contains an organic substance, and more preferably contains a natural polymer.
  • soluble microcarriers are preferable, but the present invention is not limited thereto.
  • the "soluble microcarrier” means a microcarrier that can be decomposed by means such as an enzyme to the extent that the adhered cell can be released from the microcarrier.
  • a cationic functional group may be introduced on the surface of the microcarrier.
  • the cationic functional group include groups containing a substituted or unsubstituted amino group such as a dimethylamino group, a diethylamino group and an amino group.
  • a cell adhesion polymer may be arranged on the surface of the microcarrier.
  • the cell adhesion polymer may be a polypeptide or polysaccharide exhibiting cell adhesion, and may be collagen, gelatin, alginic acid, Matrigel TM (BD Biosciences), hyaluronic acid, laminin, fibronectin, vitronectin, elastin, heparan sulfate. , Dextran, dextran sulfate, chondroitin sulfate and the like.
  • the cell adhesion polymer may be a partial peptide or oligosaccharide exhibiting cell adhesion.
  • microcarriers examples include a spherical shape, a flat shape, a columnar shape, a plate shape, and a prismatic shape.
  • the microcarriers preferably include spherical microcarriers.
  • the microcarrier may be a porous microcarrier having pores inside or a microcarrier having no pores inside.
  • the average particle size (D50) of the microcarrier is, for example, 50 to 1,000 ⁇ m, preferably 100 to 500 ⁇ m, and more preferably 150 to 250 ⁇ m from the viewpoint of promoting cell proliferation.
  • the average particle size of the microcarrier is a value measured as the median diameter (D50) in physiological saline or a medium.
  • the average particle size of the microcarriers can be measured by a laser diffraction / scattering type particle size distribution measuring device.
  • a fresh microcarrier or a used microcarrier can be used, and it is preferable to use a fresh microcarrier.
  • fresh microcarrier means a microcarrier that has never been used as a carrier (scaffold) for cell culture, that is, an unused microcarrier.
  • used microcarrier means a microcarrier that has already been used as a carrier for cell culture.
  • the concentration of the microcarriers in the suspension can be appropriately adjusted based on the shape, size, surface area, etc. of the microcarriers, and is, for example, 0.01 to 100 g / L and 0.5 to 50 g / L. It can be L, or 1 to 20 g / L.
  • the medium a liquid medium is used in the production method according to the present disclosure.
  • the medium preferably contains inorganic salts, amino acids, sugars, and water.
  • the medium may further contain any component such as serum, nucleosides and / or nucleotides, vitamins, hormones, antibiotics, growth factors, adhesion factors and the like.
  • a medium known as a basal medium for cell culture can be used as the medium.
  • any medium known to be used for culturing selected cells can be used without particular limitation.
  • DMEM Dynabeco modified Eagle's medium
  • MEM Eagle's minimum essential medium
  • ⁇ MEM medium Eagle's minimum essential medium ⁇ modified type
  • GMEM Grasgo's minimum essential medium
  • IMDM Iskov modified Dalveco medium
  • Ham's F12 nutrition mixture F-12 ham
  • RPMI-1640 RPMI-1640 medium
  • McCoy's 5A McCoy 5A medium
  • MSC glow medium 2 manufactured by Promocell
  • Prime XV XSFM manufactured by Irvine Scientific
  • known media can be used, and in particular, media known to be used for culturing stem cells can be used.
  • the medium used for culturing can be free of heterologous components.
  • the medium containing no heterologous component is a substitute additive for serum (for example, Knockout Serum Replacement (KSR) (manufactured by Invitrogen), Chemically-defined Lipid concentrated (manufactured by Gibco), Glutamax (manufactured by Gibco), instead of the serum derived from animals. (Manufactured by the company), etc.) can be included.
  • KSR Knockout Serum Replacement
  • the medium preferably contains a nucleoside and / or a nucleotide, and more preferably contains a nucleoside.
  • the nucleoside may be a ribonucleoside, a deoxyribonucleoside, or a mixture thereof.
  • Nucleotides can be ribonucleotides, deoxyribonucleotides, or mixtures thereof.
  • Examples of the base contained in the nucleoside and the nucleotide include purine bases such as adenine and guanine, and pyrimidine bases such as cytosine, thymine and uracil. If the medium contains nucleosides and / or nucleotides, these concentrations in the medium can be 1-20 mg / L, or 5-10 mg / L.
  • the conditions for culturing adherent cells may be adjusted so as to be suitable for cell proliferation according to the type of cells.
  • the culture temperature can be, for example, 20 to 45 ° C, preferably 30 to 40 ° C.
  • the carbon dioxide concentration can be, for example, 1 to 20% by volume, preferably 3 to 15% by volume. In the case of mammalian cells, a temperature of 37 ° C. and a carbon dioxide concentration of 5% (v / v) are generally used.
  • Examples of the culture container include flasks, bioreactors, tanks, culture bags and the like.
  • Culturing can be performed by stirring or shaking the cell suspension. Each step may include time to stop stirring or shaking.
  • the stirring may be intermittent stirring only, continuous stirring only, or a combination of intermittent stirring and continuous stirring.
  • the explanations, modes, examples, conditions, etc. of the intermittent stirring and the continuous stirring described later can be independently applied to the steps (A), (B) and / or (C), respectively.
  • Culturing is preferably carried out in a state where the cell suspension placed in the culture vessel is stirred or shaken and the microcarriers are suspended in the cell suspension.
  • the stirring method can be appropriately selected depending on the type and size of the culture vessel selected according to the volume of the cell suspension described later, and for example, magnetic stirrer, mechanical stirrer, homomixer, homogenizer, vortex. A method using a mixer or the like can be mentioned. Examples of the shaking method include a method using a shaking machine. According to one embodiment, it is preferable to stir the cell suspension from the viewpoint of obtaining a suspension in which the microcarriers are well dispersed in the cell suspension.
  • the stirring speed at which the microcarriers can be well dispersed in the cell suspension depends on the shape and volume of the culture vessel, but is generally in the case of a 1 to 50 L stirred tank bioreactor (Stirred Tank Bioreactor). It can be 30 to 200 rpm, preferably 40 to 100 rpm. The stirring rate may be changed during culturing depending on the floating state of the microcarriers or cells.
  • step (A) adherent cells are cultured in a cell suspension containing adherent cells, microcarriers, and a medium and having a volume of 0.3 L or more.
  • the adherent cells adhere to the microcarriers and proliferate, thereby obtaining a cell suspension containing the adherent cells adhered to the microcarriers.
  • the adherent cells obtained through the step (A) include a population of adherent cells adhered to the microcarriers. Culturing can be carried out for a predetermined time.
  • the volume of the cell suspension can be, for example, 0.3 L or more, 0.5 L or more, or 1 L or more from the viewpoint of homogeneous and efficient mass culture of adherent cells.
  • the upper limit of the volume of the cell suspension is not particularly limited, but may be, for example, 10 L or less, or 5 L or less from the viewpoint of efficiency and economy.
  • step (A) a cell suspension containing adherent cells, fresh microcarriers, and a medium and having a volume of 0.3 L or more is obtained, and the adherent cells are cultured.
  • the cell suspension obtained in the step (A) and before culturing may be referred to as a cell suspension (A1).
  • the cell suspension after culturing in the step (A) may be referred to as a cell suspension (A2).
  • the cell suspension (A1) can be obtained, for example, by mixing at least adherent cells, fresh microcarriers, and medium. More specifically, the cell suspension (A1) can be obtained by mixing at least adherent cells that are not adhered to the microcarriers, fresh microcarriers, and fresh medium.
  • the volume of the cell suspension (A1) can be, for example, 0.3 L or more, 0.5 L or more, or 1 L or more from the viewpoint of homogeneous and efficient mass culture of adherent cells.
  • the upper limit of the volume of the cell suspension (A1) is not particularly limited, but may be, for example, 10 L or less, or 5 L or less from the viewpoint of efficiency and economy.
  • the concentration of adherent cells in the cell suspension (A1) is, for example, 1 ⁇ 10 3 to 2 ⁇ 10 5 cells / mL, preferably 5 ⁇ 10 3 to 1 ⁇ 10 5 cells / mL, more preferably. is 1 ⁇ 10 4 ⁇ 5 ⁇ 10 4 cells / mL.
  • the concentration of fresh microcarriers in the cell suspension (A1) is, for example, 0.1-50 g / L, preferably 0.5-10 g / L, more preferably 1-5 g / L. ..
  • the culture period in the step (A) varies depending on the cell seeding density, cell type, culture conditions, etc., but is generally in a state where the cells are sufficiently grown, for example, the cell adhesion region of the microcarrier (adhesion cells adhere).
  • the period until it becomes 80% or more, 90% or more, 95% or more, or 100% confluent can be set based on the possible region).
  • the growth state of cells in the cell adherent region can be observed with a fluorescence microscope (manufactured by KEYENCE CORPORATION).
  • the growth state of cells can be confirmed by determining the percentage (degree of confluence) of the spread area of cells adhering to the surface of microcarriers based on the surface area of microcarriers.
  • the culture period can be, for example, 2 to 14 days.
  • the adherent cells may be cultured by stirring the cell suspension (A1) under appropriate culture conditions in which the temperature, carbon dioxide concentration, etc. are adjusted. can.
  • the stirring may be intermittent stirring only, continuous stirring only, or a combination of intermittent stirring and continuous stirring.
  • the step (A) comprises intermittently stirring the cell suspension (A1) and continuously stirring the cell suspension obtained through intermittent stirring. Intermittent stirring and continuous stirring will be described later. After the continuous stirring, the intermittent stirring may be performed, or the combination of the intermittent stirring and the continuous stirring may be repeated.
  • the agitation in step (A) consists only of intermittent agitation of the cell suspension (A1).
  • the volume of the cell suspension (A2) obtained in the step (A) can be 0.3 L or more, 0.5 L or more, or 1 L or more.
  • the medium is added to the cell suspension (A1) in the middle of culturing, and / or a part or all of the medium contained in the cell suspension (A1) in the middle of culturing is a fresh medium. Can be exchanged for.
  • two or more cell suspensions (A1) in the middle of culturing may be combined.
  • adherent cells that are not adhered to the microcarriers may be added to the cell suspension (A1) in the middle of culturing.
  • the upper limit of the volume of the cell suspension (A2) is not particularly limited, but may be, for example, 10 L or less, or 5 L or less from the viewpoint of efficiency and economy.
  • the adherent cells are cultured in a cell suspension containing the adherent cells, microcarriers, and medium obtained through the step (A) and having a volume of 5 L or more.
  • the adherent cells adhered to the microcarriers obtained in step (A) migrate to another microcarrier, preferably a fresh microcarrier, and proliferate, thereby adhering to the microcarrier.
  • a cell suspension containing cells is obtained. That is, the adherent cells obtained through the step (B) include a population of adherent cells adhered to the microcarriers. Culturing can be carried out for a predetermined time.
  • the volume of the cell suspension can be, for example, 5 L or more, 8 L or more, or 10 L or more.
  • the volume of the cell suspension can be, for example, 50 L or less, 40 L or less, or 30 L or less from the viewpoint of efficiently mass-culturing adherent cells.
  • step (B) a cell suspension containing the adherent cells obtained through step (A), fresh microcarriers, and a medium and having a volume of 5 L or more is obtained, and the adherent cells are cultured. ..
  • the cell suspension obtained in step (B) and before culturing may be referred to as cell suspension (B1).
  • the cell suspension after culturing in the step (B) may be referred to as a cell suspension (B2).
  • the cell suspension (B1) can be obtained, for example, by mixing at least the adherent cells obtained through the step (A), fresh microcarriers, and a medium. More specifically, the cell suspension (B1) can be obtained by mixing at least part or all of the cell suspension (A2), fresh microcarriers, and fresh medium. For mixing, it is also possible to use a combination of two or more cell suspensions (A2) obtained by separately performing the independent step (A). In this case, the cell suspension (B1) contains adherent cells derived from two or more cell suspensions (A2).
  • the culture vessel used in the step (A) and containing the cell suspension (A1) may be continuously used, or a culture vessel different from the culture vessel used in the step (A) may be used. You may use it.
  • a cell suspension (B1) can be obtained by adding a fresh microcarrier and a medium to the culture vessel.
  • the cell suspension (B1) can be obtained by adding the cell suspension (A1), fresh microcarriers, and medium to the culture vessel. In either case, the order of addition is not particularly limited.
  • the volume of the cell suspension (B1) can be, for example, 5 L or more, 8 L or more, or 10 L or more from the viewpoint of efficiently mass-culturing adherent cells.
  • the volume of the cell suspension (B1) can be, for example, 50 L or less, 40 L or less, or 30 L or less from the viewpoint of efficiently mass-culturing adherent cells.
  • the volume of the cell suspension (B1) is preferably larger than the volume of the cell suspension (A2).
  • the ratio of the volume of the cell suspension (B1) to the volume of the cell suspension (A2) ([volume of the cell suspension (B1)] / [volume of the cell suspension (A2)]) is the cell suspension.
  • the turbid liquid (A2) to the cell suspension (B1) it is, for example, 1.5 to 20, preferably 2 to 10, and more preferably 3 to 6.
  • the ratio of the volume of cell suspension (B1) to the total volume of cell suspension (A2) used for mixing [Cell suspension (B1)).
  • Volume] / [total volume of cell suspension (A2) used for mixing]) is, for example, 1.5 to 20, preferably 2 to 10, and more preferably 3 to 8.
  • “scale-up" means increasing the volume of the culture environment.
  • the concentration of adherent cells in the cell suspension (B1) is, for example, 1 ⁇ 10 3 to 2 ⁇ 10 5 cells / mL, preferably 5 ⁇ 10 3 to 1 ⁇ 10 5 cells / mL, more preferably. is 1 ⁇ 10 4 ⁇ 5 ⁇ 10 4 cells / mL.
  • the concentration of fresh microcarriers in the cell suspension (B1) is, for example, 0.1-50 g / L, preferably 0.5-10 g / L, more preferably 1-5 g / L. ..
  • the culture period in the step (B) varies depending on the cell seeding density, cell type, culture conditions, etc., but is generally 80 based on a sufficiently grown cell state, for example, a cell adhesionable region of a microcarrier. It can be a period until it becomes% or more, 90% or more, 95% or more, or 100% confluent.
  • the culture period can be, for example, 2 to 10 days.
  • the adherent cells may be cultured by stirring the cell suspension (B1) under appropriate culture conditions in which the temperature, carbon dioxide concentration, etc. are adjusted. can.
  • the stirring may be intermittent stirring only, continuous stirring only, or a combination of intermittent stirring and continuous stirring.
  • step (B) comprises intermittently stirring the cell suspension (B1) and continuously stirring the cell suspension obtained through intermittent stirring. Intermittent stirring and continuous stirring will be described later. After the continuous stirring, the intermittent stirring may be performed, or the combination of the intermittent stirring and the continuous stirring may be repeated.
  • the agitation in step (B) consists only of intermittent agitation of the cell suspension (B1).
  • the volume of the cell suspension (B2) obtained in the step (B) can be, for example, 5 L or more, 8 L or more, or 10 L or more.
  • the medium is added to the cell suspension (B1) in the middle of culturing, and / or a part or all of the medium contained in the cell suspension (B1) in the middle of culturing is fresh. Can be replaced with medium.
  • two or more cell suspensions (B1) in the middle of culturing may be combined.
  • adherent cells that are not adhered to the microcarriers may be added to the cell suspension (B1) in the middle of culturing.
  • the upper limit of the volume of the cell suspension (B2) is not particularly limited, but can be, for example, 50 L or less, 40 L or less, or 30 L or less from the viewpoint of efficiently mass-culturing adherent cells.
  • the adherent cells are cultured in a cell suspension containing the adherent cells, microcarriers, and medium obtained through the step (B) and having a volume of 10 L or more.
  • the adherent cells adhered to the microcarriers obtained in step (B) migrate to another microcarrier, preferably a fresh microcarrier, and proliferate, thereby adhering to the microcarrier.
  • a cell suspension containing cells is obtained. That is, the adherent cells obtained through the step (C) include a population of adherent cells adhered to the microcarriers. Culturing can be carried out for a predetermined time.
  • the volume of the cell suspension can be, for example, 10 L or more, 20 L or more, or 30 L or more from the viewpoint of efficiently mass-culturing adherent cells.
  • the volume of the cell suspension can be, for example, 500 L or less, 300 L or less, 150 L or less, 100 L or less, or 80 L or less from the viewpoint of efficiently mass-culturing adherent cells.
  • step (C) a cell suspension containing the adherent cells obtained through step (B), fresh microcarriers, and a medium and having a volume of 10 L or more is obtained, and the adherent cells are cultured. ..
  • the cell suspension obtained in the step (C) and before culturing may be referred to as a cell suspension (C1).
  • the cell suspension after culturing in the step (C) may be referred to as a cell suspension (C2).
  • the cell suspension (C1) can be obtained, for example, by mixing at least the adherent cells obtained through the step (B), fresh microcarriers, and a medium. More specifically, the cell suspension (C1) can be obtained by mixing at least some or all of the cell suspension (B2), fresh microcarriers, and fresh medium. For mixing, it is also possible to use a combination of two or more cell suspensions (B2) obtained by separately performing an independent step (B). In this case, the cell suspension (C1) contains adherent cells derived from two or more cell suspensions (B2).
  • the volume of the cell suspension (C1) can be, for example, 10 L or more, 20 L or more, or 30 L or more from the viewpoint of efficiently mass-culturing adherent cells.
  • the volume of the cell suspension (C1) can be, for example, 500 L or less, 300 L or less, 150 L or less, 100 L or less, or 80 L or less from the viewpoint of efficiently mass-culturing adherent cells.
  • the volume of the cell suspension (C1) is preferably larger than the volume of the cell suspension (B2).
  • the ratio of the volume of the cell suspension (C1) to the volume of the cell suspension (B2) ([volume of the cell suspension (C1)] / [volume of the cell suspension (B2)]) is the cell suspension.
  • the turbid liquid (B2) to the cell suspension (C1) it is, for example, 1.5 to 20, preferably 2 to 10, and more preferably 2 to 6.
  • a cell suspension (C1) is obtained by mixing a part or all of the cell suspension (B2), fresh microcarriers, and a fresh medium, cells are obtained from the cell suspension (B2).
  • the ratio of the volume of cell suspension (C1) to the total volume of cell suspension (B2) used for mixing is, for example, 1.5 to 10, preferably 1.8 to 6, and more preferably 2 to 3. ..
  • the concentration of adherent cells in the cell suspension (C1) is, for example, 1 ⁇ 10 3 to 2 ⁇ 10 5 cells / mL, preferably 5 ⁇ 10 3 to 1 ⁇ 10 5 cells / mL, more preferably. is 1 ⁇ 10 4 ⁇ 5 ⁇ 10 4 cells / mL.
  • the concentration of fresh microcarriers in the cell suspension (C1) is, for example, 0.1-50 g / L, preferably 0.5-10 g / L, more preferably 1-5 g / L. ..
  • the culture period in step (C) varies depending on the cell seeding density, cell type, culture conditions, etc., but is generally 80 based on a sufficiently grown cell state, for example, a cell adherent region of a microcarrier. It can be a period until it becomes% or more, 90% or more, 95% or more, or 100% confluent.
  • the culture period can be, for example, 3 to 20 days.
  • the adherent cells may be cultured by stirring the cell suspension (C1) under appropriate culture conditions in which the temperature, carbon dioxide concentration, etc. are adjusted. can.
  • the stirring may be intermittent stirring only, continuous stirring only, or a combination of intermittent stirring and continuous stirring.
  • step (C) comprises intermittently stirring the cell suspension (C1) and continuously stirring the cell suspension obtained through intermittent stirring. Intermittent stirring and continuous stirring will be described later. After the continuous stirring, the intermittent stirring may be performed, or the combination of the intermittent stirring and the continuous stirring may be repeated.
  • the agitation in step (C) consists only of intermittent agitation of the cell suspension (C1).
  • the volume of the cell suspension (C2) obtained by the step (C) can be, for example, 10 L or more, 20 L or more, or 30 L or more.
  • the medium is added to the cell suspension (C1) in the middle of culturing, and / or a part or all of the medium contained in the cell suspension (C1) in the middle of culturing is fresh. Can be replaced with medium.
  • two or more cell suspensions (C1) in the middle of culturing may be combined.
  • adherent cells that are not adhered to the microcarriers may be added to the cell suspension (C1) in the middle of culturing.
  • the upper limit of the volume of the cell suspension (C2) is not particularly limited, but may be, for example, 500 L or less, 300 L or less, 150 L or less, 100 L or less, or 80 L or less from the viewpoint of economy.
  • the method for producing a cell suspension includes steps (A), (B) and (C).
  • steps (A), (B) and (C) By carrying out steps (A), (B) and (C), a cell suspension containing adherent cells (for example, cell suspension (C2)) is obtained.
  • adherent cells are subcultured.
  • the method for producing a cell suspension may include each step independently twice or more. Any step may be included between each step.
  • the total number of steps (A), steps (B), and steps (C) included in the method for producing a cell suspension is preferably 6 times or less, and preferably 5 times or less. It is more preferable, and it is more preferable that it is 3 times or 4 times.
  • the total number of times of "scale-up to (C1)” is preferably 5 times or less, more preferably 4 times or less, further preferably 2 times or 3 times, and 2 times. Is particularly preferable.
  • a manufacturing method including one step (A), one step (B), and one step (C); a plurality of separately independent steps (A), 2 obtained above.
  • a production method including one step (B) and one step (C) using a combination of two or more cell suspensions (A2) (a total of four or more steps); one step (A).
  • any step included in the method for producing a cell suspension includes, for example, thawing frozen cells, washing cells, seeding cells, adding microcarriers, or containing in a medium or medium. Add at least one component to the cell suspension, replace at least a portion of the medium contained in the cell suspension, allow the cell suspension to stand, separate the cells from the microcarriers. This includes performing media exchange, cell culture, or media exchange and cell culture before or after step (A).
  • the method for producing a cell suspension does not include separating the adherent cells from the microcarriers, such as exfoliating the adherent cells from the microcarriers.
  • a method for producing a cell suspension is to obtain a cell suspension containing adherent cells, microcarriers, and a medium, and intermittently stir the cell suspension.
  • the cell suspension obtained through the intermittent stirring is continuously stirred.
  • the cell suspension here include, but are not limited to, the above-mentioned cell suspension (A1), cell suspension (B1), cell suspension (C1) and the like.
  • Adherent cells, microcarriers, medium and the like are as described above.
  • the method for producing a cell suspension is to thaw frozen cells, wash the cells, seed the cells, and add the medium and at least one component contained in the medium to the cell suspension. , The exchange of at least a part of the medium contained in the cell suspension, the standing of the cell suspension, the separation of the cells and the microcarriers, and the like may be included.
  • mass culture of adherent cells can be realized by a production method including intermittent stirring and continuous stirring.
  • continuous stirring is performed in order to obtain a good dispersed state of the microcarriers in the cell suspension for culturing.
  • the precipitation of microcarriers that may occur during continuous stirring and intermittent stirring is due to the variation in the weight of microcarriers due to the amount of adhesion of adherent cells, and the microcarriers to which the adherent cells are not adhered. It is considered that this is due to the aggregation of the cells. It can be inferred that such precipitation was eliminated by a simple method of performing intermittent stirring and continuous stirring in combination, making it possible to obtain a good suspension state of microcarriers and realizing mass culture of adherent cells. ..
  • intermittent stirring means that stirring is performed or not performed at predetermined time intervals.
  • intermittent stirring means that stirring is performed for a predetermined time and that stirring is not performed for a predetermined time alternately.
  • Alternately performing may be a combination of stirring and not stirring being repeated two or more times ("stirring" followed by "not stirring" 1 of the combination. Count as times.)
  • the time for stirring and the time for not stirring may be the same or different.
  • the stirring time and / or the non-stirring time may be the same or different between each time.
  • the time without stirring may be the time for allowing the cell suspension to stand.
  • the time for stirring is, for example, 0.5 to 60 minutes, preferably 1 to 20 minutes, and more preferably 3 to 10 minutes.
  • the time without stirring is, for example, 0.1 to 10 hours, preferably 0.5 to 6 hours, and more preferably 1 to 3 hours. Examples of combinations include “'stir for 0.5-60 minutes' followed by'0.1-10 hours, no stir'", followed by "" stir for 1-20 minutes “and subsequent” "No stirring for 0.5 to 6 hours” is preferable, and "" Stir for 3 to 10 minutes “followed by" No stirring for 1 to 3 hours "” is more preferable.
  • the time for performing intermittent stirring is, for example, 1 to 80 hours, preferably 10 to 40 hours, and more preferably. Is 20 to 30 hours.
  • Continuous stirring performed after intermittent stirring means continuous stirring for a predetermined time.
  • the stirring time is, for example, 1 to 14 days, preferably 2 to 10 days, and more preferably 3 to 7 days.
  • stirring may be temporarily stopped due to, for example, addition of medium to the cell suspension, medium exchange, and the like.
  • Temporary arrest here is usually not just the time to allow the cell suspension to stand.
  • the time for performing the stirring does not have to be the sum of the time for stirring before the temporary stop and the time for performing the stirring after the temporary stop. That is, the time for stirring may be divided and measured by stopping the stirring.
  • the intermittent stirring and the continuous stirring may be performed continuously, or may include not stirring for a predetermined time between the intermittent stirring and the continuous stirring.
  • the predetermined time is, for example, 0.1 to 24 hours, preferably 0.5 to 5 hours, and more preferably 1 to 2 hours. Not stirring may be allowing the cell suspension to stand.
  • the intermittent stirring may be performed, or the combination of the intermittent stirring and the continuous stirring may be repeated.
  • the total culture time including intermittent stirring and continuous stirring can be appropriately set according to the type, purpose, and culture conditions of the adherent cells to be cultured.
  • the method for producing adherent cells is to prepare a cell suspension obtained by any of the above embodiments, and to obtain adherent cells from the cell suspension.
  • the method for producing adherent cells may include any step.
  • the cell suspension obtained by the method for producing a cell suspension contains adherent cells adhered to microcarriers.
  • a state in which the adherent cells adhered to the microcarriers by collecting the adherent cells adhered to the microcarriers in the cell suspension from the cell suspension using a known separation method such as removal of supernatant and centrifugation. Can be obtained at. Then, the adherent cells can be obtained by exfoliating the adherent cells from the microcarriers by a known exfoliation method such as enzyme treatment and collecting them. Alternatively, the adherent cells can be obtained by lysing the microcarriers by a known lysing method and collecting the adherent cells.
  • a predetermined enzyme or the like is added to a cell suspension containing adherent cells adhered to the microcarriers to detach the adherent cells from the microcarriers, lyse the microcarriers, or both. By doing so, only adherent cells can be made recoverable. Then, the adherent cells can be obtained by collecting the adherent cells in a recoverable state using a known separator such as a filter.
  • the cell suspension obtained by the method for producing a cell suspension may contain microcarriers, adherent cells adhered to the microcarriers, and useful substances.
  • the useful substance may be a substance secreted from adherent cells, and examples thereof include extracellular vesicles such as exosomes, microvesicles, and apoptotic bodies; and functional proteins such as cytokines, hormones, and antibodies.
  • the method for producing a useful substance-containing liquid is to prepare a cell suspension obtained by any of the above embodiments, and to prepare a useful substance-containing liquid from the cell suspension. Including getting.
  • the method for producing a useful substance-containing liquid may further include any step.
  • An arbitrary step in the method for producing a useful substance-containing liquid includes, for example, an additional culture step for recovering the useful substance.
  • a recovery medium for efficiently recovering the useful substance produced inside the cells by the above-mentioned method for producing a cell suspension may be used.
  • the recovery medium include a medium that does not contain additives containing exosomes such as FBS and can maintain the growth of cells, and can be appropriately selected depending on the type of useful substance, the type of cells, and the like.
  • FBS additives containing exosomes
  • the useful substance contains exosomes
  • DMEM / F12 containing FGF-2, insulin, transferrin, and selenium can be mentioned.
  • a useful substance-containing liquid can be obtained by separating the microcarriers and adherent cells in the cell suspension from the useful substance and recovering the liquid containing the useful substance. For the separation, a known separation method such as recovery of the supernatant or centrifugation can be used.
  • a useful substance-containing liquid can be obtained by removing microcarriers and adherent cells from the cell suspension.
  • the useful substance-containing liquid does not have to contain substantially no microcarriers and adherent cells.
  • the useful substance-containing liquid does not substantially contain microcarriers and cells” means, for example, that the number of cells is 2 or less and the particle size is 50 ⁇ m or more per 100 mL of the useful substance-containing liquid. It means that there is one or less carriers.
  • the method for producing a useful substance is to prepare a cell suspension obtained by any of the above embodiments or a useful substance-containing liquid obtained by the above embodiment. , And obtaining useful substances from the cell suspension or the useful substance-containing liquid.
  • the method for producing a useful substance may further include any step.
  • the useful substance can be obtained by separating the useful substance and other components in the useful substance-containing liquid and recovering the useful substance.
  • a known separation method such as removal of the supernatant or centrifugation can be used.
  • a useful substance can be obtained by isolating the useful substance from a cell suspension.
  • the useful substance comprises an exosome.
  • Exosomes are vesicles that contain a lipid bilayer.
  • the diameter of the exosome is, for example, 50-1000 nm, 50-300 nm, or 50-200 nm. Since exosomes contain various physiologically active substances such as proteins, nucleic acids, sugars, and lipids, they are expected to be used in therapeutic and diagnostic methods for diseases, pharmaceuticals, cosmetics, and the like.
  • Exosomes are secreted from adherent cells into the cell suspension when adherent cells are present in the cell suspension.
  • the exosome derived from the adherent cell is not particularly limited as long as it is obtained from the above-mentioned adherent cell.
  • the exosomes described in WO 2009/105044 can be mentioned.
  • the useful substance-containing liquid may be filtered, concentrated, or filtered and concentrated.
  • a membrane having a size or molecular weight cutoff value can be used to filter the useful substance-containing liquid.
  • the useful substance-containing liquid can be filtered or concentrated using tangential flow filtration or ultrafiltration.
  • the useful substance in the useful substance-containing liquid can be isolated from the useful substance-containing liquid and used.
  • the useful substance in the useful substance-containing liquid can be isolated from the useful substance-containing liquid by subjecting the useful substance-containing liquid to a known treatment such as spray drying or freeze-drying.
  • the useful substance-containing liquid is an exosome-containing liquid
  • the exosome and other components in the exosome-containing liquid can be separated based on the properties of the exosome.
  • Exosomes can be isolated from exosome-containing solutions based on the properties of exosomes.
  • exosomes can be isolated based on molecular weight, size, shape, composition, or biological activity. Specifically, precipitation by ultracentrifugation, fractionation by density gradient ultracentrifugation, fractionation using size exclusion chromatography, ion exchange chromatography (eg, manufactured by CIMmultus TM EV (BIA separations)). ), Precipitation using a protein (for example, MagCapture TM Exosome Isolation Kit PS (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.)), Precipitation using an antibody, Polymers such as polyethylene glycol These methods can be carried out by one or a combination of two or more.
  • a protein for example, MagCapture TM Exosome Isolation Kit PS (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.)
  • Precipitation using an antibody for example, MagCapture TM Exosome Isolation Kit PS (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.)
  • exosomes can be used to track the activity of exosomes in methods of producing exosome-containing liquids and methods of producing exosomes.
  • exosome activity can be confirmed using static light scattering, dynamic light scattering, UV-visible detectors, fluorescence detectors, or differential refractive index detectors.
  • a method for producing a cell suspension which comprises the following (A), (B), and (C).
  • the above (A). ) Contains adherent cells, fresh microcarriers, and medium to obtain a cell suspension having a volume of 0.3 L or more, and culturing the adherent cells.
  • the (B) contains the adherent cells obtained through the (A), fresh microcarriers, and a medium to obtain a cell suspension having a volume of 5 L or more, and the adherent cells are cultured.
  • the (C) contains the adherent cells obtained through the (B), fresh microcarriers, and a medium to obtain a cell suspension having a volume of 10 L or more, and the adherent cells are cultured. Including doing, The method for producing a cell suspension according to the above [1]. [3] In the above [1] or [2], the adherent cells obtained through the above (A) and the adherent cells obtained through the above (B) include a population of cells adhered to microcarriers. The method for producing a cell suspension according to the above. [4] The above (B) comprises mixing at least the adherent cells obtained through the above (A), fresh microcarriers, and fresh medium to obtain a cell suspension.
  • the (C) comprises mixing at least the adherent cells obtained through the (B), fresh microcarriers, and fresh medium to obtain a cell suspension.
  • a cell suspension containing adherent cells, microcarriers, and a medium is obtained, and the cell suspension is intermittently stirred, and the cell suspension obtained through the intermittent stirring is continuously stirred.
  • a method for producing a cell suspension which comprises the process of making a cell suspension.
  • the cell suspension containing the adherent cells, microcarriers, and medium may be the cell suspension in at least one selected from the group consisting of (A), (B), and (C).
  • a method for producing adherent cells which comprises obtaining adherent cells from the cell suspension.
  • a method for producing a useful substance-containing liquid which comprises obtaining a useful substance-containing liquid from the cell suspension.
  • hMSCs Second-generation human bone marrow-derived mesenchymal stem cells obtained from were prepared.
  • the hMSCs were seeded in tissue culture flasks at a density of 3,000 cells / cm 2 and cultured in an incubator at 37 ° C. and 5% by volume CO 2 for 7 days.
  • Thermo Fisher Scientific Using an enzyme solution (TrypLE Select, manufactured by Thermo Fisher Scientific), cells were detached from the flask to obtain a third passage hMSC.
  • the 3rd passage hMSC was seeded in a multi-layer cell culture vessel (10-layer Nunc EasyFill Cell Factory, manufactured by Thermo Fisher Scientific) at a density of 3,000 cells / cm 2 , cultured for 7 days, and then treated with an enzyme. Subculture cells were obtained.
  • the obtained 4th generation hMSC was cryopreserved in liquid nitrogen.
  • FIG. 1 shows the concept of a method for producing a cell suspension including steps (A), (B) and (C).
  • the lower figures of steps (B) and (C) show the migration of adherent cells between microcarriers, the so-called bead-to-bead cell transfer.
  • unused microcarriers were used as fresh microcarriers, that is, microcarriers in which adherent cells did not adhere to 100% of the area of the cell adherent area.
  • the cell adhesion area (surface area / mass) of Cytodex 1 is 4,400 cm 2 / g.
  • Step (A) Two disposable 2L bioreactors (UniVessel (R) SU, manufactured by Sartorius Stedim Biotech) were used for culturing hMSC. Place 1 L of the same medium as above in each bioreactor and use a controller (BIOSTAT (R) B, manufactured by Sartorius Stedim Biotech) for temperature (37 ° C), pH (7.4), and dissolved oxygen concentration DO (100). %) was controlled over 4 hours. The hMSC cryopreserved above was thawed and washed in a water bath at 37 ° C.
  • BIOSTAT (R) B manufactured by Sartorius Stedim Biotech
  • Each bioreactor was inoculated with fresh microcarriers (Cytodex 1, manufactured by GE Healthcare Bio-Sciences Corp.) 2.27 g and 4 subculture of hMSC 3 ⁇ 10 7 cells, to obtain a cell suspension.
  • the resulting cell suspension was stirred intermittently. The conditions for intermittent stirring are "stirring for 5 minutes” and subsequent "not stirring for 25 minutes (standing)” as one cycle, and this is performed for 6 cycles. That was. Subsequently, the cell suspension was allowed to stand (without stirring) overnight. 1 L of fresh medium was added to each bioreactor to increase the volume of cell suspension to 2 L.
  • the stirring speed was increased from 70 rpm to 85 rpm, and continuous stirring was performed.
  • 50% by volume of medium was exchanged, and the cells were cultured until the 9th day.
  • step (A) the cell suspension having a volume of 1 L is intermittently stirred for 3 hours, and after standing overnight (15 hours), the cell suspension having a volume of 2 L is continuously stirred. Stirring was performed for 7 days.
  • Step (B) A 50 L culture bag (Flexsafe STR, manufactured by Sartorius Stedim Biotech) was set in a bag holder, and 15 L of the same medium as above was added to the culture bag. Medium temperature, pH, and dissolved oxygen concentration DO were controlled overnight using a control tower (BIOSTAT (R) STR, manufactured by Sartorius Stedim Biotech).
  • a control tower BIOSTAT (R) STR, manufactured by Sartorius Stedim Biotech.
  • To a 50 L culture bag 1 L of a suspension in which 18.2 g of fresh microcarriers are dispersed in a medium and 4 L of the cell suspension obtained in the above step (A) on the 9th day of culture are added to a volume of 20 L. A cell suspension was obtained. The resulting cell suspension was stirred intermittently for 25 hours. The conditions for intermittent stirring are "stirring for 5 minutes” and subsequent "not stirring for 2 hours (standing)” as one cycle, and this is performed for 12 cycles. That was. The cell suspension was then continuously
  • step (B) intermittent stirring was performed for 25 hours on the cell suspension having a volume of 20 L, and continuous stirring was performed for 4 days following the intermittent stirring.
  • Step (C) Then, on the 13th day, 1 L of a suspension in which 34.1 g of fresh microcarriers were dispersed in a medium and 29 L of warmed fresh medium were added to a culture bag. The volume of the cell suspension increased to 50 L. Similar to step (B), the cell suspension was stirred intermittently for 25 hours. The cell suspension was then continuously stirred for 7 days and 50% by volume of medium exchange was performed on day 20. After the medium exchange, the cell suspension was continuously stirred for 7 days.
  • step (C) intermittent stirring was performed for 25 hours on a cell suspension having a volume of 50 L, followed by intermittent stirring, and continuous stirring was performed for a total of 14 days.
  • step (A)-(C) samples were taken daily from the cell suspension in cell culture. The collected samples were used to evaluate cell density (cells / mL), viability (%), and total cell number (cells). The evaluation results are shown in Table 1. In the column of step (A) in the table, the results in the cell suspension having a total volume of 4 L are shown. The cell density on "0 days” was also calculated assuming that the total volume was 4 L.
  • ⁇ Recovery of hMSC (manufacturing of adherent cells)> From the agitated cell suspension on day 27, a sample for collecting cells was collected in a culture vessel. The sample was allowed to stand, the supernatant was removed after the microcarriers had precipitated, and the microcarriers were washed twice with phosphate buffered saline (PBS) containing neither magnesium nor calcium ions. An enzyme solution (TrypLE Select, manufactured by Thermo Fisher Scientific) was added to the microcarrier, and the culture vessel was vibrated for 12 minutes using a small constant temperature shaking incubator (Bioshaker, manufactured by Titec Co., Ltd.).
  • PBS phosphate buffered saline
  • the recovered cells had proliferative ability, expressed surface markers (CD73, CD90, and CD105), and maintained the ability to differentiate into three lineages into adipocytes, osteoocytes, and chondrocytes. bottom.
  • Example 2 Production of useful substance-containing liquid
  • the cells to be cultured are adipose-derived mesenchymal stem cells (manufactured by Lonza), and in step (C), intermittent stirring is performed for 25 hours on a cell suspension having a volume of 50 L to obtain intermittent stirring.
  • adipose-derived stem cells were cultured using the methods described in Step (A), Step (B) and Step (C) of Example 1 except that continuous stirring was performed for a total of 12 days.
  • a cell suspension having a volume of 50 L containing adipose-derived stem cell line stem cells is obtained.
  • the step (C) is completed on the 12th day from the start of the step (C), the cells after the completion of the culture are collected, and then PBS 50L is added to wash the cells. After removing the PBS, add 10 ng / mL FGF-2 (manufactured by Bio Vision) and 50 L DMEM / F12 (manufactured by Gibco) containing 1 ⁇ ITS (insulin, transferrin, selenium: manufactured by InVitria), and rotate appropriately. Incubate for 48 hours with stirring in numbers to obtain a cell suspension.
  • FGF-2 manufactured by Bio Vision
  • 50 L DMEM / F12 manufactured by Gibco
  • the cell suspension was removed with adherent cells, microcarriers, and cell debris floating in a 0.65 ⁇ m module, and the exosome-containing solution was removed. obtain.
  • exosome purified liquid (useful substance-containing liquid) 1> 50 mL of the exosome concentrate obtained above is centrifuged at 35,000 rpm for 70 minutes using an ultracentrifuge XE-90 and a swing rotor SW 41 Ti (both manufactured by Beckmann Coulter) to precipitate exosomes. After removing the supernatant, 10 mL of PBS is added to the precipitated exosomes, and the mixture is stirred with a vortex mixer, and then the exosomes are precipitated at 35,000 rpm for 70 minutes. After removing the supernatant, 30 ⁇ L of PBS is added and pipetting is performed to recover the exosomes.
  • exosome purified liquid (useful substance-containing liquid) 3> 50 mL of the exosome concentrate obtained above is purified using a chromatography system FPLC AKTA pure 150 (manufactured by AKTA) and a 1 mL volume monolith column (manufactured by BIA separations).
  • a chromatography system FPLC AKTA pure 150 manufactured by AKTA
  • a 1 mL volume monolith column manufactured by BIA separations.
  • exosomes are carried on a monolith column pretreated according to the protocol, and the mobile phase is allowed to flow for 1 hour to wash the column. After washing, the supported exosomes are recovered by changing the mobile phase to 50 mM HEPES buffer and 2.0 M NaCl aqueous solution and allowing the mobile phase to flow in at 1 mL / min.
  • the particle size distribution and concentration of exosomes in the exosome purification solution purified above are measured using a nanoparticle tracking device Zeta View (manufactured by Particle Metalix) according to the EV measurement method attached to the software.
  • Sensitivity is set to 82 and Shooter is set to 100.
  • the particle size distribution and concentration of exosomes can be confirmed.
  • the particle size distribution is 20 to 500 nm
  • the concentration is 10 10 to 10 11 parts / mL.
  • the mixed solution was applied to a 10% polyacrylamide gel (manufactured by ATTO), and the protein was electrophoresed and separated under the conditions of a protein amount of 500 ng / lane, 150 V, and 30 minutes using an electrophoresis device (manufactured by ATTO). do.
  • the proteins in the gel are transferred to a polyvinylidene fluoride (PVDF) membrane (manufactured by ATTO) at 100 V for 15 minutes using a transfer device (both manufactured by ATTO).
  • PVDF polyvinylidene fluoride
  • a blocking buffer manufactured by Nacalai Tesque
  • 1 ⁇ g / mL anti-human CD9 mouse IgG, anti-human CD63 mouse IgG, anti-human CD81 mouse IgG antibody (all manufactured by Cosmo Bio Co., Ltd.) and at 4 ° C. React each for 18 hours.
  • the membrane is washed with TBS buffer, and 0.2 ⁇ g / mL mouse-HRP antibody is reacted at room temperature for 1 hour.

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WO2022080461A1 (ja) * 2020-10-16 2022-04-21 富士フイルム和光純薬株式会社 細胞外小胞の産生用培地、培地キット、添加剤及び細胞外小胞の産生方法
JP7160294B1 (ja) 2022-03-31 2022-10-25 ヒューマン・メタボローム・テクノロジーズ株式会社 脂質二重膜に覆われた微粒子又は細胞外小胞の回収方法および脂質二重膜に覆われた微粒子又は細胞外小胞を回収するための回収キット
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WO2025197916A1 (ja) * 2024-03-18 2025-09-25 大日本印刷株式会社 新規細胞培養方法

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