WO2021200744A1 - 増殖細胞の生産方法、細胞生産物の生産方法、間葉系幹細胞集団およびその生産方法、幹細胞の培養上清およびその生産方法、並びに治療剤 - Google Patents

増殖細胞の生産方法、細胞生産物の生産方法、間葉系幹細胞集団およびその生産方法、幹細胞の培養上清およびその生産方法、並びに治療剤 Download PDF

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WO2021200744A1
WO2021200744A1 PCT/JP2021/013099 JP2021013099W WO2021200744A1 WO 2021200744 A1 WO2021200744 A1 WO 2021200744A1 JP 2021013099 W JP2021013099 W JP 2021013099W WO 2021200744 A1 WO2021200744 A1 WO 2021200744A1
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Prior art keywords
culture
cells
cell
mesenchymal stem
laminin
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English (en)
French (fr)
Japanese (ja)
Inventor
康友 柳田
中豪 李家
武文 石田尾
南　一成
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Cell Exosome Therapeutics
Cell Exosome Therapeutics Inc
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Cell Exosome Therapeutics
Cell Exosome Therapeutics Inc
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Priority to EP22207135.9A priority Critical patent/EP4155389A3/en
Priority to JP2022512159A priority patent/JPWO2021200744A1/ja
Priority to CA3173721A priority patent/CA3173721A1/en
Priority to CN202180024893.1A priority patent/CN115362252A/zh
Priority to US17/995,095 priority patent/US20230172992A1/en
Priority to AU2021247879A priority patent/AU2021247879B2/en
Priority to CN202310081275.1A priority patent/CN116179480A/zh
Priority to EP21779739.8A priority patent/EP4130240A4/en
Application filed by Cell Exosome Therapeutics, Cell Exosome Therapeutics Inc filed Critical Cell Exosome Therapeutics
Publication of WO2021200744A1 publication Critical patent/WO2021200744A1/ja
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Priority to JP2022160399A priority patent/JP2022181218A/ja
Priority to AU2022259729A priority patent/AU2022259729B2/en
Priority to JP2023206347A priority patent/JP2024015245A/ja
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    • C12N2501/20—Cytokines; Chemokines
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    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/50—Cell markers; Cell surface determinants
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    • C12N2533/00—Supports or coatings for cell culture, characterised by material
    • C12N2533/50—Proteins
    • C12N2533/52—Fibronectin; Laminin

Definitions

  • the present invention relates to a method for producing proliferating cells, a method for producing a cell product, a mesenchymal stem cell population and a method for producing the same, a culture supernatant of stem cells and a method for producing the same, and a therapeutic agent.
  • an object of the present invention is to provide a cell culture technique capable of proliferating cells seeded at a low cell density at a high proliferation ratio. Further, the present invention is a cell culture technique that is a further development of the above cell culture technique, and can produce a large amount of cell products by a simple method while maintaining the adhered state of the obtained proliferating cells. An object of the present invention is to provide a cell culture technique. Furthermore, the present invention provides a mesenchymal stem cell population having novel characteristics based on the above-mentioned cell culture technique, and includes a large amount of cell products such as cytokines based on the above-mentioned cell culture technique. , An object of the present invention is to provide a culture supernatant of stem cells. Another object of the present invention is to provide a therapeutic agent containing the above-mentioned mesenchymal stem cell population or the above-mentioned culture supernatant.
  • a culture substrate selected from laminin fragments having integrin-binding activity and variants thereof in a growth culture medium in which cells seeded at a cell density of 0.002-2000 cells / cm 2 is used.
  • a method for producing proliferating cells comprises culturing by adherent culture to proliferate the cells.
  • Stem cells are cultured by adhesive culture in a growth culture medium in the presence of a culture substrate selected from a laminin fragment having integrin-binding activity and a variant thereof to obtain the growth cells in an adhesive state.
  • a culture substrate selected from a laminin fragment having integrin-binding activity and a variant thereof to obtain the growth cells in an adhesive state.
  • the recovery culture solution preferably contains no protein in the above-mentioned "production culture solution", whereas it preferably contains a protein that promotes cell proliferation.
  • proteins that promote stem cell growth include bFGF (basic fibroblast growth factor), TGF ⁇ 1 (transforming growth factor ⁇ 1), EGF (epidermal growth factor), IGF (insulin-like growth factor), and VEGF (vascular). Endophilic cell growth factor), HGF (hepatocellular growth factor), insulin, albumin, transferase and the like.
  • the recovery culture medium is a medium prepared by adding growth factors to basal medium for cell culture (for example, MEM, DMEM, IMDM, Ham's F-12, DMEM / F12, RPMI1640, etc.). You may use it.
  • the method according to the second embodiment since cells seeded at a low cell density can be grown at a high proliferation rate, a large amount of cell products are produced from a small amount of raw material cells. Can be done.
  • the method according to the second embodiment can maintain the adherent state of cells, it is possible to shift from proliferation culture to production culture or repeat the cycle of production culture and recovery culture simply by exchanging the culture solution. It is convenient in that it can be used. That is, the method according to the second embodiment does not require cell passage, and thus it is possible to eliminate lot differences due to passage and costs associated with passage.
  • the method according to the second embodiment is convenient in that the culture supernatant containing the cell product can be easily recovered because the adherent state of the cells can be maintained.
  • a method for producing proliferating cells which comprises culturing and proliferating the cells.
  • A4 Seeding a cell suspension containing a growth culture medium and an amount of cells that achieves a seeding density of 0.002-2000 cells / cm 2 into a culture vessel.
  • iPS cell human induced pluripotent stem cell
  • ES cell human embryonic stem cell
  • the cells are human umbilical cord-derived mesenchymal stem cells, human bone marrow-derived mesenchymal stem cells, human adipose-derived mesenchymal stem cells, human placenta-derived mesenchymal stem cells, or human umbilical cord blood-derived mesenchymal stem cells.
  • [A14] The method according to any one of [A1] to [A13], wherein the cell is a human umbilical cord-derived mesenchymal stem cell.
  • [A15] The method according to any one of [A1] to [A14], wherein the cell is a frozen cell.
  • [A16] The method according to any one of [A1] to [A15], wherein the cell is a cell prepared by thawing a frozen cell.
  • the cells are 0.002 to 1900 cells / cm 2 , preferably 0.002 to 1800 cells / cm 2 , more preferably 0.002 to 1700 cells / cm 2 , and even more preferably 0.002 to 1600.
  • Cells / cm 2 more preferably 0.002-1500 cells / cm 2 , still more preferably 0.002-1400 cells / cm 2 , still more preferably 0.002-1300 cells / cm 2 , still more preferably 0.002.
  • the cells are 1 to 2000 cells / cm 2 , preferably 1 to 1900 cells / cm 2 , more preferably 1 to 1800 cells / cm 2 , still more preferably 1 to 1700 cells / cm 2 , still more preferably. 1-1600 cells / cm 2 , more preferably 1-1500 cells / cm 2 , even more preferably 1-1400 cells / cm 2 , even more preferably 1-1300 cells / cm 2 , even more preferably 1-1200 cells / cm.
  • [A26] The method according to [A25], wherein the laminin fragment is a human-derived laminin fragment.
  • [A27] The method according to [A25] or [A26], wherein the laminin fragment is a laminin E8 fragment.
  • the laminin fragments are laminin 511 E8 fragment, laminin 521 E8 fragment, laminin 411 E8 fragment, laminin 421 E8 fragment, laminin 332 E8 fragment, laminin 311 E8 fragment, laminin 321 E8 fragment, laminin 211 E8 fragment, laminin 221.
  • [A31] The method according to [A30], wherein the variant is a complex of a laminin fragment having integrin-binding activity and another functional molecule.
  • [A32] The method according to [A30] or [A31], wherein the variant is a complex of a laminin fragment having integrin-binding activity and a growth factor-binding molecule.
  • [A33] The method according to [A31] or [A32], wherein the laminin fragment is a laminin E8 fragment.
  • the laminin E8 fragment is laminin 511 E8 fragment, laminin 521 E8 fragment, laminin 411 E8 fragment, laminin 421 E8 fragment, laminin 332 E8 fragment, laminin 311 E8 fragment, laminin 321 E8 fragment, laminin 211 E8 fragment, laminin. 221 E8 Fragment, Laminin 213 E8 Fragment, Laminin 111 E8 Fragment, or Laminin 121 E8 Fragment [A33]. [A35] The method according to [A33] or [A34], wherein the laminin E8 fragment is a laminin 511 E8 fragment. [A36] The method according to [A33] or [A34], wherein the laminin E8 fragment is a laminin 421 E8 fragment.
  • [A40] The method according to any one of [A1] to [A39], wherein the concentration of the laminin fragment or a variant thereof in the growth culture solution is 0.005 ⁇ g to 2 ⁇ g per 1 cm 2 of the culture area of the culture vessel. .. [A41] Described in any one of [A1] to [A40], wherein the concentration of the laminin fragment or a variant thereof in the growth culture solution is 0.01 ⁇ g to 0.5 ⁇ g per 1 cm 2 of the culture area of the culture vessel. the method of.
  • a method for producing a cell product which comprises culturing the proliferating cells in a production culture medium while maintaining the adherent state, and causing the cells to produce a cell product.
  • a method for producing a cell product which comprises culturing the proliferating cells in a production culture medium while maintaining the adherent state, and causing the cells to produce a cell product.
  • B2 To obtain the proliferated cells in an adherent state by culturing the cells in a proliferative culture medium according to the method according to any one of [A1] to [A42]. By culturing the proliferating cells in a production culture medium while maintaining the adherent state, the cells are allowed to produce a cell product.
  • mesenchymal stem cells are known to be HLA-ABC positive and CD105 positive.
  • the positiveness of the cell surface marker can be represented by the value of the positive rate of the cell surface marker.
  • the positive rate of the cell surface marker can be determined by a flow cytometer using a fluorescently labeled antibody of the cell surface marker as described in Examples described later.
  • the term "mesenchymal stem cell” includes both the mesenchymal stem cell before culturing by the above-mentioned method and the mesenchymal stem cell obtained by culturing by the above-mentioned method. That is, in the present specification, the term “mesenchymal stem cell” refers to mesenchymal stem cells that are HLA-ABC positive, CD105-positive mesenchymal stem cells, and HLA-ABC-negative mesenchymal stem cells. It also includes stem cells and mesenchymal stem cells that are CD105 negative. Therefore, as used herein, the term “mesenchymal stem cell” can be defined as a mesenchymal stem cell that is positive for CD44, CD73, CD90 and negative for CD45, CD34, CD31, HLA-DR. ..
  • a method for producing a culture supernatant of stem cells is a method for producing a culture supernatant of stem cells.
  • Stem cells are cultured by adhesive culture in a growth culture medium in the presence of a culture substrate selected from a laminin fragment having integrin-binding activity and a variant thereof to obtain the growth cells in an adhesive state.
  • a culture substrate selected from a laminin fragment having integrin-binding activity and a variant thereof to obtain the growth cells in an adhesive state.
  • the proliferating cells in a production culture medium while maintaining the adherent state
  • the cells are allowed to produce a cell product.
  • the culture in the production culture solution and the culture in the recovery culture solution are alternately repeated while maintaining the adhesive state of the cells, and the method is the culture in the production culture solution.
  • a method is provided that further comprises collecting the supernatant of the production culture broth
  • This culture supernatant contains, for example, 5000-1000000 pg / mL, preferably 10000-1000000 pg / mL, more preferably 15000-1000000 pg / mL HGF.
  • HGF is known to have angiogenic and wound healing effects.
  • a culture supernatant of stem cells containing a CD9 / CD63 EC domain fusion protein of 50 pg / mL or more can be obtained (see FIG. 42).
  • this culture supernatant is also referred to as a culture supernatant according to the second embodiment.
  • This culture supernatant contains, for example, 50 pg / mL or more, preferably 100 pg / mL or more, more preferably 200 pg / mL or more of the CD9 / CD63 EC domain fusion protein.
  • This culture supernatant contains, for example, 50-100,000 pg / mL, preferably 100-100,000 pg / mL, more preferably 200-100,000 pg / mL of CD9 / CD63 EC domain fusion protein.
  • the CD9 / CD63 EC domain fusion protein is a marker protein for exosomes, and a high content of the CD9 / CD63 EC domain fusion protein in the culture supernatant indicates a high content of exosomes in the culture supernatant. Exosomes are known to have angiogenic and wound healing effects.
  • the culture supernatant according to the first embodiment and the culture supernatant according to the second embodiment are each of MCP-1 (monocyte chemotactic protein-1) of 3000 pg / mL or more and GRO of 1000 pg / mL or more. (Growth-related oncogene), and fibronectin of 5 ⁇ g / mL or more can be further contained (see FIGS. 18 to 20).
  • MCP-1 monocyte chemotactic protein-1
  • GRO 1000 pg / mL or more.
  • fibronectin 5 ⁇ g / mL or more can be further contained (see FIGS. 18 to 20).
  • Each of the culture supernatant according to the first embodiment and the culture supernatant according to the second embodiment is, for example, 3000 pg / mL or more, preferably 4000 pg / mL or more, more preferably 6000 pg / mL or more MCP-1; for example, 1000 pg. Further containing fibronectin of / mL or more, preferably 2000 pg / mL or more, more preferably 4000 pg / mL or more; and, for example, 5 ⁇ g / mL or more, preferably 6 ⁇ g / mL or more, more preferably 8 ⁇ g / mL or more. Can be done.
  • Each of the culture supernatant according to the first embodiment and the culture supernatant according to the second embodiment is, for example, 3000 to 1000000 pg / mL, preferably 4000 to 1000000 pg / mL, and more preferably 6000 to 1000000 pg / mL MCP-1.
  • 1000-1000000 pg / mL preferably 2000-1000000 pg / mL, more preferably 4000-1000000 pg / mL GRO; and, for example, 5-1000 ⁇ g / mL, preferably 6-1000 ⁇ g / mL, more preferably 8-1000 ⁇ g. It can further contain / mL fibronectin.
  • MCP-1, GRO and fibronectin are all known to have angiogenic and wound healing effects. Since GRO is also known as CXCL1, it is also referred to as GRO / CXCL1 in this specification.
  • each of the culture supernatant according to the first embodiment and the culture supernatant according to the second embodiment has TGF-1b (transforming growth factor-1b) of 200 pg / mL or more and IL of 5 pg / mL or more.
  • TGF-1b transforming growth factor-1b
  • IL IL of 5 pg / mL or more.
  • -4 interleukin-4
  • IL-10 interleukin-10
  • Each of the culture supernatant according to the first embodiment and the culture supernatant according to the second embodiment is TGF-1b having, for example, 200 to 100,000 pg / mL, preferably 300 to 100,000 pg / mL, and more preferably 500 to 100,000 pg / mL.
  • IL-4 of 5 to 100,000 pg / mL, preferably 10 to 100,000 pg / mL, more preferably 20 to 100,000 pg / mL; and, for example, 8 to 100,000 pg / mL, preferably 10 to 100,000 pg / mL, more preferably 12. It can further contain ⁇ 100,000 pg / mL IL-10.
  • TGF-1b, IL-4 and IL-10 are all known to have anti-inflammatory effects.
  • each of the culture supernatant according to the first embodiment and the culture supernatant according to the second embodiment does not contain at least one of insulin, transferrin, and albumin. It is more preferable that each of the culture supernatant according to the first embodiment and the culture supernatant according to the second embodiment does not contain any of insulin, transferrin, and albumin.
  • the culture supernatant not containing the above-mentioned component can be obtained. Considering that the culture supernatant is used as a therapeutic agent, it is desirable that the culture supernatant does not contain the above components.
  • each of the culture supernatant according to the first embodiment and the culture supernatant according to the second embodiment does not contain heterologous components.
  • heterologous refers to a species different from the species of stem cells used to obtain the culture supernatant. Considering that the culture supernatant is used as a therapeutic agent, it is desirable that the culture supernatant does not contain heterologous components.
  • each of these cytokines has been demonstrated to have a concentration of 10 pg / mL or higher (see FIGS. 16-19, 21-29, 33-41). ).
  • concentration of each cytokine of 20 or more cytokines varies depending on the type of cytokine, but is, for example, 1,000,000 pg / mL or less.
  • mesenchymal stem cells other than mesenchymal stem cells, such as pluripotent stem cells such as ES cells and iPS cells, nerve stem cells, skin stem cells, and hepatic stem cells, Somatic stem cells such as muscle stem cells and adipose stem cells are also known to secrete cytokines, extracellular matrix, and exosomes into the culture medium.
  • pluripotent stem cells such as ES cells and iPS cells
  • nerve stem cells such as ES cells and iPS cells
  • skin stem cells and hepatic stem cells
  • Somatic stem cells such as muscle stem cells and adipose stem cells are also known to secrete cytokines, extracellular matrix, and exosomes into the culture medium.
  • stem cells other than mesenchymal stem cells such as pluripotent stem cells such as ES cells and iPS cells, nerve stem cells, skin stem cells, hepatic stem cells, muscle stem cells, and fat.
  • somatic stem cells such as stem cells
  • a culture supernatant containing a large amount of cytokines, extracellular matrix, and exosomes can be obtained as in the case of "culture supernatant of mesenchymal stem cells”.
  • a therapeutic agent containing the mesenchymal stem cell population of the present invention is provided.
  • the mesenchymal stem cell population of the present invention is ⁇ 1.
  • Proliferating cells can be obtained by culturing mesenchymal stem cells in a proliferative culture medium in the presence of a laminin fragment having integrin-binding activity according to the method described in the column of>.
  • the mesenchymal stem cell population of the present invention is characterized by a low positive rate of a specific cell surface marker HLA-ABC, unlike the conventional findings regarding the mesenchymal stem cell population.
  • the culture supernatant of the present invention is characterized by containing a large amount of cytokines, extracellular matrix and exosomes as compared with the conventional culture supernatant.
  • the mesenchymal stem cell population of the present invention includes cytokines (eg, HGF, MCP-1, GRO / CXCL1, PDGF-AA, VEGF, TGF-1b, IL-4, IL-10, IL-13, IL-7, IL-15, IL-9, IL-8, EOTAXIN, IL-6, G-CSF, GM-CSF, MCP-3, IL-12P40, IP-10, and MIP-1 ⁇ ), extracellular matrix (eg, It has been demonstrated to secrete Fibronectin) and exosomes, and the culture supernatant of the present invention has been demonstrated to contain cytokines, extracellular matrix and exosomes secreted by mesenchymal stem cells.
  • cytokines eg, HGF, MCP-1, GRO / CXCL1, PDGF-AA, VEGF, TGF-1b, IL-4, IL-10, IL-13, IL-7, IL-15, IL-9, IL-8, E
  • the therapeutic agent containing the mesenchymal stem cell population of the present invention or the culture supernatant of the present invention is HGF, MCP-1, MCP-3, GRO / CXCL1, Fibronectin, PDGF-AA, VEGF, IL-8, EOTAXIN. , IL-6, IP-10, and exosomes are known to have angiogenic effects, so ischemic diseases such as lower limb ischemia, myocardial infarction, cerebral infarction, spinal cord infarction, and chronic arterial occlusion; epithelium Wounds, burns and other wounds; can be used to treat senchymal ischemia.
  • IL-7, IL-15, GM-CSF, and G-CSF are considered to be involved in immunoregulation in the therapeutic agent containing the mesenchymal stem cell population of the present invention or the culture supernatant of the present invention.
  • immune disorders such as GVHD (graft-versus-host disease), Sjogren's syndrome, atopic dermatitis, collagen disease, multiple sclerosis, autoimmune disorders; it can be used for the treatment of cancer disorders.
  • the therapeutic agent containing the mesenchymal stem cell population of the present invention and the therapeutic agent containing the culture supernatant of the present invention are, for example, liquid preparations, preferably liquid preparations for injection or liquid preparations for external use. Is.
  • the therapeutic agent may be diluted with a pharmaceutically acceptable medium.
  • a pharmaceutically acceptable medium is, for example, a medium or infusion preparation of mesenchymal stem cells.
  • Therapeutic agents may include additives to increase storage stability, isotonicity, absorbability, and / or viscosity.
  • the therapeutic agent may be in the form of a cell sheet if it comprises a mesenchymal stem cell population.
  • the dose of the therapeutic agent can be appropriately determined according to the target disease, age, body weight, symptoms, and the like.
  • a single dose of the therapeutic agent is, for example, 1000-100000000 cells / kg, preferably 100,000,000-10000000 cells / kg. This dose may be administered as a single dose in multiple doses, or the dose may be administered in multiple doses.
  • the therapeutic agent contains a culture supernatant
  • a single dose of the therapeutic agent is expressed in terms of the amount (mL) of the culture supernatant before dilution, for example, 0.01 to 100 mL / kg, preferably 0.1 to 10 mL. / Kg.
  • This dose may be administered in multiple doses as a single dose, or this dose may be administered in multiple doses.
  • the mesenchymal stem cells are umbilical cord-derived mesenchymal stem cells, bone marrow-derived mesenchymal stem cells, adipose-derived mesenchymal stem cells, placenta-derived mesenchymal stem cells, or cord blood-derived mesenchymal stem cells [C1]. ] The method described in. [C3] The method according to [C1] or [C2], wherein the mesenchymal stem cells are umbilical cord-derived mesenchymal stem cells or adipose-derived mesenchymal stem cells, preferably umbilical cord-derived mesenchymal stem cells.
  • the mesenchymal stem cells are human umbilical cord-derived mesenchymal stem cells, human bone marrow-derived mesenchymal stem cells, human adipose-derived mesenchymal stem cells, human placenta-derived mesenchymal stem cells, or human cord blood-derived mesenchymal stem cells.
  • the mesenchymal stem cell is a human umbilical cord-derived mesenchymal stem cell or a human adipose-derived mesenchymal stem cell, preferably a human umbilical cord-derived mesenchymal stem cell.
  • the method of. [C7] The method according to any one of [C1] to [C6], wherein the growth culture solution is a growth culture solution containing a protein that promotes the growth of the stem cells.
  • the growth culture solution is a culture solution containing a basal medium for cell culture to which a growth factor has been added.
  • [C23] The method according to [C21] or [C22], wherein the laminin E8 fragment is a laminin 511 E8 fragment.
  • [C24] The method according to [C21] or [C22], wherein the laminin E8 fragment is a laminin 421 E8 fragment.
  • [C25] The method according to any one of [C20] to [C24], wherein the growth factor-binding molecule is heparan sulfate.
  • [C29] Described in any one of [C1] to [C28], wherein the concentration of the laminin fragment or a variant thereof in the growth culture solution is 0.01 ⁇ g to 0.5 ⁇ g per 1 cm 2 of the culture area of the culture vessel. the method of. [C30] Described in any one of [C1] to [C29], wherein the concentration of the laminin fragment or a variant thereof in the growth culture solution is 0.05 ⁇ g to 0.25 ⁇ g per 1 cm 2 of the culture area of the culture vessel. the method of.
  • Stem cell culture supernatant and its production method A method for producing a culture supernatant of stem cells.
  • Stem cells are cultured by adhesive culture in a growth culture medium in the presence of a culture substrate selected from a laminin fragment having integrin-binding activity and a variant thereof to obtain the growth cells in an adhesive state.
  • a culture substrate selected from a laminin fragment having integrin-binding activity and a variant thereof to obtain the growth cells in an adhesive state.
  • the culture in the production culture solution and the culture in the recovery culture solution are alternately repeated while maintaining the adhesive state of the cells, and the method is the culture in the production culture solution.
  • the method further comprises collecting the supernatant of the production culture broth after the above.
  • the supernatant of the production culture solution is collected after the second and subsequent cultures in the production culture solution, preferably after the third and subsequent cultures in the production culture solution.
  • the stem cells are somatic stem cells such as mesenchymal stem cells, nerve stem cells, skin stem cells, hepatic stem cells, muscle stem cells, and adipose stem cells; or induced pluripotent stem cells (iPS cells) and embryonic stem cells (ES cells).
  • stem cell is an umbilical cord-derived mesenchymal stem cell or an adipose-derived mesenchymal stem cell, preferably an umbilical cord-derived mesenchymal stem cell.
  • the stem cell is a human stem cell.
  • the stem cells are human somatic stem cells such as human mesenchymal stem cells, human neural stem cells, human skin stem cells, human hepatic stem cells, human muscle stem cells, and human adipose stem cells; or human induced pluripotent stem cells (human iPS cells).
  • the stem cells are human umbilical cord-derived mesenchymal stem cells, human bone marrow-derived mesenchymal stem cells, human adipose-derived mesenchymal stem cells, human placenta-derived mesenchymal stem cells, or human umbilical cord blood-derived mesenchymal stem cells.
  • the stem cell is a human umbilical cord-derived mesenchymal stem cell or a human adipose-derived mesenchymal stem cell, preferably a human umbilical cord-derived mesenchymal stem cell.
  • the growth culture solution is a growth culture solution containing a protein that promotes the growth of the stem cells.
  • the growth culture medium is a culture medium containing a basal medium for cell culture to which a growth factor has been added.
  • the laminin fragments are laminin 511 E8 fragment, laminin 521 E8 fragment, laminin 411 E8 fragment, laminin 421 E8 fragment, laminin 332 E8 fragment, laminin 311 E8 fragment, laminin 321 E8 fragment, laminin 211 E8 fragment, laminin 221.
  • the laminin E8 fragment is laminin 511 E8 fragment, laminin 521 E8 fragment, laminin 411 E8 fragment, laminin 421 E8 fragment, laminin 332 E8 fragment, laminin 311 E8 fragment, laminin 321 E8 fragment, laminin 211 E8 fragment, laminin. 221 E8 Fragment, Laminin 213 E8 Fragment, Laminin 111 E8 Fragment, or Laminin 121 E8 Fragment [E26].
  • [E31] The method according to any one of [E23] to [E30], wherein the variant is a complex of laminin 511 E8 fragment and heparan sulfate.
  • [E32] The method according to any one of [E23] to [E30], wherein the variant is a complex of laminin 421 E8 fragment and heparan sulfate.
  • [E33] The method according to any one of [E1] to [E32], wherein the concentration of the laminin fragment or a variant thereof in the growth culture solution is 0.005 ⁇ g to 2 ⁇ g per 1 cm 2 of the culture area of the culture vessel. ..
  • [E41] The method according to any one of [E1] to [E40], wherein the production culture medium is a basal medium for cell culture or a basal medium for cell culture to which a nutritional component of cells is added.
  • the production culture medium is a basic medium for cell culture to which a nutritional component of cells, preferably amino acids, is added.
  • the production culture solution does not contain a laminin fragment or a variant thereof.
  • [E46] The method according to any one of [E1] to [E45], wherein the recovery culture medium is a culture medium containing a protein that promotes the proliferation of the stem cells.
  • the recovery culture medium is a culture medium containing a basal medium for cell culture to which a growth factor has been added.
  • the recovery culture medium is a basic medium for cell culture to which a growth factor has been added.
  • the recovery culture solution has the same composition as the growth culture solution.
  • [E50] The method according to any one of [E1] to [E49], wherein the recovery culture solution does not contain a laminin fragment or a variant thereof.
  • [E51] The method according to any one of [E1] to [E50], wherein the culture in the recovery culture solution is carried out for 0.5 to 10 days, preferably 2 to 5 days.
  • [E52] The method according to any one of [E1] to [E51], wherein the cycle of the culture in the production culture solution and the culture in the recovery culture solution is repeated 2 to 10 times.
  • [F1] A culture supernatant of stem cells containing HGF of 5000 pg / mL or more.
  • [F2] The culture supernatant according to [F1], wherein the culture supernatant contains HGF of 10,000 pg / mL or more, preferably 15,000 pg / mL or more.
  • [F3] The culture supernatant according to [F1] or [F2], wherein the culture supernatant contains HGF of 5000 to 1000000 pg / mL, preferably 10,000 to 1,000,000 pg / mL, more preferably 15,000 to 1,000,000 pg / mL.
  • the culture supernatant contains 50 to 100,000 pg / mL, preferably 100 to 100,000 pg / mL, more preferably 200 to 100,000 pg / mL of CD9 / CD63 EC domain fusion protein.
  • the culture supernatant further contains a CD9 / CD63 EC domain fusion protein of 50-100,000 pg / mL, preferably 100-100,000 pg / mL, more preferably 200-100,000 pg / mL [F1]-[F3].
  • the culture supernatant is 3000-1000000 pg / mL, preferably 4000-1000000 pg / mL, more preferably 6000-1000000 pg / mL MCP-1; 1000-1000000 pg / mL, preferably 2000-1000000 pg / mL, More preferably 4000-1000000 pg / mL GRO; and any of [F1]-[F9] further containing 5-1000 ⁇ g / mL, preferably 6-1000 ⁇ g / mL, more preferably 8-1000 ⁇ g / mL fibronectin.
  • the culture supernatant according to 1.
  • the culture supernatant according to any one of [F1] to [F20], wherein the culture supernatant contains 20 or more types of cytokines, and each cytokine has a concentration of 10 pg / mL or more.
  • the culture supernatant is a culture supernatant of somatic stem cells such as mesenchymal stem cells, nerve stem cells, skin stem cells, hepatic stem cells, muscle stem cells, and adipose stem cells; or artificial pluripotent stem cells (iPS cells), embryos.
  • the culture supernatant according to any one of [F1] to [F21] which is a culture supernatant of pluripotent stem cells such as sex stem cells (ES cells).
  • the culture supernatant according to any one of [F1] to [F23], wherein the culture supernatant is a culture supernatant of human mesenchymal stem cells.
  • the culture supernatant is a culture supernatant of human umbilical cord-derived mesenchymal stem cells, a culture supernatant of human bone marrow-derived mesenchymal stem cells, a culture supernatant of human adipose-derived mesenchymal stem cells, and a human placenta-derived mesenchymal stem cell.
  • [F29] A culture supernatant obtained by the method according to any one of [E1] to [E52].
  • [F30] The culture supernatant according to any one of [F1] to [F28], wherein the culture supernatant is obtained by the method according to any one of [E1] to [E52].
  • Therapeutic agent > [G1] A therapeutic agent containing the mesenchymal stem cell population according to any one of [D1] to [D6] or the culture supernatant according to any one of [F1] to [F30]. [G2] A therapeutic agent containing the mesenchymal stem cell population according to any one of [D1] to [D6]. [G3] A therapeutic agent containing the culture supernatant according to any one of [F1] to [F30].
  • the therapeutic agent is an ischemic disease such as lower limb ischemia, myocardial infarction, cerebral infarction, spinal cord infarction, chronic arterial occlusion; wounds such as epithelial wounds and burns; Arthritis such as arthroplasty; Inflammatory diseases such as nephritis, keratitis, cytokine storm; Psychiatric diseases such as autism and insomnia that are considered to be one of the causes; GVHD (transplant-to-host disease), Schegren The following one of [G1] to [G3], which is for treating an immune disease such as a syndrome, atopic dermatitis, collagen disease, multiple sclerosis, autoimmune disease; or a cancer disease. Therapeutic agent.
  • the therapeutic agent is for treating ischemic disease; preferably lower limb ischemia, myocardial infarction, cerebral infarction, spinal cord infarction, or chronic arterial occlusion; more preferably lower limb ischemia [G1]. ] To [G4].
  • the therapeutic agent according to any one.
  • [G6] The subject is administered with the mesenchymal stem cell population according to any one of [D1] to [D6] or the culture supernatant according to any one of [F1] to [F30]. How to treat injuries and illnesses.
  • [G7] A method for treating an injury or illness, which comprises administering the mesenchymal stem cell population according to any one of [D1] to [D6] to a subject.
  • [G8] A method for treating an injury or illness, which comprises administering the culture supernatant according to any one of [F1] to [F30] to a subject.
  • the injuries include ischemic diseases such as lower limb ischemia, myocardial infarction, cerebral infarction, spinal cord infarction, and chronic arterial occlusion; wounds such as epithelial wounds and burns; Arthritis such as arthritis; Inflammatory diseases such as cytokine storm, nephritis, and keratitis; Psychiatric diseases such as autism and insomnia that are considered to be one of the causes; GVHD (transplant-to-host disease), Schegren's syndrome , An inflammatory disease such as atopic dermatitis, collagen disease, multiple sclerosis, autoimmune disease; or the method according to any one of [G6] to [G8], which is a cancer disease.
  • ischemic diseases such as lower limb ischemia, myocardial infarction, cerebral infarction, spinal cord infarction, and chronic arterial occlusion
  • wounds such as epithelial wounds and burns
  • Arthritis such as arthritis
  • Inflammatory diseases such
  • the therapeutic agent is an ischemic disease such as lower limb ischemia, myocardial infarction, cerebral infarction, spinal cord infarction, chronic arterial occlusion; wounds such as epithelial wounds and burns; Arthritis such as arthritis; inflammatory diseases such as cytokine storm, nephritis, and keratitis; mental diseases such as autism and insomnia that are considered to be caused by neuroinflammation; GVHD (transplant-to-host disease), Schegren The following one of [G12] to [G14], which is for treating an immune disease such as a syndrome, atopic dermatitis, collagen disease, multiple sclerosis, autoimmune disease; or a cancer disease. use.
  • the therapeutic agent is for treating ischemic disease; preferably lower limb ischemia, myocardial infarction, cerebral infarction, spinal cord infarction, or chronic arterial occlusion; more preferably lower limb ischemia [G12]. ] To [G15].
  • the therapeutic agent according to any one of.
  • Example 1 Proliferation culture (1) Method In Example 1, stem cell proliferation culture was performed. As stem cells, umbilical cord-derived mesenchymal stem cells (UCMSC) cryopreserved in a cryopreservation solution Stem Cell Banker (Xenoac) were used.
  • UMSC umbilical cord-derived mesenchymal stem cells
  • Xenoac Stem Cell Banker
  • each flask After 5 days (day5), 10 days (day10), 15 days (day15), and 20 days (day20) after sowing, each flask is treated with TrypLE TM Select (Thermo Fisher Scientific) for 10 to 20 minutes, and is single. After dispersing in cells, the number of cells was counted with a cell counter.
  • TrypLE TM Select Thermo Fisher Scientific
  • the present inventors seeded umbilical cord-derived mesenchymal stem cells (UCMSC) at a cell density of 2 cells / cm 2 according to the same method as in Experiment 1-2, and proliferated containing the iMatrix-511 laminin fragment. It has been demonstrated that cells can proliferate until they reach a confluent state even when cultured in a culture medium.
  • UMSC umbilical cord-derived mesenchymal stem cells
  • Umbilical cord-derived mesenchymal stem cells are hung in 100 ml of MSC Expansion XSFM B (Fuji Film Wako Pure Chemical Industries, Ltd.) (hereinafter also referred to as MSC medium B) with a cell number of 5x10e5 (seed density 1000 cells / cm 2).
  • MSC medium B MSC Expansion XSFM B
  • 5x10e5 seed density 1000 cells / cm 2
  • the cell suspension obtained after turbidity was seeded in a peel-off T512 flask (Sumitomo Bakelite Co., Ltd.).
  • MSC medium A protein-free medium
  • the amino acids added here are MEM essential amino acid solution (Fujifilm Wako Pure Chemical Industries, Ltd.) and MEM non-essential amino acid solution (Fujifilm Wako Pure Chemical Industries, Ltd.).
  • MSC medium A was added and cultured for 3 days (production culture). After culturing for 3 days, two images of cells were taken with an Olympus microscope. The two images were taken at different locations within the flask. Micrographs are shown in FIGS. 7A and 7B.
  • Umbilical cord-derived mesenchymal stem cells are hung in 100 ml of MSC Expansion XSFM B (Fuji Film Wako Pure Chemical Industries, Ltd.) (hereinafter also referred to as MSC medium B) with a cell number of 5x10e5 (seed density 1000 cells / cm 2).
  • MSC medium B MSC Expansion XSFM B
  • iMatrix-511 Laminine Fragment was added at 50 ⁇ l / 100 ml, and the obtained cell suspension was seeded in a peel-off T512 flask (Sumitomo Bakelite Co., Ltd.).
  • the medium was changed every 5 days. Five days after sowing, the iMatrix-511 laminin fragment was added again at 50 ⁇ l / 100 ml. No laminin fragment was added 10 days after sowing.
  • Example 3 Long-term production culture (1) Method In Example 3, long-term production culture was performed after proliferative culture of stem cells. The culture step performed in Example 3 is schematically shown in FIG. As stem cells, umbilical cord-derived mesenchymal stem cells (UCMSC) cryopreserved in a cryopreservation solution Stem Cell Banker (Xenoac) were used.
  • UMSC umbilical cord-derived mesenchymal stem cells
  • Xenoac Stem Cell Banker
  • Umbilical cord-derived mesenchymal stem cells are suspended in 100 ml of MSC Expansion XSFM B (Fujifilm Wako Pure Chemical Industries, Ltd.) (hereinafter also referred to as MSC medium B) with a cell number of 5x10e5 (seed density 1000 cells / cm2).
  • MSC medium B MSC Expansion XSFM B
  • IMatrix-511 Laminine Fragment was added at 50 ⁇ l / 100 ml, and the obtained cell suspension was seeded in a peel-off T512 flask (Sumitomo Bakelite Co., Ltd.).
  • the medium was changed every 5 days. Five days after sowing, the iMatrix-511 laminin fragment was added again at 50 ⁇ l / 100 ml. No laminin fragment was added 10 days after sowing.
  • VEGF vascular endothelial growth factor
  • IL-7 interleukin-7
  • HGF hepatocyte growth factor
  • the cells were returned to MSC medium B and cultured for 3 days (recovery culture). After the recovery culture for 3 days, the medium was replaced with MSC medium A again, and the culture was carried out for 4 days. This culture is referred to as "second production culture" in FIG.
  • FIGS. 10 to 12 Results The results of quantification of cytokines are shown in FIGS. 10 to 12.
  • the graphs of FIGS. 10 to 12 are shown in order from the left.
  • the amount of cytokines in the culture supernatant recovered after the first production culture in the example of the present invention, The amount of cytokines in the culture supernatant recovered after the second production culture in the example of the present invention, The amount of cytokines in the culture supernatant recovered after the third production culture in the example of the present invention and the amount of cytokines in the culture supernatant recovered in the comparative example are shown.
  • cytokines in an amount equal to or higher than that of the comparative example could be produced, and the production amount decreased even after repeated production culture. It has been shown to not or increase.
  • VEGF and HGF are cytokines with angiogenic effects.
  • the production amount of VEGF may be slightly smaller than that of the comparative example, it can be said that VEGF is produced in a sufficient amount because the absolute amount of VEGF production is large.
  • the production of HGF tended to increase as the production culture was repeated.
  • IL-7 is a cytokine involved in immune cell activation and inflammation. The production of IL-7 tended to increase as the production culture was repeated.
  • Example 3 a total of 3 production cultures were performed, and a total of 3 times of culture supernatant recovery and cytokine analysis were performed. However, the present inventors performed a total of 6 production cultures to obtain cytokines. It demonstrates that it can be produced.
  • the obtained proliferating cells are cultured in a protein-free culture medium containing no foreign components, and then produced and cultured while maintaining the adherent state of the proliferating cells. It can be performed.
  • recovery culture can be performed while maintaining the adherent state of the cells, and the ability of the cells to produce cell products can be restored.
  • a plurality of production cultures can be repeated while maintaining the cell adhesion state, and as a result, a large amount of cell products can be produced over a long period of time by a simple method.
  • Example 4 Mesenchymal stem cell population
  • mesenchymal stem cells were cultured in the presence of a laminin fragment, and the cell surface marker positive rate of the obtained mesenchymal stem cell population was analyzed.
  • mesenchymal stem cells were cultured in the presence of laminin fragments, and the expression of cell surface markers in the obtained mesenchymal stem cell population was confirmed by immunostaining images.
  • Method Experiment 4-1 (Example of the present invention) Umbilical cord-derived mesenchymal stem cells (UCMSC) or adipose-derived mesenchymal stem cells (ADMSC) with a cell number of 5x10e5 (seed density 1000 cells / cm 2 ), MSC Expansion XSFM B medium (Fujifilm Wako Pure Chemical Industries, Ltd.) 100 ml The iMatrix-511 laminin fragment (Nippi Co., Ltd.) was added at 50 ⁇ l / 100 ml, and the obtained cell suspension was seeded in a peel-off T512 flask (Sumitomo Bakelite Co., Ltd.). Umbilical cord-derived mesenchymal stem cells were cultured for a period of 1 week, 2 weeks, and 3 weeks, and adipose-derived mesenchymal stem cells were cultured for a period of 1 week and 2 weeks.
  • UMSC Umbilical cord-derived me
  • the cells were detached by treating with TrypLE Select solution (Thermo Fisher Scientific). The obtained cells were stained with the cell surface markers CD44, CD73, CD90, CD105, and HLA-ABC antibody with PE (phycoerythrin) (BioLegend), and each marker was positive using a flow cytometer (Sony). The rate was analyzed.
  • umbilical cord-derived mesenchymal stem cells were seeded on a 24-well plate (Corning) in the presence of a laminin fragment and cultured for 1 week. After that, mesenchymal stem cells adhered to 24 wells were immunostained with PE-equipped antibodies of cell surface markers CD73, CD90, CD105, and HLA-ABC, and bright-field imaging and fluorescence imaging were performed with a fluorescence microscope (Keyence). went.
  • UMSC Umbilical cord-derived mesenchymal stem cells
  • MSC Expansion XSFM B medium Flujifilm Wako Pure Chemical Industries, Ltd.
  • 5x10e5 seed density 1000 cells / cm 2
  • iMatrix-511 laminin fragment was added.
  • the seeds were sown in a peel-off T512 flask (Sumitomo Bakelite Co., Ltd.) without addition. Culturing was carried out in the same procedure as in Experiment 4-1 except that no laminin fragment was added.
  • Example 5 Culture supernatant of mesenchymal stem cells
  • the mesenchymal stem cells were proliferated and cultured according to the method described in Example 3, followed by multiple production cultures, and each production culture was performed. After that, the culture supernatant was collected. The amounts of cytokines, extracellular matrix, and exosome markers contained in the obtained culture supernatant of mesenchymal stem cells were analyzed.
  • cytokines and extracellular matrix contained in the culture supernatant were analyzed using an ELISA analysis kit (R & D Systems).
  • cytokines HGF (hepatocellular growth factor), MCP-1, GRO / CXCL1, PDGF-AA, VEGF (vascular endothelial growth factor), TGF-1b, IL-4, IL-10, IL-13, IL-7 , IL-15, IL-9, IL-1 ⁇ , IL-1 ⁇ , TNF- ⁇ , IL-8, EOTAXIN, IL-6, G-CSF, GM-CSF, MCP-3, IL-12P40, IP-10 , MIP-1 ⁇ was analyzed, and Fibronectin was analyzed as an extracellular matrix.
  • the amount of exosomes contained in the culture supernatant was analyzed using the CD9 / CD63 ELISA kit (Cosmo Bio) using the exosome marker protein (CD9 / CD63 fusion protein) as an index.
  • the amount of cytokine, the amount of extracellular matrix, and the amount of exosome marker protein refer to the values measured by the ELISA method using a specific antibody.
  • FIGS. 16 to 19 and 21 to 41 The results of the amount of cytokines contained in the culture supernatant are shown in FIGS. 16 to 19 and 21 to 41, and the results of the amount of fibronectin contained in the culture supernatant are shown in FIG. The result of the amount of is shown in FIG.
  • first refers to the amount of cytokine, fibronectin, or exosome marker protein in the culture supernatant recovered after the first production culture (4 days) in the example of the present invention.
  • second time indicates the amount of cytokine, fibronectin, or exosome marker protein in the culture supernatant recovered after performing the second production culture (4 days) in the example of the present invention, and "3". The same meanings apply to “the 4th time”, “the 4th time”, and “the 5th time”.
  • “Comparative Example” refers to the amount of cytokine, fibronectin, or exosome marker protein in the culture supernatant recovered 2 days after the start of the first production culture in Comparative Example. show.
  • proteins other than IL-1 ⁇ , IL-1 ⁇ , and TNF- ⁇ that is, HGF, MCP-1, GRO / CXCL1, PDGF-AA, VEGF, TGF-1b, IL-4, IL-10, IL-13, IL-7, IL-15, IL-9, IL-8, EOTAXIN, IL-6, G-
  • the amount of CSF, GM-CSF, MCP-3, IL-12P40, IP-10, MIP-1 ⁇ , Fibronectin, and exosome marker protein tended to increase.
  • the culture supernatant of mesenchymal stem cells is obtained by culturing in a protein-free culture medium containing no foreign components, all the proteins contained in this culture supernatant are interstitial. Since it is derived from mesenchymal stem cells, it has the advantage of being easy to apply as a therapeutic agent. Moreover, since the culture supernatant of mesenchymal stem cells can increase the content of cytokines such as HGF, fibronectin and exosomes by repeatedly collecting them, it is excellent in that therapeutic effects by these proteins and exosomes can be expected. ing.
  • the culture supernatant of mesenchymal stem cells is inflamed because the contents of IL-1 ⁇ , IL-1 ⁇ , and TNF- ⁇ , which are known to cause inflammation, do not increase when the culture supernatant is repeatedly collected. It is excellent in that the content of system cytokines is relatively low.
  • Example 6 Therapeutic agent In Example 6, the mesenchymal stem cells obtained according to the method of the present invention were administered to rats of a lower limb ischemia model, and the therapeutic effect was confirmed.
  • LDPI laser Doppler perfusion imaging
  • Umbilical cord-derived mesenchymal stem cells were cultured in the same manner as in Example 4 in the presence and absence of the laminin fragment, respectively.
  • ADMSC adipose-derived mesenchymal stem cells
  • Obtained mesenchymal stem cells ie, umbilical cord-derived mesenchymal stem cells cultured in the presence of laminin fragment, umbilical cord-derived mesenchymal stem cells cultured in the absence of laminin fragment, fat cultured in the presence of laminin fragment.
  • the cell suspension was intravenously administered from the tail vein with a 27-gauge needle.
  • the administration was performed 4 times in total on the 4th, 5th, 6th, and 10th days after the induction of lower limb ischemia.
  • As a control only DMEM / F12 medium was administered.
  • the mesenchymal stem cells obtained according to the method of the present invention are effective as a therapeutic agent for lower limb ischemia. Since this therapeutic effect is considered to be brought about by cytokines, extracellular matrix, and exosomes secreted by mesenchymal stem cells, the culture supernatant of mesenchymal stem cells obtained according to the method of the present invention is also treated in the same manner. It is considered to have an effect.
  • the mesenchymal stem cells and mesenchymal stem cells obtained according to the method of the present invention Culture supernatants are used for diseases in which cytokines are known to have therapeutic effects, diseases in which extracellular matrices are known to have therapeutic effects, and diseases in which exosomes are known to have therapeutic effects. On the other hand, it is considered to have a therapeutic effect.

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CN202310081275.1A CN116179480A (zh) 2020-03-31 2021-03-26 增殖细胞和细胞生产物的生产法、间充质干细胞群和干细胞培养上清及其生产法、治疗剂
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CN202180024893.1A CN115362252A (zh) 2020-03-31 2021-03-26 增殖细胞和细胞生产物的生产法、间充质干细胞群和干细胞培养上清及其生产法、治疗剂
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