WO2022158152A1 - 成熟軟骨細胞および成熟軟骨細胞含有組成物の製造方法 - Google Patents
成熟軟骨細胞および成熟軟骨細胞含有組成物の製造方法 Download PDFInfo
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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
- C12N5/0655—Chondrocytes; Cartilage
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- 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
- A61K35/32—Bones; Osteocytes; Osteoblasts; Tendons; Tenocytes; Teeth; Odontoblasts; Cartilage; Chondrocytes; Synovial membrane
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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
- C12N2501/00—Active agents used in cell culture processes, e.g. differentation
- C12N2501/10—Growth factors
- C12N2501/115—Basic fibroblast growth factor (bFGF, FGF-2)
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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
- C12N2501/00—Active agents used in cell culture processes, e.g. differentation
- C12N2501/30—Hormones
- C12N2501/38—Hormones with nuclear receptors
- C12N2501/39—Steroid hormones
Definitions
- the present invention relates to methods for producing mature chondrocytes and compositions containing mature chondrocytes.
- Vacanti et al. invented a tissue engineering method that regenerates cartilage from a limited number of chondrocytes obtained by enzymatically treating animal cartilage tissue.
- This method is an invention in which cartilage tissue is regenerated by seeding chondrocytes on a scaffold with a network structure made of artificially synthesized degradable polymer, and the cells adhere and proliferate on the scaffold.
- a scaffold with a network structure made of artificially synthesized degradable polymer
- An object of the present invention is to provide a method for producing mature chondrocytes suitable for transplantation and a composition containing mature chondrocytes.
- the first invention described in this specification relates to a method for producing mature chondrocytes.
- This mature chondrocyte can be used, for example, for cartilage regeneration as described later.
- This method for producing mature chondrocytes includes a cartilage culture step of culturing cartilage using a first culture medium to obtain mature chondrocytes.
- the first culture medium is a medium containing hydrocortisone and FGF-2.
- cartilage is auricular cartilage.
- the cartilage may be shredded cartilage (microcartilage).
- the cartilage culturing step includes, for example, a step of culturing cartilage in an environment of 2% to 15% carbon dioxide gas and 1% to 10% oxygen gas.
- different media may be used for primary culture and subculture.
- media containing autologous serum and FGF-2 may be used for primary culture.
- a medium containing autologous serum and FBS, hydrocortisone and FGF-2 may be used.
- Another invention described in this specification relates to a method for producing a mature chondrocyte-containing composition.
- This method A cartilage culture step of culturing cartilage using the first culture medium to obtain mature cartilage cells;
- the first culture medium is a medium containing hydrocortisone and FGF-2.
- cartilage is auricular cartilage.
- the cartilage may be shredded cartilage.
- the cartilage culturing step includes, for example, a step of culturing cartilage in an environment of 2% to 15% carbon dioxide gas and 1% to 10% oxygen gas.
- different media may be used for primary culture and subculture.
- media containing autologous serum and FGF-2 may be used for primary culture.
- a medium containing FBS, hydrocortisone and FGF-2 may be used.
- Conditioned medium contains, for example, interleukin-8 (IL-8), growth-related genes (GRO), monocyte chemoattractant-1 (MCP-1), and VEGF.
- IL-8 interleukin-8
- GRO growth-related genes
- MCP-1 monocyte chemoattractant-1
- VEGF vascular endothelial growth factor-1
- a mature chondrocyte-containing composition is a composition used for transplantation for cartilage regeneration.
- the composition containing mature chondrocytes is used for auricular formation, ear canal formation, rhinoplasty, mandibular formation, zygoma hard tissue recession, cranial hard tissue recession, trachea, funnel chest, etc. It is a composition used in surgery.
- Angiogenic agents include mature chondrocyte-containing compositions containing angiogenic factors and VEGFR2-positive cells. This method includes the step of producing a mature chondrocyte-containing composition by the method for producing a mature chondrocyte-containing composition described above.
- a method for producing mature chondrocytes suitable for transplantation can be provided. For example, by adding cultured chondrocytes and conditioned medium (e.g., supernatant of mature chondrocytes) at the time of transplantation at the same time, the regeneration of blood vessels around the cartilage is increased, and the engraftment rate of chondrocyte transplantation is improved. It can increase the size of cartilage and enable long-term survival. As a result, the transplanted regenerated cartilage is sufficiently supplied with blood and nutrients, suppresses cell death and necrosis, and then maintains its shape and function. In addition, since nutrients are supplied to the cartilage, a large amount of chondrocytes are engrafted and cartilage resorption is suppressed.
- conditioned medium e.g., supernatant of mature chondrocytes
- CM supernatant fluid
- GRO GRO
- IL8 IL8
- MCP-1 vascular endothelial growth factor-1
- VEGF vascular endothelial growth factor
- FIG. 1 is a photograph in place of a drawing showing a phase-contrast microscopic photograph of chondrocytes in Example 1.
- FIG. 2 is a graph in place of a drawing showing analysis results of cytokines and chemokines contained in a conditioned medium obtained by culturing cultured mature chondrocytes.
- FIG. 3 is a photograph in lieu of a drawing showing regenerated cartilage taken from the abdomen one year after transplantation.
- FIG. 4 is a photograph in place of a drawing showing the results of histopathological analysis.
- FIG. 5 is a photograph in lieu of a drawing showing changes in VEGFR2 in cultured chondrocytes during passage.
- FIG. 6 is a graph in place of a drawing showing the number of chondrocytes that can be subcultured and PDL.
- the first invention described in this specification relates to a method for producing mature chondrocytes.
- This mature chondrocyte can be used, for example, for cartilage regeneration as described later.
- This method for producing mature chondrocytes includes a cartilage culture step of culturing cartilage using a first culture medium to obtain mature chondrocytes.
- the first culture medium is a medium containing hydrocortisone and FGF-2. This medium may contain either or both autologous serum and FBS.
- cartilage is auricular cartilage.
- the cartilage may be shredded cartilage (microcartilage).
- Cartilage may be shredded, washed, and blood components removed.
- the cartilage culturing step includes, for example, a step of culturing cartilage in an environment of 2% to 15% carbon dioxide gas and 1% to 10% oxygen gas.
- different media may be used for primary culture and subculture.
- media containing autologous serum and FGF-2 may be used for primary culture.
- a medium containing autologous serum and FBS, hydrocortisone and FGF-2 may be used.
- a mature chondrocyte-containing composition is a composition used for transplantation for cartilage regeneration.
- the composition containing mature chondrocytes is used for auricular formation, ear canal formation, rhinoplasty, mandibular formation, zygoma hard tissue recession, cranial hard tissue recession, trachea, funnel chest, etc. It is a composition used in surgery.
- the cartilage culture step is a step for culturing cartilage using the first culture medium to obtain mature chondrocytes.
- the collected cartilage (after washing) may be cultured as it is.
- the cartilage culturing step may be a step of culturing cut microcartilage or a step of culturing finely chopped and filtered microcartilage. The process of culturing the finely chopped and filtered microcartilage will be described below.
- the first culture medium is a medium containing hydrocortisone and FGF-2.
- This medium may contain either or both autologous serum and FBS.
- the first culture medium is a medium containing autologous serum, hydrocortisone and FGF-2, or a medium containing FBS, hydrocortisone and FGF-2.
- the necessary elements can be added to the basal medium as appropriate.
- basal media are ⁇ -MEM medium, Eagle's basal medium, and DMEM.
- reagents well-known reagents used in the medium may be added as appropriate.
- reagents are fetal bovine serum (FBS), HC (hydrocortisone), FGF2, IGF (insulin-like growth factor), insulin, PDGF (platelet derived growth factor), ACTH (adrenocorticotropic hormone), LIF (leukemia inhibitory factor) ), TGF ⁇ , BMP, steroids, chondroitin sulfate, soybean trypsin inhibitor, ascorbic acid, hyaluronic acid, proline, dexamethasone, insulin, transferrin, selenite.
- FBS fetal bovine serum
- HC hydrocortisone
- FGF2 fetal bovine serum
- IGF insulin
- PDGF platelet derived growth factor
- ACTH adrenocorticotropic hormone
- LIF leukemia inhibitory factor
- Each may be added to the medium at a concentration of 0.1 ng/mL or more and 20 ⁇ g/mL or less (or 0.2 ng/mL or more and 10 ⁇ g/mL or less). These may be adjusted and added appropriately according to the degree of purification and the required amount.
- Autologous serum may be added instead of FBS.
- a serum-free medium may also be used. In any case, it is preferable to add HC (hydrocortisone) and FGF-2 to the medium.
- a culture medium is ⁇ -MEM medium containing 1 to 10% fetal bovine serum (FBS), 20 ng/ml to 100 ng/ml hydrocortisone, 5 ng/ml to 20 ng/ml (or 50 ng/ml).
- FGF2 Fibroblast Growth Factor 2
- 1-10% autologous serum may be added along with or in place of 1-10% fetal bovine serum (FBS). These amounts may be adjusted as appropriate.
- FBS and autologous serum may be included in the medium at 0.1%-20%.
- Micro cartilage may be cultured and grown under normal culture conditions.
- the amount of cells can range from around 10% to 100% confluent, and high-density, multi-layer culture exceeding 100% confluent is also possible.
- Hypoxia may be initiated immediately after transplantation or after a period of rest.
- a commercially available hypoxic incubator that lowers the partial pressure of oxygen by mixing nitrogen gas can be used, or nitrogen gas can be blown into an appropriate space to lower the partial pressure of oxygen. may be cultured using
- the cartilage culturing step includes, for example, a step of culturing cartilage in an environment of 2% to 15% carbon dioxide gas and 1% to 10% oxygen gas.
- different media may be used for primary culture and subculture.
- media containing autologous serum and FGF-2 may be used for primary culture.
- a medium containing FBS, hydrocortisone and FGF-2 may be used.
- the mature chondrocyte-containing composition obtaining step is a step for obtaining a mature chondrocyte-containing composition containing mature chondrocytes and a conditioned medium obtained through a cartilage culturing step. is.
- the mature chondrocyte-containing composition may contain mature chondrocytes and culture supernatant.
- Preferred examples of mature chondrocytes include the chemokine/cytokine interleukin-8 (IL-8), growth-related genes (GRO), monocyte chemoattractant-1 (MCP-1), and VEGF, respectively. cells expressing 4-fold more than 6. Any one or two or more of IL-8, GRO and MCP-1 may be expressed 10 times or more than IL-6, or may be expressed 20 times or more than IL-6 Alternatively, it may be expressed 20-fold or more than IL-6.
- cytokines/chemokines with high angiogenesis-promoting ability and low inflammation in this way, the mature chondrocyte-containing composition exhibits a high angiogenic-promoting ability without causing inflammation when transplanted.
- the mature chondrocyte-containing composition preferably contains cartilage collected from a patient to whom the transplant is to be performed.
- TNF -It is expressed 10 times or more than TNF- ⁇ , preferably 50 times or more than TNF- ⁇ , preferably 100 times or more than TNF- ⁇ , and is preferably 100 times or more expressed than TNF- ⁇ Those expressing 500-fold or more are preferred.
- IL-8 interleukin 8
- GRO growth-related genes
- MCP-1 monocyte chemotactic factor-1
- VEGF vascular endothelial growth factor-1
- -10-fold or more expression than IL-1- ⁇ preferably 50-fold or more expression than IL-1- ⁇ , preferably 100-fold or more expression than IL-1- ⁇ , Those that express 500-fold or more compared to IL-1- ⁇ are preferred.
- chondrocytes include interleukin 8 (IL-8), growth-related genes (GRO), monocyte chemotactic factor-1 (MCP-1), and VEGF, or two or more of them, and INF - is expressed 10 times or more than INF- ⁇ , preferably 50 times or more than INF- ⁇ , preferably 100 times or more than INF- ⁇ , and is preferably 100 times or more expressed than INF- ⁇ Those expressing 500-fold or more are preferred.
- IL-8 interleukin 8
- GRO growth-related genes
- MCP-1 monocyte chemotactic factor-1
- VEGF vascular endothelial growth factor-1
- the mature chondrocyte-containing composition may contain appropriate amounts of mature chondrocytes and conditioned medium (or culture supernatant) depending on the purpose. Also, this composition may be stored in a necessary container in the same manner as a normal composition so that it can be used as needed.
- the amount of mature chondrocytes may be, for example, 1 ⁇ 10 4 to 1 ⁇ 10 10 cells per application, or 1 ⁇ 10 5 to 1 ⁇ 10 8 cells per application.
- compositions containing culture supernatant as an active ingredient are known, for example, as disclosed in JP-A-2013-18756, JP-A-2013-18756, JP-A-5139294, and JP-A-5526320. Accordingly, compositions containing conditioned medium can be manufactured using known methods.
- Examples of the culture supernatant of mature chondrocytes are processed products, evaporators, etc. obtained by removing water from the culture supernatant, which is the supernatant component obtained by solid-liquid separation of the culture supernatant by centrifugation
- the supernatant obtained by culturing the mature chondrocytes of the present invention is centrifuged (e.g., 1,000 x g, 10 minutes) and then fractionated with ammonium sulfate (e.g., 65% saturated ammonium sulfate) to remove the precipitate.
- ammonium sulfate e.g., 65% saturated ammonium sulfate
- dialysis treatment may be performed and filtered through a syringe filter (eg, 0.2 ⁇ m) to obtain a sterile culture supernatant.
- the collected culture supernatant can be used as it is, or it can be stored frozen and thawed before use.
- a pharmaceutically acceptable carrier may be added to dispense into sterilized containers so that the liquid volume becomes easy to handle.
- the culture supernatant may be treated with a virus clearance filter or ultraviolet irradiation.
- the mature chondrocyte-containing composition preferably contains 1 mL or more and 1,000 mL or less, more preferably 30 mL or more and 300 mL or less of the culture supernatant as one dosage unit.
- chondrocytes and conditioned medium e.g., supernatant of mature chondrocytes
- vascular regeneration around the cartilage is increased and the engraftment rate of chondrocyte transplantation is improved, resulting in regenerated cartilage. can be greatly increased and transplanted successfully.
- the composition is used, for example, in auroplasty, rhinoplasty, mandibular plasty, zygoma hard-tissue recessoplasty, cranial hard-tissue recessoplasty, funnel chest hard-tissue recessoplasty.
- the invention described in this specification which is different from the above, relates to an angiogenesis promoter.
- This angiogenesis promoting agent includes, for example, a mature chondrocyte-containing composition obtained by any of the above-described methods for producing a mature chondrocyte-containing composition. That is, this angiogenesis promoting agent is the same as the mature chondrocyte-containing composition described above.
- the angiogenesis promoter may be, for example, an injection.
- the above-described mature chondrocyte-containing composition may be contained in a syringe, mixed with the patient's tissue as necessary, and transplanted to the patient's required site.
- Angiogenic agents include mature chondrocyte-containing compositions that include duct-inducing factors and VEGFR2-positive cells. This method includes the step of producing a mature chondrocyte-containing composition by the method for producing a mature chondrocyte-containing composition described above.
- chondrocytes were isolated.
- chondrocytes were seeded at a cell density of 1 ⁇ 10 3 cells/cm 2 , and 10% autologous serum and FGF-2 (5-10 ng/ml, FIBRAST®, Kaken Pharmaceuticals, Tokyo) were added.
- Primary culture was performed in DMEM medium.
- chondrocytes were seeded at 1 ⁇ 10 3 cells/cm 2 per 175 cm 2 culture flask.
- the subcultured cells were seeded, and a graft material containing chondrocytes and CM was obtained as the final product (1 ⁇ 10 7 cells/1 cc CM).
- the medium used was a High glucose-DME medium supplemented with 5% FBS (Fetal Bovine Serum), 40 ng/ml Hydrocortisone, and 10 ng/ml Fibroblast Growth Factor 2.
- the culture conditions were 5-10% carbon dioxide gas. Cultured cells of 2 to 3 ⁇ 10 6 cells were collected on the 12th day of the primary culture.
- Some of the cells were frozen and other cells were seeded at a density of 4-5 ⁇ 10 4 /cm 2 and cultured for 14 days. After 15 days, the cells were washed three times with PBS(-) and cultured for 2 days in the above-mentioned medium without FBS. This medium was used for analysis as a conditioned medium.
- the medium used was High glucose-DME medium supplemented with 5% fetal bovine serum (FBS), 40 ng/ml hydrocortisone, and 10 ng/ml FGF2 (Fibroblast Growth Factor 2).
- the culture conditions were 10% carbon dioxide gas and 5% oxygen gas.
- 1 is a photograph in place of a drawing showing a phase-contrast microscopic photograph of chondrocytes in Example 1.
- Cytokine/chemokine analysis was performed on the conditioned medium of cultured mature chondrocytes using the Antibody-Immobilized Magnetic Beads method. The supernatant obtained by centrifuging the conditioned medium sample at 13,000 G, 4°C for 5 minutes was used for the measurement. Concentrations of 40 target proteins in Conditioned Medium were measured using the Luminex® system. Pretreated samples were used at 25 ⁇ L per well and measurements were performed in triplicate. One point of standard solution was added based on the manual, and 7 points of 5-fold dilution series were prepared and measured 3 times.
- the 40 target proteins are EGF, FGF-2, eotaxin, TGF- ⁇ , G-CSF, Flt-3L, GM-CSF, fractalkine, IFN ⁇ 2, IFN ⁇ , GRO, IL-10, MCP-3, IL-12P40 , MDC, IL-12P70, PDGF-AA, IL-13, PDGF-AB/BB, IL-15, sCD40L, IL-17A, IL-1RA, IL-1 ⁇ , IL-9, IL-1 ⁇ , IL-2 , IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IP-10, MCP-1, MIP-1 ⁇ , MIP-1 ⁇ , RANTES, TNF ⁇ , TNF ⁇ , and VEGF there were.
- FIG. 2 is a graph in place of a drawing showing analysis results of cytokines and chemokines contained in a conditioned medium obtained by culturing cultured mature chondrocytes.
- IL-8, GRO, and MCP-1 which are cytokines with high angiogenic potential, were produced in large amounts (ng/ml).
- TNF- ⁇ which causes inflammation
- IL-1- ⁇ and INF- ⁇ which have strong inflammation-inducing power
- Chondrocyte transplantation to humans [chondrogenesis for auricle reconstruction) [Case 1]: A 22-year-old man underwent reconstructive treatment with cultured cartilage for bilateral microtia (no external ear) and zygoma hypoplasia. Cartilage tissue of 15mm x 15mm was collected from the right remnant auricle cartilage, sterilized, and cultured for seeding. did. 4.3 ⁇ 10 6 cells were collected on the 11th day of the primary culture, and 2.2 ⁇ 10 ⁇ 7 cells on the 13th day of the primary culture. The cultured cells were temporarily cryopreserved. Before transplantation, thawed cells were cultured in 30 flasks with a base area of 150 cm ⁇ 2 for 40 days.
- FIG. 3 is a photograph in lieu of a drawing showing regenerated cartilage taken from the abdomen one year after transplantation.
- the size of the regenerated cartilage was 80x200x8mm.
- the cut surface of the cartilage was a white cartilage tissue throughout the thickness.
- FIG. 4 is a photograph in place of a drawing showing the results of histopathological analysis.
- FIG. 4A shows the results of HE staining.
- FIG. 4B shows the results of toluidine blue staining.
- FIG. 4C shows the results of Alcian blue staining.
- FIG. 4D shows the results of Elastica Fungieson staining.
- a perichondrium is formed around the cartilage, and many vascular cavities are observed therein. It has been shown that nutrients are supplied from the perichondrium to the cartilage. Further, from FIGS.
- cartilage is formed because each is stained with a pigment that specifically stains cartilage.
- mature regenerated cartilage and regenerated perichondrium were formed after transplantation, and the traits and characteristics of elastic cartilage derived from auricle were maintained.
- Chondrocyte transplantation to humans Reconstruction of funnel thoracic recess An 18-year-old man was found to have a residual chest recess (around the midline xiphoid process to hypochondrium) after funnel thorax surgery. A 10 mm ⁇ 7 mm cartilage tissue was collected from the left auricle, sterilized, and cultured. The medium used was High glucose-DME medium supplemented with autologous serum, Hydrocortisone, and Fibroblast Growth Factor 2. Cultured cells of 4.2 ⁇ 10 6 cells were collected on the 21st day of the primary culture, and 3.3 ⁇ 10 6 cells on the 23rd day of the primary culture. The cultured cells were temporarily cryopreserved.
- VEGFR2 Changes in VEGFR2 in cultured chondrocytes during passage VEGFR2 is expressed only in vascular endothelial cells and their progenitor cells. Therefore, if VEGFR2 is mixed, it is found that a composition containing cultured mature chondrocytes and a conditioned medium produces blood vessels. Cells from P2 to P4 were used for transplantation. As for VEGFR2, cells expressing VEGFR2 were present in all from P1 to P4. In this chondrocyte culture system, it was revealed that all cells from P1 to P4 contained endothelium cells.
- FIG. 5 is a photograph in lieu of a drawing showing changes in VEGFR2 in cultured chondrocytes during passage.
- FIG. 6 is a graph in place of a drawing showing the number of chondrocytes that can be subcultured and PDL. As shown in FIG. 6, it was confirmed that aging-induced growth arrest occurred.
- Senescence-associated galactosidase an enzyme associated with senescence-associated senescence-associated galactosidase, was found in senescence-associated ⁇ -galactosidase (SA- ⁇ -gal)-positive staining of senescence-associated senescence-associated ⁇ -galactosidase (SA- ⁇ -gal) in four auricular chondrocytes at passages P13 and P14.
- activity staining senescence-associated galactosidase activity staining was performed at each stage. As a result, the above enzyme activity appeared before growth arrest, so it was confirmed that growth arrest due to aging occurred.
- D Chondrocytes from a 63-year-old woman (proliferation stopped at P-13)
- PDL 0.2388 (P-12:2.5x10 ⁇ 5 ⁇ 2.95x10 ⁇ 5/dish)
- C. Chondrocytes of a 29-year-old female PDL 0.2013 (P-14: 1x10 ⁇ 5 ⁇ 1.15x10 ⁇ 5/dish) Total PDL: 47.1421 SA X-Gal: 100% positive
- DISCUSSION Formation of perichondrium is required to inhibit the formation of regenerated cartilage and its resorption.
- Artificial cartilage structures containing large numbers of cells usually require a high oxygen environment for their growth and proliferation. Therefore, if cartilage does not form to the center of the structure, the perichondrium will not form [5]. Their blood and nutrient supply was therefore considerably restricted. This was found to cause cell death and inevitable necrosis of the artificial construct, followed by loss of shape and function [6-8].
- the mature chondrocyte-containing composition contains cytokines/chemokines that have high angiogenesis-promoting ability and low inflammation, so that when transplanted, it exhibits a high angiogenic-promoting ability without causing inflammation.
- Cartilage conditioned medium contains 7 times more IL6 than VEGF, 20 times more GRO than IL6, 80 times more IL8 than IL6, and 100 times more MCP-1 than IL6.
- TNF- ⁇ and IL-1- ⁇ and INF- ⁇ which have a strong inflammatory-inducing power, are hardly produced in these cells, so it was also shown to be safe.
- the aging test confirmed that growth arrest occurred, it was confirmed that the cells were not tumorigenic, indicating that the cells were safe.
- IL-8 The angiogenic potential of IL-8 has also been documented (Mikula-Pietrasik J Kuczmarska A, Kucin ⁇ ska M. Muriaset M. al. Resveratrol and its synthetic derivatives exert opposite effects on mesothelial cell-dependent angiogenesis via modulating secretion of VEGF and IL-8 /CXCL8. Angiogenesis 2012; 15:361-376 and Keglowich1 et al., supra).
- MCP-1 has been reported as an angiogenic chemokine and has angiogenic potential as well, according to another publication (6. Niu J, Azfer A, Zhelyabovska O, Fatma S, and Kolattukudy PE. Monocyte Chemotactic Protein (MCP)-1 Promotes Angiogenesis via a Novel Transcription Factor, MCP-1-induced Protein (MCPIP). J Biol Chem2008 May 23;283(21): 14542-51.doi: 10.1074/jbc.M802139200). In addition, MCP-1 mediates the angiogenic ability of VEGF (3. Hong KH, Ryu J, Han KH.
- Monocyte Chemoattractant protein-1-induced Angiogenesis Is Mediated by Vascular Endothelial Growth factor-A. Blood 2005; 105:1405-1407 DOI:10,1182/blood-2004-08-3178). Activation of macrophages is essential for angiogenesis, and MCP-1 is the essential factor to do so.
- This invention can be used in fields such as medical equipment.
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Abstract
Description
この成熟軟骨細胞の製造方法は,第1の培養用培地を用いて軟骨を培養し,成熟軟骨細胞を得る軟骨培養工程を含む。
そして,第1の培養用培地は,ヒドロコルチゾン及びFGF-2を含む培地である。
この方法は,
第1の培養用培地を用いて軟骨を培養し,成熟軟骨細胞を得る軟骨培養工程と,
成熟軟骨細胞と,軟骨養工程を経て得られる馴化培地(conditioned medium)とを含む成熟軟骨細胞を含む組成物を得る成熟軟骨細胞含有組成物取得工程とを含む。
そして,第1の培養用培地は,ヒドロコルチゾン及びFGF-2を含む培地である。
成熟軟骨細胞含有組成物は,耳介形成,外耳道形成,鼻形成,下顎形成,頬骨の硬組織陥凹形成術,頭蓋の硬組織陥凹形成術,気管,漏斗胸などの硬組織陥凹形成術に用いられる組成物である。
血管再生促進剤は,血管誘導因子と,VEGFR2陽性細胞とを含む,成熟軟骨細胞含有組成物を含む。
そして,この方法は,上記した成熟軟骨細胞含有組成物の製造方法により,成熟軟骨細胞含有組成物を製造する工程を含む。
この成熟軟骨細胞の製造方法は,第1の培養用培地を用いて軟骨を培養し,成熟軟骨細胞を得る軟骨培養工程を含む。
そして,第1の培養用培地は,ヒドロコルチゾン及びFGF-2を含む培地である。この培地は,自己血清およびFBSのいずれか又は両方を含んでもよい。
軟骨培養工程は,第1の培養用培地を用いて軟骨を培養し,成熟軟骨細胞を得るための工程である。軟骨培養工程は,採取した軟骨を(洗浄後)そのまま培養してもよい。また,軟骨培養工程は,裁断した微小軟骨を培養する工程であってもよいし,細断ろ過した微小軟骨を培養する工程であってもよい。以下,細断ろ過した微小軟骨を培養する工程に基づいて説明する。
成熟軟骨細胞含有組成物取得工程は,成熟軟骨細胞と,軟骨培養工程を経て得られる馴化培地(conditioned medium)とを含む成熟軟骨細胞含有組成物を得るための工程である。成熟軟骨細胞含有組成物は,成熟軟骨細胞と培養上清を含むものであってもよい。
培地はHigh glucose-DME培地に5%ウシ胎児血清(FBS:Fetal Bovine Serum), 40ng/ml ヒドロコルチゾン(Hydrocortisone), 10ng/ml FGF2(Fibroblast Growth Factor 2)を添加したものを使用した。培養条件は,10%炭酸ガス,5%酸素ガス下で行った。図1は,実施例1の軟骨細胞の位相差顕微鏡写真を示す図面に代わる写真である。
[症例 1]:22歳男性 両側小耳症(外耳がない)および頬骨低形成のために培養軟骨による再建治療を行った。右遺残耳介軟骨より軟骨組織を15mm×15mm採取し,除菌して播種培養を行った培地はHigh glucose-DME培地に自家血清および5%FBS,ヒドロコルチゾン,FGF2 2を添加したものを使用した。初代培養11日目に4.3×106cells ,初代培養13日目に2.2×10^7 cellsの培養細胞を回収した。この培養細胞を一旦凍結保存した。移植前に,解凍した細胞を,底面積150cm^2のフラスコ30枚に40日間培養した。培養成熟軟骨細胞及び馴化培地(Conditioned Medium)を含む組成物を総量230cc (1 X 107 cells / 1 cc CM)準備した 。両側下腹部に培養成熟軟骨細胞及び馴化培地(Conditioned Medium)を含む組成物を注入移植し,移植後1年後に採取した再生軟骨を利用して両側耳介と頬骨を再建した。
図3は,移植1年後,腹部から採取された再生軟骨を示す図面に代わる写真である。図3において,再生軟骨の大きさは80x200x8mmであった。軟骨の割面は全層性に白い軟骨組織であった。
18歳男性に対し,漏斗胸術後胸部陥凹(正中剣状突起周辺~季肋部)残存を認めた。左耳介より軟骨組織を10mm×7mm採取し,除菌して播種培養を行った。培地はHigh glucose-DME培地に自家血清,Hydrocortisone, Fibroblast Growth Factor 2を添加してものを使用した。初代培養21日目に4.2×106cells,初代培養23日目に3.3×106cellsの培養細胞を回収した。この培養細胞を一旦凍結保存した。移植前に,解凍した細胞を,底面積150cm2のフラスコ15枚に40日間培養した。培養成熟軟骨細胞とcondition mediumの移植総量は84mL1X107 cells / 1ccCM)であった。移植後2年,軟骨は吸収されず,胸部の外貌形態は良好であった。
20歳女性,鞍鼻,短鼻,前額部の陥凹変形を認めたために前額部と鼻部に培養軟骨による再建治療を行った。左耳介より軟骨組織を10mm×15mm採取し,除菌して播種培養を行った。培地はHigh glucose-DME培地に自家血清, Hydrocortisone, Fibroblast Growth Factor 2を添加してものを使用した。初代培養18日目に1.1×107 cellsの培養細胞を回収した。この培養細胞を一旦凍結保存した。移植前に,解凍した細胞を,底面積150cm2のフラスコ7枚に40日間培養した。培養成熟軟骨細胞とcondition mediumの移植総量は27.3mL(1X107 cells / 1ccCM)であった。移植後3年,軟骨は吸収されず,外貌形態は良好であった。
VEGFR2は,血管内皮細胞とその前駆細胞にしか発現しない。このためVEGFR2が混在すれば,培養成熟軟骨細胞及び馴化培地(Conditioned Medium)を含む組成物は,血管を造成することがわかる。移植に用いたのはP2からP4の細胞である。VEGFR2はP1からP4まで全てにVEGFR2を発現する細胞が存在していた。このchondrocyteの培養系では,P1からP4まで全てにendothelium 系の細胞が含まれていることが明らかになった。したがって,この細胞系を移植すれば,軟骨組織と共に血管内皮細胞が出現し,その結果軟骨組織の周囲に血管網が構築され,移植後の軟骨組織の維持が期待できる。図5は,継代における培養軟骨細胞のVEGFR2 の変化を示す図面に代わる写真である。
4名の耳介軟骨細胞を3か月間培養して増殖停止するまでのPDL(CPD)を計測した。(何回分裂したかの意味)。その結果を図6に示す。図6は,軟骨細胞の継代可能数とPDLを示す図面に代わるグラフである。図6に示されるように,Agingによる増殖停止が起こっていることが確認された。
P14=total 84 day culture:縦軸CDL(=PDL). A(20歳,B(37歳,C(29歳,D(63歳)
A. 20歳女性軟骨細胞 (P-13にて増殖停止)
PDL=0.8952 (P-12:2.5x10^5⇒4.65x10^5/dish)
Total PDL: 27.6676 SA X- Gal: 92%陽性
D.63歳女性軟骨細胞 (P-13にて増殖停止)
PDL=0.2388 (P-12:2.5x10^5⇒2.95x10^5/dish)
Total PDL: 27.6676 SA X- Gal: 95%陽性
B.37歳女性軟骨細胞
PDL=0.5109 (P-14:1x10^5⇒1.425x10^5/dish)
Total PDL: 44.0962 SA X- Gal: 100%陽性
C.29歳女性軟骨細胞
PDL=0.2013 (P-14:1x10^5⇒1.15x10^5/dish)
Total PDL: 47.1421 SA X- Gal: 100%陽性
再生軟骨の形成とその吸収を抑制するためには軟骨膜の形成が必要とされる。大量の細胞を含む人工軟骨構造は,通常,その成長と増殖のために高酸素環境を必要とする。したがって,軟骨が構造の中心まで形成されない場合,軟骨膜は形成されない [5]。したがって,それらの血液と栄養素の供給はかなり制限された。これは,細胞死と人工構築物の不可避の壊死を引き起こし,その後,形状と機能が失われることがわかった[6-8]。
他方,成熟軟骨細胞含有組成物は,血管再生促進能が高く炎症性の少ないサイトカイン/ケモカインを含むことで,移植した際に,炎症を惹き起こさずに高い血管促進能を発揮するため,再生軟骨周囲に再生軟骨膜が形成されるので再生軟骨の移植に適している。軟骨の馴化培地はVEGFがIL6の7倍,GROがIL6の20倍,IL8はIL6の80倍,MCP-1はIL6の100倍を含む。TNF-αや炎症惹起力の強いIL-1-β,INF-γはこの細胞では殆ど産生されていないので,安全であることも示された。また、Aging試験では増殖停止が起こることが確認されたので腫瘍化した細胞ではないことが確認され、安全であることも示された。
Claims (9)
- 第1の培養用培地を用いて軟骨を培養し,成熟軟骨細胞を得る軟骨培養工程を含む,成熟軟骨細胞の製造方法であって,
第1の培養用培地は,
ドロコルチゾン及びFGF-2を含む培地である
成熟軟骨細胞の製造方法。 - 請求項1に記載の成熟軟骨細胞の製造方法であって,
前記軟骨は耳介軟骨である,方法。 - 請求項1に記載の成熟軟骨細胞の製造方法であって,
前記軟骨培養工程は,2%以上15%以下の炭酸ガス及び1%以上10%以下の酸素ガスの環境下において前記軟骨を培養する工程を含む,
方法。 - 第1の培養用培地を用いて軟骨を培養し,成熟軟骨細胞を得る軟骨培養工程と,
前記成熟軟骨細胞と,前記軟骨培養工程を経て得られる馴化培地(conditioned medium)とを含む成熟軟骨細胞を含む組成物を得る成熟軟骨細胞含有組成物取得工程とを含む,
成熟軟骨細胞含有組成物の製造方法であって,
第1の培養用培地は,
ヒドロコルチゾン及びFGF-2を含む培地である
成熟軟骨細胞含有組成物の製造方法。 - 請求項4に記載の成熟軟骨細胞含有組成物の製造方法であって,
前記軟骨培養工程は,2%以上15%以下の炭酸ガス及び1%以上10%以下の酸素ガスの環境下において前記軟骨を培養する工程を含む,
成熟軟骨細胞含有組成物の製造方法。 - 請求項4に記載の成熟軟骨細胞含有組成物の製造方法であって,
前記馴化培地は,インターロイキン8(IL-8),成長関連遺伝子(GRO),単球走化性因子-1(MCP-1),及びVEGFを含む,方法。 - 請求項4に記載の成熟軟骨細胞含有組成物の製造方法であって,
前記成熟軟骨細胞含有組成物は,軟骨再生の為の移植に用いられる組成物である,方法。 - 請求項4に記載の成熟軟骨細胞含有組成物の製造方法であって,
前記成熟軟骨細胞含有組成物は,
耳介形成,外耳道形成,鼻形成,下顎形成,頬骨の硬組織陥凹形成術,頭蓋の硬組織陥凹形成術,気管,漏斗胸などの硬組織陥凹形成術に用いられる組成物である,方法。 - 血管再生促進剤の製造方法であって,
前記血管再生促進剤は,血管誘導因子と,VEGFR2陽性細胞とを含む,成熟軟骨細胞含有組成物を含み,
請求項4に記載の成熟軟骨細胞含有組成物の製造方法により,前記成熟軟骨細胞含有組成物を製造する工程を含む,方法。
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