WO2011070974A1 - Préparation de cellules pour traiter le cancer de la prostate comprenant des cellules souches mésenchymateuses dérivées de tissu adipeux - Google Patents

Préparation de cellules pour traiter le cancer de la prostate comprenant des cellules souches mésenchymateuses dérivées de tissu adipeux Download PDF

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WO2011070974A1
WO2011070974A1 PCT/JP2010/071633 JP2010071633W WO2011070974A1 WO 2011070974 A1 WO2011070974 A1 WO 2011070974A1 JP 2010071633 W JP2010071633 W JP 2010071633W WO 2011070974 A1 WO2011070974 A1 WO 2011070974A1
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cells
cell
asc
adipose tissue
prostate cancer
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PCT/JP2010/071633
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Japanese (ja)
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徳則 山本
直史 小出
百万 後藤
佳史 武井
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国立大学法人名古屋大学
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Priority to JP2011545191A priority Critical patent/JP5035737B2/ja
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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
    • C12N5/06—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
    • C12N5/0667—Adipose-derived stem cells [ADSC]; Adipose stromal stem cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P13/00—Drugs for disorders of the urinary system
    • A61P13/08—Drugs for disorders of the urinary system of the prostate
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells

Definitions

  • the present invention relates to a cell preparation. Specifically, the present invention relates to a cell preparation effective for treating prostate cancer.
  • This application claims priority based on Japanese Patent Application No. 2009-277437 filed on Dec. 7, 2009, the entire contents of which are incorporated by reference.
  • Patent Document 1 It has been shown to be effective for the reconstruction of adipose tissue (Patent Document 1).
  • ASC adipose-derived stem cells
  • Adipose adipose-derived stem cells for ischemic diseases, renal dysfunction, wounds, urinary incontinence and osteoporosis.
  • -derived regeneration cells ADRC
  • Adipose-derived mesenchymal stem cells AT-MSC, AD-MSC, etc.
  • An object of the present invention is to provide a new medical use of adipose tissue-derived mesenchymal stem cells (ASC).
  • ASC adipose tissue-derived mesenchymal stem cells
  • LNCaP cells were cultured in a medium supplemented with ASC culture supernatant under the expectation that the humoral component secreted by ASC exhibits antitumor activity, and changes in PSA production were examined.
  • ASC suppresses the growth of LNCaP cells or decreases the activity by direct action, that is, cell-to-cell interaction.
  • ASC suppresses the growth of LNCaP cells or decreases the activity by direct action, that is, cell-to-cell interaction.
  • Non-patent Document 2 When mature adipocytes and prostate cancer cells are co-cultured, the proliferation of cancer cells is promoted (Non-patent Document 2), and the proliferation of cancer cells is suppressed by stroma fibroblasts (Non-patent Document 3).
  • Non-patent Document 3 When mature adipocytes and prostate cancer cells are co-cultured, the proliferation of cancer cells is promoted (Non-patent Document 2), and the proliferation of cancer cells is suppressed by stroma fibroblasts.
  • Non-patent Document 3 has been reported.
  • the above findings clarified after the study by the present inventors are not predictable from these reports, but also indicate facts that may be contradictory, and their academic and clinical significance is extremely great. .
  • a cell preparation for treating prostate cancer comprising adipose tissue-derived mesenchymal stem cells.
  • adipose tissue-derived mesenchymal stem cell is positive for a cell surface marker CD44.
  • the adipose tissue-derived mesenchymal stem cell is (1) Adherent cells or passage cells thereof included in a sedimented cell population that settles when a cell population separated from adipose tissue is centrifuged.
  • [5] The cell preparation according to any one of [1] to [4], wherein the adipose tissue is human adipose tissue.
  • [6] The cell preparation according to any one of [1] to [5], further comprising a substance that promotes contact between cells.
  • [8] A method for treating prostate cancer, comprising administering to the patient with prostate cancer the cell preparation according to any one of [1] to [6].
  • [9] The treatment method according to [8], wherein the cell preparation is administered to or around a lesion site of prostate cancer.
  • a method for treating prostate cancer comprising administering a therapeutically effective amount of adipose tissue-derived mesenchymal stem cells to a patient with prostate cancer.
  • adipose tissue-derived mesenchymal stem cells are administered to or around a lesion site of prostate cancer.
  • ASC adipose tissue-derived stem cells
  • Lane 1 is PSA concentration (ng / ml) when cultured in medium supplemented with 10% (v / v) inactivated FCS (Fetalcalf serum)
  • Lane 2 is inactivated FCS (10% (v / v)) and PSA concentration (ng / ml) when cultured in medium supplemented with ASC culture supernatant
  • Lane 3 is medium supplemented with 20% (v / v) KSR (Knockoutockserum replacement) PSA concentration (ng / ml) when cultured (control)
  • lane 4 is PSA concentration (ng / ml) when cultured in a medium supplemented with KSR (20% (v / v)) and ASC culture supernatant .
  • PSA was detected by EIA method.
  • PSA prostate specific antigen.
  • the PSA concentration when ASC and LNCaP cells were co-cultured (48 hours) was compared with the PSA concentration when LNCaP cells were cultured alone. From left to right, PSA concentration when only ASC is cultured, PSA concentration when only LNCaP cells are cultured, PSA concentration when LNCaP cells and ASC are co-cultured (cell number ratio 1: 1), and LNCaP cells PSA concentration when ASC is co-cultured (cell number ratio 1: 2), LNCaP cells and ASC are co-cultured (cell ratio 2: 1.
  • the number of LNCaP cells is twice that of the former two) ) Is the PSA concentration.
  • the culture was performed using 10% (v / v) inactivated FCS-containing knockout DMEM (KODMEM, Invitrogen).
  • the PSA was detected by the EIA method.
  • PSA prostate specific antigen.
  • N.D . Not detected.
  • Anti-tumor effect by co-culture of ASC The state of the cells when ASC and LNCaP cells were co-cultured (48 hours) was observed with a phase contrast microscope.
  • a Phase contrast microscopic image when only ASC was cultured
  • b Phase contrast microscopic image when only LNCaP cells were cultured
  • c Phase contrast microscopic image when only LNCaP cells were cultured
  • c Co-cultured LNCaP cells and ASC (ratio of cell number 1: 1) Phase contrast microscopic image
  • e, f Phase contrast microscopic image when LNCaP cells and ASC are cocultured (cell number ratio 1: 2)
  • g, h Coculture of NCLNCaP cells and ASC (cell number (2: 1 ratio) Phase contrast microscope image when the number of LNCaP cells is twice that of the former.
  • the culture was performed using 10% (v / v) inactivated FCS-containing knockout DMEM (KODMEM, Invitrogen).
  • LoVo cells Changes in tumor markers by co-culture with ASC. Whether colorectal cancer cell lines (LoVo cells and LS180 cells) and prostate cancer cell lines (LNCaP cells) were co-cultured with ASC was examined to determine whether the tumor marker value was changed.
  • a Experimental results of LoVo cells (48 hours culture). Lane 1 is when ASC alone is cultured (cell count 1.8 ⁇ 10 5 ), Lane 2 is when only LoVo cells are cultured (cell count 1.8 ⁇ 10 5 ), and Lane 3 is co-cultured with LoVo cells and ASC (cell count) When the number of cells is 1.8 ⁇ 10 5 ), lane 4 is co-cultured with LoVo cells and ASC (ratio of cell number is 1: 2.
  • LoVo cell number is 1.8 ⁇ 10 5 ).
  • 5 shows a case where only LoVo cells are cultured (cell number 3.6 ⁇ 10 5 ), and lane 6 shows a co-culture of LoVo cells and ASC (ratio of cell number 2: 1; LoVo cell number 3.6 ⁇ 10 5 ).
  • b Experimental results of LoVo cells (96 hours culture). The conditions for each lane are the same as a.
  • c Experimental results of LS180 cells (cultured for 48 hours).
  • Lane 1 shows the case where only ASC is cultured (number of cells: 1.8 ⁇ 10 5 )
  • Lane 2 shows the case where only LS180 cells are cultured (number of cells: 1.8 ⁇ 10 5 )
  • Lane 3 shows co-culture of LS180 cells and ASC (number of cells) When the number of cells is 1.8 ⁇ 10 5 ), lane 4 is when LS180 cells and ASC are co-cultured (cell number ratio is 1: 2, the number of LS180 cells is 1.8 ⁇ 10 5 ).
  • Lane 1 is when only ASC is cultured (cell count 1.8 ⁇ 10 5 )
  • Lane 2 is when only LNCaP cells are cultured (cell count 1.8 ⁇ 10 5 )
  • Lane 3 is co-cultured with LNCaP cells and ASC (cell count)
  • the ratio of each cell number was 1.8 ⁇ 10 5
  • lane 4 was cultured with LNCaP cells only (cell number 3.6 ⁇ 10 5 )
  • lane 5 was co-cultured with LNCaP cells and ASC (cell number The ratio is 2: 1.
  • the number of LNCaP cells is 3.6 ⁇ 10 5 ).
  • f LNCaP cell experimental results (96 hours culture). The conditions for each lane are the same as e.
  • CEA Carcinoembryonic antigen.
  • PSA prostate specific antigen.
  • ND Not detected. Changes in tumor markers by co-culture with ASC. Whether or not the tumor marker value was changed by co-culturing a colon cancer cell line (LoVo cell) with ASC (commercially available ASC (Invitrogen) and patient-derived ASC) was examined. a: Experimental results of commercially available ASC (culture for 48 hours).
  • Lane 1 is when ASC alone is cultured (cell count 1.8 ⁇ 10 5 )
  • Lane 2 is when only LoVo cells are cultured (cell count 1.8 ⁇ 10 5 )
  • Lane 3 is co-cultured with LoVo cells and ASC (cell count)
  • the ratio is 1: 1, each cell number is 1.8 ⁇ 10 5
  • lane 4 is when only LoVo cells are cultured (cell number 3.6 ⁇ 10 5 )
  • lane 5 is co-cultured with LoVo cells and ASC (number of cells)
  • the ratio is 2: 1.
  • the number of LoVo cells is 3.6 ⁇ 10 5 ).
  • b Results of commercial ASC experiment (96 hours culture). The conditions for each lane are the same as a.
  • the conditions for each lane are the same as for c. Changes in tumor markers by co-culture with ASC. It was examined whether or not the tumor marker value was changed by co-culturing a colon cancer cell line (LS180 cell) with ASC (commercially available ASC (Invitrogen) and patient-derived ASC). a: Experimental results of commercially available ASC (culture for 48 hours).
  • Lane 1 shows the case where only ASC is cultured (number of cells: 1.8 ⁇ 10 5 )
  • Lane 2 shows the case where only LS180 cells are cultured (number of cells: 1.8 ⁇ 10 5 )
  • Lane 3 shows co-culture of LS180 cells and ASC (number of cells)
  • the ratio is 1: 1, each cell number is 1.8 ⁇ 10 5
  • lane 4 is when only LS180 cells are cultured (cell number 3.6 ⁇ 10 5 )
  • lane 5 is LS180 cells and ASC co-cultured (cell number The ratio is 2: 1.
  • the number of LS180 cells is 3.6 ⁇ 10 5 ).
  • b Results of commercial ASC experiment (96 hours culture). The conditions for each lane are the same as a.
  • Lane 1 shows the case where only ASC is cultured (number of cells: 1.8 ⁇ 10 5 )
  • Lane 2 shows the case where only LS180 cells are cultured (number of cells: 1.8 ⁇ 10 5 )
  • Lane 3 shows co-culture of LS180 cells and ASC (number of cells) The ratio is 1: 1, each cell number is 1.8 ⁇ 10 5 )
  • lane 4 is when only LS180 cells are cultured (cell number 3.6 ⁇ 10 5 )
  • lane 5 is LS180 cells and ASC co-cultured (cell number The ratio is 2: 1.
  • the number of LS180 cells is 3.6 ⁇ 10 5 ).
  • d Experimental results of patient-derived ASC (96 hours culture).
  • Lane 1 is when only ASC is cultured (cell count 1.8 ⁇ 10 5 )
  • Lane 2 is when only LNCaP cells are cultured (cell count 1.8 ⁇ 10 5 )
  • Lane 3 is co-cultured with LNCaP cells and ASC (cell count)
  • the ratio of each cell number was 1.8 ⁇ 10 5
  • lane 4 was cultured with LNCaP cells only (cell number 3.6 ⁇ 10 5 )
  • lane 5 was co-cultured with LNCaP cells and ASC (cell number The ratio is 2: 1.
  • the number of LNCaP cells is 3.6 ⁇ 10 5 ).
  • b Results of commercial ASC experiment (96 hours culture). The conditions for each lane are the same as a.
  • Lane 1 is when only ASC is cultured (cell count 1.8 ⁇ 10 5 )
  • Lane 2 is when only LNCaP cells are cultured (cell count 1.8 ⁇ 10 5 )
  • Lane 3 is co-cultured with LNCaP cells and ASC (cell count)
  • the ratio of each cell number was 1.8 ⁇ 10 5
  • lane 4 was cultured with LNCaP cells only (cell number 3.6 ⁇ 10 5 )
  • lane 5 was co-cultured with LNCaP cells and ASC (cell number The ratio is 2: 1.
  • the number of LNCaP cells is 3.6 ⁇ 10 5 ).
  • d Experimental results of patient-derived ASC (96 hours culture).
  • the present invention relates to a cell preparation applied to a specific disease and its use.
  • the cell preparation of the present invention contains adipose tissue-derived mesenchymal stem cells (sometimes abbreviated as “ASC” in the present specification).
  • ASC adipose tissue-derived mesenchymal stem cell
  • ASC somatic stem cell contained in an adipose tissue.
  • the somatic stem cell culture Cells obtained by subculture
  • ASC adipose tissue-derived mesenchymal stem cells
  • ASC is prepared in an “isolated state” as a cell constituting a cell population (including cells other than ASC derived from adipose tissue) using adipose tissue separated from a living body as a starting material.
  • isolated state as used herein means a state extracted from its original environment (that is, a state constituting a part of a living body), that is, a state different from the original existence state by an artificial operation.
  • Adipose tissue-derived mesenchymal stem cells are also referred to as ADRC (Adipose-derived regeneration cells), AT-MSC (Adipose-derived mesenchymal stem cells), AD-MSC (Adipose-derived mesenchymal stem cells), and the like.
  • ADRC Adipose-derived regeneration cells
  • AT-MSC Adipose-derived mesenchymal stem cells
  • AD-MSC Adipose-derived mesenchymal stem cells
  • ASC preparation method ASC is prepared through steps such as separation, washing, concentration, and culture of stem cells from adipose matrix.
  • a method for preparing ASC is not particularly limited. For example, a known method (Fraser JK et al. (2006), Fat tissue: an underappreciated source of stem cells for biotechnology. Trends in Biotechnology; Apr; 24 (4): 150-4. Epub 2006 Feb 20. Review .; Zuk PA et al. (2002), Human adipose tissue is a source of multipotent stem cells.Molecular Biology of the Cell; Dec; 13 (12): 4279-95 .; Zuk PA et al. (2001), Multilineage cells from human Adipose tissue: implications for cell-based therapies.
  • Adipose tissue is collected from animals by means such as excision and suction.
  • the term “animal” herein includes humans and non-human mammals (pet animals, domestic animals, laboratory animals. Specifically, for example, mice, rats, guinea pigs, hamsters, monkeys, cows, pigs, goats, sheep, Dogs, cats, etc.).
  • adipose tissue self-adipose tissue
  • this does not preclude the use of adipose tissue of the same species (other family) or adipose tissue of different species.
  • adipose tissue examples include subcutaneous fat, visceral fat, intramuscular fat, and intermuscular fat.
  • subcutaneous fat can be collected very easily under local anesthesia, so that the burden on the patient at the time of collection is small and it can be said that it is a preferable cell source.
  • one type of adipose tissue is used, but two or more types of adipose tissue can be used in combination.
  • adipose tissue collected in multiple times may be mixed and used for subsequent operations.
  • the amount of adipose tissue collected can be determined in consideration of the type of donor, the type of tissue, or the amount of ASC required, for example, about 0.5 to 500 g.
  • the amount collected at a time is preferably about 10 to 20 g or less in consideration of the burden on the donor.
  • the collected adipose tissue is subjected to the following enzyme treatment after removal of blood components adhering to it and fragmentation as necessary.
  • the blood component can be removed by washing the adipose tissue in an appropriate buffer or culture solution.
  • Enzyme treatment is performed by digesting adipose tissue with enzymes such as collagenase, trypsin, dispase and the like. Such enzyme treatment may be carried out by methods and conditions known to those skilled in the art (for example, see RI Freshney, Culture of Animal Cells: A Manual of Basic Technique, 4th Edition, A John Wiley & Sones Inc., Publication). . Preferably, the enzyme treatment here is performed according to the methods and conditions described in the Examples described later.
  • the cell population obtained by the above enzyme treatment includes pluripotent stem cells, endothelial cells, stromal cells, blood cells, and / or precursor cells thereof. The type and ratio of the cells constituting the cell population depend on the origin and type of the adipose tissue used.
  • the cell population is subsequently subjected to centrifugation.
  • the sediment by centrifugation is collected as a sedimented cell population (also referred to herein as “SVF fraction”).
  • the conditions for centrifugation vary depending on the type and amount of cells, but are, for example, 1 to 10 minutes and 800 to 1500 rpm.
  • the cell population after the enzyme treatment is preferably subjected to filtration or the like, and the enzyme undigested tissue contained therein is preferably removed.
  • “SVF fraction” obtained here includes ASC. Therefore, the cell preparation of the present invention can be prepared using the SVF fraction. That is, in one embodiment of the cell preparation of the present invention, the SVF fraction is contained.
  • the type and ratio of cells constituting the SVF fraction depend on the origin and type of the adipose tissue used, the conditions for enzyme treatment, and the like. In addition, the characteristics of the SVF fraction are shown in the pamphlet of International Publication No. 2006 / 006692A1.
  • the SVF fraction contains other cell components (endothelial cells, stromal cells, blood cells, progenitor cells thereof, etc.) in addition to ASC. . Therefore, in one embodiment of the present invention, the following selective culture is performed to remove unnecessary cell components from the SVF fraction. The resulting cells are used as ASC in the cell preparation of the present invention.
  • the SVF fraction After suspending the SVF fraction in an appropriate medium, it is seeded on a culture dish and cultured overnight. Suspension cells (non-adherent cells) are removed by medium exchange. Thereafter, the culture is continued while appropriately changing the medium (for example, once every 2 to 4 days). Subculture as necessary.
  • the number of passages is not particularly limited, but from the viewpoint of maintaining pluripotency and proliferation ability, it is not preferable to repeat the passages excessively (preferably to be kept to about 5 passages).
  • the culture medium a normal animal cell culture medium can be used.
  • DMEM Dulbecco's modified Eagle's Medium
  • ⁇ -MEM Dainippon Pharmaceutical Co., Ltd.
  • DMEM Ham's F12 mixed medium (1: 1) (Dainippon Pharmaceutical Co., Ltd.), Ham's F12 medium (Dainippon Pharmaceutical Co., Ltd.), MCDB201 medium (Functional Peptide Laboratory), etc.
  • a medium supplemented with serum fetal bovine serum, human serum, sheep serum, etc.
  • KSR Knockout serum replacement
  • the addition amount of serum or serum replacement can be set, for example, within a range of 5% (v / v) to 30% (v / v).
  • adherent cells selectively survive and proliferate. Subsequently, the proliferated cells are collected.
  • the collection operation may be carried out in accordance with a conventional method.
  • the cells after enzyme treatment trypsin or dispase treatment
  • a cell scraper or pipette when sheet culture is performed using a commercially available temperature-sensitive culture dish or the like, it is also possible to recover the cells as they are without performing enzyme treatment.
  • ASC a cell preparation containing ASC with high purity can be prepared.
  • Low-serum culture selective culture in low-serum medium
  • cell recovery the following low-serum culture is performed instead of or after the operation of (3) above. I do.
  • the resulting cells are used as ASC in the cell preparation of the present invention.
  • the SVF fraction (when using this step after (3), the cells collected in (3) are used) are cultured under low serum conditions, and the target pluripotent stem cell (ie ASC ) Selectively. Since a small amount of serum is used in the low serum culture method, it is possible to use the serum of the subject (patient) to whom the cell preparation of the present invention is administered. That is, culture using autoserum becomes possible.
  • autologous serum a cell preparation is provided that is capable of excluding foreign animal material from the manufacturing process, and is expected to have high safety and high therapeutic effect.
  • “under low serum conditions” is a condition containing 5% or less of serum in the medium.
  • the cells are preferably cultured in a culture solution containing 2% (V / V) or less of serum. More preferably, the cells are cultured in a culture solution containing 2% (V / V) or less of serum and 1 to 100 ng / ml of fibroblast growth factor-2 (bFGF).
  • bFGF fibroblast growth factor-2
  • Serum is not limited to fetal bovine serum, and human serum or sheep serum can be used.
  • human serum more preferably, serum of a subject to which the cell preparation of the present invention is applied (that is, autoserum) is used.
  • a normal medium for animal cell culture can be used on condition that the amount of serum contained in use is low.
  • Dulbecco's modified Eagle's Medium DMEM
  • ⁇ -MEM Disainippon Pharmaceutical Co., Ltd.
  • DMEM Ham's F12 mixed medium (1: 1) (Dainippon Pharmaceutical Co., Ltd.), Ham's F12 medium (Dainippon Pharmaceutical Co., Ltd.), MCDB201 medium (Functional Peptide Laboratory), etc.
  • DMEM Dulbecco's modified Eagle's Medium
  • ⁇ -MEM Dainippon Pharmaceutical Co., Ltd.
  • DMEM Ham's F12 mixed medium (1: 1) (Dainippon Pharmaceutical Co., Ltd.), Ham's F12 medium (Dainippon Pharmaceutical Co., Ltd.), MCDB201 medium (Functional Peptide Laboratory), etc.
  • pluripotent stem cells By culturing by the above method, pluripotent stem cells (ASC) can be selectively proliferated.
  • pluripotent stem cells (ASC) that proliferate under the above culture conditions have high proliferative activity, the number of cells required for the cell preparation of the present invention can be easily prepared by subculture.
  • International Publication No. 2006 / 006692A1 pamphlet shows the characteristics of cells that selectively proliferate by culturing the SVF fraction in low serum.
  • the cells selectively proliferated by the above low serum culture are collected.
  • the collection operation may be performed in the same manner as in the above (3).
  • ASC collected cells
  • the cells obtained from the SVF fraction, the cells obtained as a result of the selective culture (3), or the cells obtained as a result of the low serum culture (4) are added with physiological saline or an appropriate buffer (for example, phosphorous).
  • a cell preparation can be obtained by suspending in an acid buffer or the like.
  • 1 ⁇ 10 6 to 1 ⁇ 10 10 cells may be contained as a single dose so that a therapeutically effective amount of cells is administered.
  • the content of the cells can be appropriately adjusted in consideration of the purpose of use, the target disease, the sex of the application target (recipient), age, weight, the state of the affected area, the state of the cells, and the like.
  • DMSO Dimethyl sulfoxide
  • serum albumin for the purpose of cell protection
  • antibiotics for the purpose of preventing bacterial contamination
  • various components for the purpose of cell activation, proliferation or differentiation induction, etc.
  • cytokines are interleukin (IL), interferon (IFN), colony stimulating factor (CSF), granulocyte colony stimulating factor (G-CSF) and erythropoietin (EPO), activin, oncostatin M (OSM).
  • IL interleukin
  • IFN interferon
  • CSF colony stimulating factor
  • G-CSF granulocyte colony stimulating factor
  • EPO erythropoietin
  • OSM oncostatin M
  • CSF, G-CSF, EPO, etc. are also growth factors.
  • growth factors examples include hepatocyte growth factor (HGF), basic fibroblast growth factor (bFGF, FGF2), epidermal growth factor (EGF), platelet-derived growth factor (PDGF), insulin-like growth factor (IGF) Transforming growth factor (TGF), nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF).
  • HGF hepatocyte growth factor
  • bFGF basic fibroblast growth factor
  • FGF2 epidermal growth factor
  • EGF epidermal growth factor
  • PDGF platelet-derived growth factor
  • IGF insulin-like growth factor
  • TGF nerve growth factor
  • BDNF brain-derived neurotrophic factor
  • other pharmaceutically acceptable ingredients for example, carriers, excipients, disintegrants, buffers, emulsifiers, suspensions, soothing agents, stabilizers, preservatives, preservatives, physiological saline, etc.
  • the cell preparation is composed of cells grown by low serum culture of the SVF fraction, but the cell population obtained from the adipose tissue is directly (via centrifugation to obtain the SVF fraction).
  • a cell preparation may be prepared using cells grown by low serum culture as ASC. That is, in one embodiment of the present invention, cells that proliferate when a cell population obtained from adipose tissue is cultured in low serum are used as ASC.
  • a cell preparation should be constructed using the SVF fraction (containing adipose tissue-derived mesenchymal stem cells) as it is, instead of the pluripotent stem cells obtained by selective culture (above (3) and (4)). It may be.
  • the cell preparation of this embodiment comprises (a) a precipitated cell population (SVF fraction) recovered as a sediment by subjecting adipose tissue to protease treatment, followed by filtration, and then centrifuging the filtrate, or (b) fat After the tissue is treated with protease, it contains a sedimented cell population (SVF fraction) that is collected as a sediment by centrifuging without filtration.
  • SVF fraction precipitated cell population
  • the cell preparation of this embodiment comprises (a) a precipitated cell population (SVF fraction) recovered as a sediment by subjecting adipose tissue to protease treatment, followed by filtration, and then centrifuging the filtrate, or (b) fat After the tissue is treated with protease, it contains a sedimented cell population (SVF fraction) that is collected as a sediment by centrifuging without filtration.
  • “use as it is” means to use it as an active ingredient of a cell preparation without undergoing selective culture.
  • the cell-cell contact promoting substance may be any substance that can directly or indirectly promote contact between cells.
  • intercellular contact promoters include cells such as extracellular matrix, collagen, atelocollagen (eg, Koken Co., Ltd.), Matrigel (trademark, BD Biosciences), fibronectin, vitronectin, laminin, cadherin, integrin, selectin, RGD peptide Mention may be made of adhesion peptides. Two or more kinds of intercellular contact promoting substances may be used in combination.
  • the disease to be treated with the cell preparation of the present invention is prostate cancer.
  • the cell preparation of the present invention is used for the treatment of prostate cancer. Therefore, the cell preparation of the present invention is usually administered to a patient with prostate cancer.
  • the cell preparation of the present invention can also be used for experiments or research purposes such as confirmation and verification of the effect.
  • Prostate cancer is a cancer that occurs in the prostate gland, often accompanied by symptoms such as dysuria (difficulty urinating, frequent urination, residual urine sensation, urgency), and lower discomfort. Prostate cancer is known to easily metastasize to lymph nodes and bone. Age (elderly), race, family history of prostate cancer, etc. have been reported as risk factors for prostate cancer. There is a sensitive tumor marker called prostate specific antigen (PSA), which is used for early detection of prostate cancer. PSA values correlate with tumor volume (Matthew A.
  • the stage of prostate cancer is generally classified according to the TNM classification.
  • the stage is represented by a combination of three indicators consisting of the size and progression of the primary lesion (T), the status of metastasis to regional lymph nodes (N), and the presence or absence of distant metastasis (M).
  • T primary lesion
  • N regional lymph nodes
  • M distant metastasis
  • “surgical therapy”, “radiotherapy”, “hormone therapy” and the like are adopted in consideration of the stage and age of the patient.
  • the subject to which the cell preparation of the present invention is administered is typically a human male.
  • cell preparations for mammals other than humans including pet animals, domestic animals, laboratory animals, specifically mice, rats, guinea pigs, hamsters, monkeys, cows, pigs, goats, sheep, dogs, cats, etc.
  • mammals other than humans including pet animals, domestic animals, laboratory animals, specifically mice, rats, guinea pigs, hamsters, monkeys, cows, pigs, goats, sheep, dogs, cats, etc.
  • the administration route of the cell preparation of the present invention is not particularly limited.
  • the cell preparation of the present invention is administered by intravenous injection, intraarterial injection, intraportal injection, intradermal injection, subcutaneous injection, intramuscular injection, or intraperitoneal injection.
  • the cell preparation of the present invention is administered by local injection into the affected area. That is, the cell preparation of the present invention is particularly suitable for local treatment (focal therapy).
  • the injection site in this case is typically at or around the prostate cancer lesion. If metastasis is observed, it may be administered locally to or around the metastatic lesion.
  • An example of the dosage of the cell preparation is 0.1 ml to 20 ml, preferably 0.5 ml to 10 ml, for example. You may decide to administer to two or more places.
  • the administration schedule may be created in consideration of the subject's (patient) sex, age, weight, disease state, and the like. In addition to single administration, multiple administration may be performed continuously or periodically.
  • the administration interval for multiple administrations is not particularly limited, and is, for example, 1 day to 1 month.
  • count of administration is not specifically limited. An example of the administration frequency is 2 to 10 times.
  • any one of the following cells (1) to (5) may be directly administered to a patient having an autoimmune disease in a therapeutically effective amount.
  • a sedimented cell population recovered as a sediment by subjecting adipose tissue to protease treatment, followed by filtration, and then centrifuging the filtrate.
  • the conditions for the centrifugal treatment (1) are preferably 800 to 1500 rpm and 1 to 10 minutes.
  • the adipose-derived stem cell separation apparatus performs a process of separating the stem cells from the fat matrix, and at that time, the stem cells are separated and then washed and concentrated. This real-time processing is performed in a closed environment to minimize the risk of contact with contaminants and is completed within the time of a single surgical procedure.
  • ASC was prepared and transplanted as follows ((1) to (3)).
  • ASC separation Subcutaneous adipose tissue treatment
  • ASC separation device Celution (registered trademark) device.
  • the collected adipose tissue was injected into a sterilized disposable set and washed. Thereafter, the adipose tissue was thawed, digested by adding an enzyme (Celase TM ) for cell separation, and the cell suspension was automatically centrifuged and the enzyme washed in a closed circuit.
  • the collection, adjustment, and transplantation were performed in the operating room of the Nagoya University Hospital. Its cleanliness was class 100-10000, and a clean environment was maintained in all procedures.
  • ASC and LNCaP cells were seeded on a 12-well plate, and cell culture supernatant of ASC was collected 24 hours later. Next, the culture solution of LNCaP cells was divided into two groups, ASC supernatant and normal culture solution, and replaced. After 24 hours, two groups of cell culture supernatants were collected and the PSA concentration was measured. In the experiment, the above 2. ASC prepared by the method shown in 1. was used.
  • FIG. 6 shows the results of measuring the PSA concentration in the culture medium after culture. Moreover, the phase-contrast microscope image is shown in FIG. 7 about each cell after culture
  • LNCaP cells were cultured in the presence of ASC, the PSA concentration in the medium was significantly reduced (FIG. 6).
  • the decrease in PSA concentration due to ASC was dependent on the number of ASC cells (concentration dependency).
  • observation with a phase contrast microscope showed that ASC surrounded LNCaP cells when ASC was co-cultured (FIGS. 7e, f, g, h). From the above results, it was found that ASC suppresses the growth of LNCaP cells or decreases the activity by a direct action by cell-to-cell interaction.
  • the surprising fact that ASC has an antitumor effect on prostate cancer was found, and part of its mechanism of action became clear. This finding strongly suggests that ASC is effective in the treatment of prostate cancer and is noteworthy.
  • ASC exerts an antitumor effect on prostate cancer cells.
  • the effectiveness of ASC in the treatment of prostate cancer has been experimentally demonstrated.
  • a part of the mechanism of action of ASC's antitumor action was clarified.
  • the antitumor action of ASC is highly specific to cancer types.
  • the cell preparation of the present invention is used for the treatment of prostate cancer.
  • Application to patients of various stages is envisaged.
  • application to treatment targeting metastatic lesions is also envisaged.
  • application to the treatment of other cancer types is also expected.

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Abstract

La présente invention concerne une nouvelle utilisation médicale de cellules souches mésenchymateuses dérivées de tissu adipeux. Il a été identifié clairement que des cellules souches mésenchymateuses dérivées de tissu adipeux (ASC) exercent un effet anticancéreux sur le cancer de la prostate. Sur la base de cette observation, la présente invention concerne une préparation de cellules qui comprend des cellules souches mésenchymateuses dérivées de tissu adipeux et est efficace dans le traitement du cancer de la prostate.
PCT/JP2010/071633 2009-12-07 2010-12-03 Préparation de cellules pour traiter le cancer de la prostate comprenant des cellules souches mésenchymateuses dérivées de tissu adipeux WO2011070974A1 (fr)

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JP2016007161A (ja) * 2014-06-24 2016-01-18 国立大学法人名古屋大学 卵子活性化方法及びその用途
US11285194B2 (en) * 2014-10-24 2022-03-29 Calidi Biotherapeutics, Inc. Combination immunotherapy approach for treatment of cancer
US11607450B2 (en) 2015-08-11 2023-03-21 Calidi Biotherapeutics, Inc. Smallpox vaccine for cancer treatment
US10857225B2 (en) 2015-08-11 2020-12-08 Calidi Biotherapeutics, Inc. Smallpox vaccine for cancer treatment
JP2019526277A (ja) * 2016-07-29 2019-09-19 ラ，チョンチャン 癌細胞の増殖を抑制する間葉幹細胞の製造方法
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CN111727046A (zh) * 2018-07-17 2020-09-29 仿生技术支持有限公司 类二十烷酸产生促进剂
US11655455B2 (en) 2018-11-06 2023-05-23 Calidi Biotherapeutics, Inc. Enhanced systems for cell-mediated oncolytic viral therapy
CN109731096A (zh) * 2018-12-29 2019-05-10 广东维赛生物科技有限公司 一种用于治疗犬细小病毒病的组合物及其制备方法
CN112852738B (zh) * 2019-11-27 2023-02-28 四川省肿瘤医院 一种前列腺癌细胞膜片的制备方法
CN112852738A (zh) * 2019-11-27 2021-05-28 四川省肿瘤医院 一种前列腺癌细胞膜片及其制备方法

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