Method for producing mature chondrocytes

Abstract

To provide a method for producing mature chondrocytes suitable for transplantation.SOLUTION: The present invention relates to a method for producing mature chondrocytes, the method comprising a cartilage culture step for culturing cartilage with a first culture medium to obtain mature chondrocytes, wherein the first culture medium contains hydrocortisone and FGF-2.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] The present invention relates to a method for producing mature chondrocytes. [Background technology]

[0002] In the field of surgery, reconstruction procedures using bone and cartilage transplants are frequently performed. The donor areas used so far include the skull, ribs, costal cartilage, and ilium. However, harvesting a large amount of cartilage cannot rule out the possibility of functional problems or deformities occurring after the donor's bone is harvested. Cases requiring multiple reconstructive surgeries often require the sacrifice of many donor bone sites. Furthermore, there is a limit to the amount of bone that can be harvested.

[0003] Vacanti et al. invented a tissue engineering method for regenerating cartilage from a limited number of chondrocytes obtained by enzymatically treating animal cartilage tissue. In this method, chondrocytes are seeded onto a mesh-structured scaffold made of an artificially synthesized degradable polymer, where the cells attach and proliferate to regenerate cartilage [1, 2]. However, clinically applicable results have not yet been obtained because cartilage tissue is not uniformly generated within the scaffold. Furthermore, the synthetic polymer scaffold has been found to induce inflammation upon in vivo absorption, leading to resorption of the regenerated cartilage [3-4]. As later reported by Vacanti et al., artificial cartilage structures containing large numbers of cells typically require a high-oxygen environment for their growth and proliferation. Therefore, if cartilage does not form to the center of the structure, perichondrium does not form [5]. Consequently, their blood and nutrient supplies are significantly limited. This leads to cell death and inevitable necrosis of the artificial construct, resulting in subsequent loss of shape and function. [Prior art documents] [Non-patent literature]

[0004] [Non-Patent Document 1] [ PubMed ] Vacanti CA, Langer R, Schloo B, Vacanti JP. Synthetic polymers seeded with chondrocytes provide a template for new cartilage formation. Plastic Reconstr Surg. 1991; 88:753-7

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[0005] Until now, even when cultured chondrocytes were transplanted, it was difficult to obtain a sufficient quantity and size, making clinical application difficult. The purpose of this invention is to provide a method for producing mature chondrocytes suitable for transplantation. [Means for solving the problem]

[0006] The first invention described in this specification relates to a method for producing mature chondrocytes, which can be used, for example, in cartilage regeneration procedures, as described below. This method for producing mature chondrocytes includes a cartilage culturing 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.

[0007] An example of cartilage is auricular cartilage. The cartilage may be shredded cartilage (microcartilage).

[0008] The cartilage culture step includes, for example, culturing cartilage in an environment of 2% to 15% carbon dioxide gas and 1% to 10% oxygen gas. In the cartilage culture step, different media may be used for primary culture and subculture. For example, a medium containing autologous serum and FGF-2 may be used for primary culture. Furthermore, for example, when culturing cartilage or mature chondrocytes after cryopreservation for a certain period of time, a medium containing autologous serum, FBS, hydrocortisone, and FGF-2 may be used.

[0009] Another invention described in this specification relates to a method for producing a mature chondrocyte-containing composition. This method is a cartilage culture step of culturing cartilage using the first culture medium to obtain mature chondrocytes; The method includes a step of obtaining a mature chondrocyte-containing composition, which obtains a composition containing mature chondrocytes, the composition containing mature chondrocytes and a conditioned medium obtained through a cartilage cultivation step. The first culture medium is a medium containing hydrocortisone and FGF-2.

[0010] An example of cartilage is auricular cartilage. The cartilage may be shredded cartilage.

[0011] The cartilage culture step includes, for example, culturing cartilage in an environment of 2% to 15% carbon dioxide gas and 1% to 10% oxygen gas. In the cartilage culture step, different media may be used for primary culture and subculture. For example, a medium containing autologous serum and FGF-2 may be used for primary culture. Furthermore, for example, when culturing cartilage or mature chondrocytes after cryopreservation for a certain period of time, a medium containing FBS, hydrocortisone, and FGF-2 may be used.

[0012] Conditioned medium contains, for example, interleukin 8 (IL-8), growth-related genes (GRO), monocyte chemoattractant protein-1 (MCP-1), and VEGF.

[0013] The mature chondrocyte-containing composition is a composition used for transplantation for cartilage regeneration. The mature chondrocyte-containing composition is a composition used for ear formation, ear canal formation, nose formation, mandibular formation, hard tissue depression formation of the cheekbone, hard tissue depression formation of the skull, trachea, pectus excavatum, etc.

[0014] Another invention described in this specification relates to a method for producing a vascular regeneration promoter. The vascular regeneration promoter comprises a mature chondrocyte-containing composition containing a vessel-inducing factor and VEGFR2-positive cells. This method includes the step of producing a mature chondrocyte-containing composition by the above-mentioned method for producing a mature chondrocyte-containing composition. [Effects of the Invention]

[0015] This invention provides a method for producing mature chondrocytes suitable for transplantation. For example, by simultaneously adding cultured chondrocytes and conditioned medium (e.g., supernatant of mature chondrocytes) at the time of transplantation, it is possible to enhance vascular regeneration around the cartilage, improve the rate of chondrocyte transplant engraftment, and expand the size of the transplanted cartilage, enabling long-term survival. As a result, the transplanted regenerated cartilage receives a sufficient supply of blood and nutrients, suppressing cell death and necrosis and subsequently maintaining its shape and function. Furthermore, the supply of nutrients to the cartilage allows a large number of chondrocytes to engraft, suppressing cartilage resorption, and enabling the production of large regenerated cartilage that was previously not possible.

[0016] Adding cultured chondrocytes and the supernatant produced by these cells simultaneously at the time of transplantation promotes cartilage formation, significantly increasing the amount of regenerated cartilage, resulting in successful transplantation. When chondrocytes and the supernatant (conditioned medium) were transplanted together, angiogenesis occurred around the cartilage, achieving a high transplant rate. We then investigated the factors present in the cultured cells and found several cytokines / chemokines that specifically induce angiogenesis, with high concentrations. The addition of mature chondrocytes and the supernatant (conditioned medium) containing specific cytokines / chemokines that promote angiogenesis, particularly GRO, IL-8, MCP-1, and VEGF, to the chondrocytes inhibits resorption after transplantation and improves engraftment rates. [Brief explanation of the drawings]

[0017] [Figure 1] FIG. 1 is a photograph, in place of a drawing, showing a phase contrast microscope photograph of the chondrocytes of Example 1. [Figure 2] FIG. 2 is a graph, instead of a drawing, showing the results of an analysis of cytokines and chemokines contained in the conditioned medium obtained when mature chondrocytes were cultured. [Figure 3] Figure 3 is a photograph, in place of a drawing, showing regenerated cartilage harvested from the abdomen one year after transplantation. [Figure 4] FIG. 4 is a photograph, instead of a drawing, showing the results of histopathological analysis. [Figure 5] FIG. 5 is a photograph, instead of a drawing, showing changes in VEGFR2 in cultured chondrocytes during subculture. [Figure 6] FIG. 6 is a graph, instead of a drawing, showing the number of chondrocytes that can be passaged and PDL. [Figure 7] FIG. 7 is a photograph, instead of a drawing, showing cultured mature chondrocytes (P=13) from a 20-year-old woman. [Figure 8] FIG. 8 is a photograph, instead of a drawing, showing cultured mature chondrocytes (P=13) from a 63-year-old woman. [Figure 9]FIG. 9 is a photograph, instead of a drawing, showing cultured mature chondrocytes (P=14) from a 37-year-old woman. [Figure 10] FIG. 10 is a photograph, instead of a drawing, showing cultured mature chondrocytes (P=14) from a 29-year-old woman. DETAILED DESCRIPTION OF THE INVENTION

[0018] The following describes embodiments of the present invention with reference to the drawings. The present invention is not limited to the embodiments described below, and also includes appropriate modifications of the embodiments below within the scope obvious to those skilled in the art.

[0019] The first invention described in this specification relates to a method for producing mature chondrocytes, which can be used, for example, in cartilage regeneration procedures, as described below. This method for producing mature chondrocytes includes a cartilage culturing 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 of autologous serum and FBS.

[0020] An example of cartilage is auricular cartilage. The cartilage may be shredded cartilage (microcartilage). The cartilage may be shredded, washed, and then have blood components removed.

[0021] The cartilage culture step includes, for example, culturing cartilage in an environment of 2% to 15% carbon dioxide gas and 1% to 10% oxygen gas. In the cartilage culture step, different media may be used for primary culture and subculture. For example, a medium containing autologous serum and FGF-2 may be used for primary culture. Furthermore, for example, when culturing cartilage or mature chondrocytes after cryopreservation for a certain period of time, a medium containing autologous serum, FBS, hydrocortisone, and FGF-2 may be used.

[0022] Another invention described in this specification relates to a method for producing a mature chondrocyte-containing composition. The mature chondrocyte-containing composition is a composition used for transplantation for cartilage regeneration. The mature chondrocyte-containing composition is a composition used for ear formation, ear canal formation, rhinoplasty, mandibular formation, hard tissue depression formation of the zygomatic bone, hard tissue depression formation of the skull, and hard tissue depression formation of the trachea, pectus excavatum, etc.

[0023] Cartilage culture process The cartilage culture step is a step for culturing cartilage using a first culture medium to obtain mature chondrocytes. The cartilage culture step may involve culturing the collected cartilage as is (after washing). The cartilage culture step may also be a step for culturing cut microcartilage, or a step for culturing microcartilage that has been finely divided and filtered. The following description will be based on the step for culturing microcartilage that has been finely divided and filtered.

[0024] The first culture medium is a medium containing hydrocortisone and FGF-2. This medium may contain either or both of autologous serum and FBS. The first culture medium is preferably a medium containing autologous serum, hydrocortisone, and FGF-2, or a medium containing FBS, hydrocortisone, and FGF-2.

[0025] The culture medium can be prepared by adding the necessary elements to the basal medium as appropriate. Examples of basal media include α-MEM medium, Eagle's basal medium, and DMEM. Known reagents used in culture media can be added as appropriate. Examples of reagents include 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), TGFβ, BMP, steroids, chondroitin acid, soybean trypsin inhibitor, ascorbic acid, hyaluronic acid, proline, dexamethasone, insulin, transferrin, and selenite. When adding ascorbic acid or other substances to the culture medium, salt forms such as 2-phosphate salts can be added. Each should be added to the medium at a concentration of 0.1 ng / mL to 20 μg / mL (or 0.2 ng / mL to 10 μg / mL). These can be added after adjusting appropriately depending on the degree of purification and the required amount. Autologous serum can also 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.

[0026] An example of a culture medium is α-MEM medium supplemented with 1-10% fetal bovine serum (FBS), 20 ng / ml to 100 ng / ml hydrocortisone, and 5 ng / ml to 20 ng / ml (or 50 ng / ml) FGF2 (Fibroblast Growth Factor 2). 1-10% autologous serum may be added in addition to or instead of the 1-10% fetal bovine serum (FBS). The amounts of these may be adjusted appropriately. For example, the medium may contain 0.1% to 20% FBS and autologous serum.

[0027] Microcartilage may be cultured and grown under standard culture conditions. Cell volumes can range from around 10% to 100% confluence, and it is also possible to culture at high densities, even exceeding 100% confluence, in a layered state. Immediately after transplantation, or after leaving the cells to rest for a while, they may be transferred to a hypoxic state. Hypoxic culture can be achieved using, for example, a hypoxic incubator that mixes commercially available nitrogen gas to reduce the oxygen partial pressure, or by blowing nitrogen gas into an appropriate space to reduce the oxygen partial pressure.

[0028] The cartilage culture step includes, for example, culturing cartilage in an environment of 2% to 15% carbon dioxide gas and 1% to 10% oxygen gas. In the cartilage culture step, different media may be used for primary culture and subculture. For example, a medium containing autologous serum and FGF-2 may be used for primary culture. Furthermore, for example, when culturing cartilage or mature chondrocytes after cryopreservation for a certain period of time, a medium containing FBS, hydrocortisone, and FGF-2 may be used.

[0029] Step of obtaining mature chondrocyte-containing composition The step of obtaining a mature chondrocyte-containing composition is a step for obtaining a mature chondrocyte-containing composition containing mature chondrocytes and a conditioned medium obtained through a cartilage culture step. The mature chondrocyte-containing composition may contain mature chondrocytes and a culture supernatant.

[0030] A preferred example of mature chondrocytes is a cell that expresses the chemokines / cytokines interleukin-8 (IL-8), growth-related gene (GRO), monocyte chemoattractant protein-1 (MCP-1), and VEGF at levels four times higher than IL-6. The mature chondrocytes may express one or more of IL-8, GRO, and MCP-1 at levels ten times higher than IL-6, or at levels twenty times higher than IL-6, or at levels twenty times higher than IL-6. By containing cytokines / chemokines with high revascularization-promoting ability and low inflammatory potential, the mature chondrocyte-containing composition exhibits high angiogenesis-promoting ability without causing inflammation upon transplantation, making it suitable for transplantation. This mature chondrocyte-containing composition is particularly preferably one that contains cartilage harvested from the patient to be transplanted.

[0031] Preferred examples of mature chondrocytes are those that express one or more of interleukin 8 (IL-8), growth-related gene (GRO), monocyte chemoattractant protein-1 (MCP-1), and VEGF at a level 10 times or more compared to TNF-α, preferably at a level 50 times or more compared to TNF-α, preferably at a level 100 times or more compared to TNF-α, and preferably at a level 500 times or more compared to TNF-α.

[0032] Preferred examples of mature chondrocytes are those that express one or more of interleukin 8 (IL-8), growth-related gene (GRO), monocyte chemoattractant protein-1 (MCP-1), and VEGF at a level 10 times or more compared to IL-1-β, preferably at a level 50 times or more compared to IL-1-β, preferably at a level 100 times or more compared to IL-1-β, and preferably at a level 500 times or more compared to IL-1-β.

[0033] Preferred examples of mature chondrocytes are those that express one or more of interleukin 8 (IL-8), growth-related gene (GRO), monocyte chemoattractant protein-1 (MCP-1), and VEGF at a level 10 times or more compared to INF-γ, preferably at a level 50 times or more compared to INF-γ, preferably at a level 100 times or more compared to INF-γ, and preferably at a level 500 times or more compared to INF-γ.

[0034] The mature chondrocyte-containing composition may contain an appropriate amount of mature chondrocytes and conditioned medium (or culture supernatant) depending on the purpose. This composition may be stored in a required container in the same way as a normal composition, and may be used as needed. The amount of mature chondrocytes per use may be, for example, 1 x 10 4 1 x 10 cells or more 10 Cells are acceptable, or 1 x 10 per use 5 1 x 10 cells or more 8 It may also be a cell.

[0035] Agents containing a culture supernatant as an active ingredient are publicly known, as disclosed in, for example, JP 2013-18756 A, Japanese Patent No. 5139294, and Japanese Patent No. 5526320. Therefore, a composition containing a conditioned medium can be produced using a publicly known method.

[0036] Examples of culture supernatants of mature chondrocytes include a processed product obtained by freeze-drying the culture supernatant, which is the supernatant component obtained by solid-liquid separation of the culture supernatant by centrifugation, to remove water; a processed product obtained by concentrating the culture supernatant under reduced pressure using an evaporator or the like; a processed product obtained by concentrating the culture supernatant using an ultrafiltration membrane or the like; a processed product obtained by solid-liquid separation of the culture supernatant using a filter; or the undiluted culture supernatant before the above-mentioned processes. Furthermore, for example, the supernatant obtained by culturing the mature chondrocytes of the present invention may be centrifuged (e.g., 1,000 × g, 10 minutes) and then fractionated with ammonium sulfate (e.g., 65% saturated ammonium sulfate). The precipitate may be suspended in an appropriate buffer solution, dialyzed, and filtered through a syringe filter (e.g., 0.2 μm) to obtain a sterile culture supernatant. The collected culture supernatant may be used as is, or may be stored frozen and thawed before use. Alternatively, a pharmaceutically acceptable carrier may be added and dispensed into sterile containers to obtain a liquid volume that is easy to handle. Furthermore, as a countermeasure against the risk of infectious pathogens, the culture supernatant may be treated with a virus clearance filter or by ultraviolet irradiation. The mature chondrocyte-containing composition preferably contains 1 mL to 1,000 mL of culture supernatant as a single dosage unit, and more preferably 30 mL to 300 mL.

[0037] By simultaneously adding cultured chondrocytes and conditioned medium (e.g., supernatant from mature chondrocytes) at the time of transplantation, vascular regeneration around the cartilage is promoted, improving the engraftment rate of chondrocyte transplants and significantly increasing the amount of regenerated cartilage, resulting in successful transplantation. This composition is used, for example, in earplasty, rhinoplasty, mandibular augmentation, zygomatic hard tissue depressionplasty, cranial hard tissue depressionplasty, and pectus excavatum hard tissue depressionplasty.

[0038] Another invention described in this specification relates to a vascular regeneration promoter. This vascular regeneration promoter includes, for example, a mature chondrocyte-containing composition obtained by any of the above-mentioned methods for producing a mature chondrocyte-containing composition. In other words, this vascular regeneration promoter is similar to the above-mentioned mature chondrocyte-containing composition. The vascular regeneration promoter may be, for example, an injectable agent. The above-mentioned mature chondrocyte-containing composition may be contained in a syringe, mixed with patient tissue as needed, and implanted into the required location in the patient. The vascular regeneration promoter includes a mature chondrocyte-containing composition that includes a vessel-inducing factor and VEGFR2-positive cells. This method includes the step of producing a mature chondrocyte-containing composition by the above-mentioned method for producing a mature chondrocyte-containing composition. [Example]

[0039] One square centimeter of auricular cartilage was harvested from the patient's ear remnants. The fragments were minced and rinsed with phosphate-buffered saline (Ca-) supplemented with antibiotics. They were then treated with 0.3% collagenase (Worthington Biochemical, Freehold, NJ) / PBS and rotated on a stirrer at 37°C for 4–6 hours before chondrocyte isolation. Chondrocytes were cultured at 1 × 10 3 cells / cm 2 The chondrocytes were seeded at a cell density of 175 cm and primary cultured in DMEM medium supplemented with 10% autologous serum and FGF-2 (5–10 ng / ml, FIBRAST®, Kaken Pharmaceuticals, Tokyo). 2 1x10 per culture flask 3 cells / cm 2 Then, the subcultured cells were seeded, and the implant material containing chondrocytes and CM was prepared as the final product (1 × 10 7The cells were obtained at a density of 2-3 x 10 cells / 1 cc CM. The medium used was 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. On the 12th day of primary culture, 2-3 x 10 cells were obtained. 6 The cultured cells were harvested. Some of these cells were frozen and the other cells were frozen at 4-5 x 10 4 / cm 2 The cells were seeded at a density of 1000 and cultured for 14 days. After 15 days, they were washed three times with PBS(-) and cultured for two days in a medium containing the above medium components but excluding FBS. This medium was used for analysis as the conditioned medium.

[0040] Medium composition The culture 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 and 5% oxygen. Figure 1 is a photograph, in place of a drawing, showing a phase contrast microscope photograph of the chondrocytes of Example 1. [Example]

[0041] Cytokine / chemokine analysis of conditioned medium from cultured mature chondrocytes was performed using the antibody-immobilized magnetic bead method. The conditioned medium sample was centrifuged at 13,000 G at 4°C for 5 minutes, and the supernatant was used for measurement. The concentrations of 40 target proteins in the conditioned medium were measured using the Luminex® system. 25 μL of pretreated sample was used per well, and measurements were performed in triplicate. One standard solution was added according to the manual, and a 5-fold dilution series of seven points was prepared and measured in triplicate. The 40 target proteins include EGF, FGF-2, eotaxin, TGF-α, G-CSF, Flt-3L, GM-CSF, fractalkine, IFNα2, IFNg, GRO, IL-10, MCP-3, IL-12P40, MDC, IL-12P70, PDGF-AA, IL-13, PDGF-AB / BB, IL-15, sCD40L, These were IL-17A, IL-1RA, IL-1α, IL-9, IL-1b, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IP-10, MCP-1, MIP-1α, MIP-1b, RANTES, TNFα, TNFb, and VEGF.

[0042] Cytokine / chemokine analysis of the conditioned medium of cultured mature chondrocytes was performed using the antibody-immobilized magnetic beads method. The results are shown in Figure 2. Figure 2 is a graph, instead of a drawing, showing the analysis results of cytokines and chemokines contained in the conditioned medium obtained when cultured mature chondrocytes were cultured. As shown in Figure 2, it was proven that IL-8, GRO, and MCP-1, cytokines with high vascular regeneration potential, were produced in large amounts (ng / ml). On the other hand, these cells hardly produced any TNF-α, which causes inflammation, or IL-1-β and INF-γ, which have strong inflammation-inducing properties, indicating that they are safe. [Example]

[0043] Chondrocyte transplantation into humans (chondrogenesis for ear reconstruction) [Case 1]: A 22-year-old male with bilateral microtia (absence of external ears) and malar hypoplasia underwent reconstructive treatment using cultured cartilage. A 15mm x 15mm piece of cartilage tissue was harvested from the right residual auricular cartilage, sterilized, and seeded in a high glucose-DME medium supplemented with autologous serum, 5% FBS, hydrocortisone, and FGF2. On the 11th day of primary culture, 4.3 x 10 6 On the 13th day of primary culture, 2.2 x 10^7 cultured cells were harvested. These cultured cells were temporarily frozen and stored. Before transplantation, the thawed cells were cultured for 40 days in 30 flasks with a bottom area of ​​150 cm^2. A composition containing cultured mature chondrocytes and conditioned medium was added to a total volume of 230 cc (1 x 10^7). 7 A composition containing cultured mature chondrocytes and conditioned medium was injected into both lower abdominal regions, and regenerated cartilage was harvested one year after transplantation and used to reconstruct the ears and cheekbones on both sides. Figure 3 is a photograph, in place of a drawing, showing regenerated cartilage harvested from the abdomen one year after transplantation. In Figure 3, the size of the regenerated cartilage was 80 x 200 x 8 mm. The cut surface of the cartilage was full-thickness white cartilage tissue.

[0044] Next, histopathological analysis was performed to verify cartilage regeneration. The results are shown in Figure 4. Figure 4 is a photograph, instead of a drawing, showing the results of histopathological analysis. Figure 4A shows the results of HE staining. Figure 4B shows the results of toluidine blue staining. Figure 4C shows the results of Alcian blue staining. Figure 4D shows the results of Elastica van Gieson staining. As shown in Figure 4A, perichondrium was formed around the cartilage, and numerous vascular cavities were observed within it. This indicates that nutrients are supplied to the cartilage from the perichondrium. Furthermore, Figures 4B to 4D show that cartilage was formed, as each was stained with a dye that specifically stains cartilage. As shown in Figure 4, mature regenerated cartilage and regenerated perichondrium were formed after transplantation, and it was clear that the characteristics and properties of the elastic cartilage tissue derived from the auricular origin were maintained. [Example]

[0045] Chondrocyte transplantation in humans: reconstruction of pectus excavatum and chest cavity An 18-year-old male was found to have a residual chest cavity (from the midline around the xiphoid process to the hypochondrium) after surgery for pectus excavatum. A 10mm x 7mm piece of cartilage tissue was taken 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. On the 21st day of primary culture, 4.2 x 10 6 cells, 3.3 × 10 on day 23 of primary culture 6 The cultured cells were harvested and temporarily frozen. Before transplantation, the thawed cells were placed in a 150cm2 tube with a base area of ​​150cm2. 2 The total volume of cultured mature chondrocytes and conditioned medium transplanted was 84 mL (1 x 10 7 Two years after transplantation, the cartilage had not been absorbed and the external appearance of the chest was good. [Example]

[0046] Chondrocyte transplantation in humans: nasal deformity and skull depression deformity A 20-year-old woman with a saddle nose, short nose, and depressed forehead deformity underwent reconstructive treatment using cultured cartilage for the forehead and nose. A 10mm x 15mm piece of cartilage tissue was harvested from the left auricle, sterilized, and seeded for culture. The medium used was high glucose-DME medium supplemented with autologous serum, hydrocortisone, and fibroblast growth factor 2. On the 18th day of primary culture, 1.1 x 10 7 The cultured cells were harvested and temporarily frozen. Before transplantation, the thawed cells were placed in a 150cm2 tube with a base area of ​​150cm2. 2 The total volume of cultured mature chondrocytes and conditioned medium transplanted was 27.3 mL (1 x 10 7 Three years after transplantation, the cartilage had not been absorbed and the external appearance was good. [Example]

[0047] Changes in VEGFR2 in cultured chondrocytes during passage VEGFR2 is expressed only in vascular endothelial cells and their precursor cells. Therefore, if VEGFR2 is expressed, a composition containing cultured mature chondrocytes and conditioned medium will produce blood vessels. Cells from P2 to P4 were used for transplantation. VEGFR2-expressing cells were present in all cells from P1 to P4. It was revealed that this chondrocyte culture system contained endothelial cells in all cells from P1 to P4. Therefore, if this cell line is transplanted, vascular endothelial cells will appear along with the cartilage tissue, resulting in the construction of a vascular network around the cartilage tissue, which is expected to maintain the cartilage tissue after transplantation. Figure 5 is a photograph (in lieu of a drawing) showing the changes in VEGFR2 in cultured chondrocytes during passage. [Example]

[0048] Next, in order to investigate the number of possible passages of cultured chondrocytes, the number of possible passages and PDL of chondrocytes were measured. Tumorous cells have the characteristic of continuing to grow without stopping their proliferation. In order to confirm the safety of the cultured chondrocytes created in this study, an aging test of the chondrocytes was conducted. The cells were cultured and continuously passaged to see whether proliferation stopped. Auricular chondrocytes from four individuals were cultured for three months and the PDL (CPD) until proliferation stopped was measured (meaning the number of times they divided). The results are shown in Figure 6. Figure 6 is a graph in place of a drawing showing the number of possible passages of chondrocytes and PDL. As shown in Figure 6, it was confirmed that proliferation stopped due to aging. P14 = total 84-day culture: vertical axis CDL (= PDL). A (20 years old), B (37 years old), C (29 years old), D (63 years old)

[0049] Senescence-associated β-galactosidase (SA-β-gal) staining is positive when cells become senescent. Therefore, we performed senescence-associated galactosidae activity staining on auricular chondrocytes at passages P13 and P14 of four individuals to examine cellular senescence. Senescence-associated galactosidae activity staining was performed at each stage, and the above enzyme activity appeared before growth arrest, confirming that growth arrest due to aging had occurred. For senescence-associated galactosidae activity staining, we used the SA X-Gal staining Cellular Senescence Kit: OZBIOSCIENCES (Catalog Number: GXS0003).

[0050] FIG. 7 is a photograph, instead of a drawing, showing cultured mature chondrocytes (P=13) from a 20-year-old woman. A. Chondrocytes from a 20-year-old woman (proliferation stopped at P-13) PDL=0.8952 (P-12:2.5x10^5⇒4.65x10^5 / dish) Total PDL: 27.6676 SA X-Gal: 92% positive

[0051] FIG. 8 is a photograph, instead of a drawing, showing cultured mature chondrocytes (P=13) from a 63-year-old woman. 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) Total PDL: 27.6676 SA X-Gal: 95% positive

[0052] FIG. 9 is a photograph, instead of a drawing, showing cultured mature chondrocytes (P=14) from a 37-year-old woman. B. Chondrocytes from a 37-year-old woman PDL=0.5109 (P-14:1x10^5⇒1.425x10^5 / dish) Total PDL: 44.0962 SA X-Gal: 100% positive

[0053] FIG. 10 is a photograph, instead of a drawing, showing cultured mature chondrocytes (P=14) from a 29-year-old woman. C. Chondrocytes from a 29-year-old woman PDL=0.2013 (P-14:1x10^5⇒1.15x10^5 / dish) Total PDL: 47.1421 SA X-Gal: 100% positive

[0054] Consideration The formation of perichondrium is required to inhibit the formation and resorption of regenerated cartilage. 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 does not form [5]. Therefore, their blood and nutrient supplies are significantly limited. This has been shown to cause cell death and inevitable necrosis of the artificial construct, followed by loss of shape and function [6-8]. On the other hand, mature chondrocyte-containing compositions contain cytokines / chemokines that are highly capable of promoting vascular regeneration and have little inflammation. When transplanted, these cells exhibit high vascular regeneration without causing inflammation, leading to the formation of a regenerated perichondrium around the regenerated cartilage, making them suitable for transplantation. Cartilage-conditioned medium contains 7 times more VEGF than IL6, 20 times more GRO than IL6, 80 times more IL8 than IL6, and 100 times more MCP-1 than IL6. These cells produce very little TNF-α or IL-1-β or INF-γ, which have strong proinflammatory properties, demonstrating their safety. Furthermore, aging tests confirmed that the cells stopped growing, confirming that they were not tumorigenic and demonstrating their safety.

[0055] The angiogenic potential of IL-8 has also been demonstrated (Mikula-Pietrasik J, Kuczmarska A, Kucin´ska M, Muriaset M. et 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 Keglowich et al. supra).

[0056] MCP-1 has been reported as an angiogenic chemokine and its angiogenic potential has been demonstrated in another paper (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 Chem 2008 May 23;283(21): 14542-51. doi: 10.1074 / jbc.M802139200). Furthermore, it has been shown in the literature that MCP-1 mediates the angiogenic activity 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). Macrophage recruitment is essential for angiogenesis, and MCP-1 is an essential factor for this.

[0057] Therefore, it was shown that all four cytokines (MCP-1, IL-8, GRO, VEGF) present in the conditioned medium secreted by the cultured chondrocytes in the examples have angiogenic potential. [Industrial Applicability]

[0058] This invention can be used in the field of medical equipment, etc.

Claims

1. A method for producing mature chondrocytes, comprising a cartilage culture step of culturing cartilage using a first culture medium to obtain mature chondrocytes, The first culture medium comprises: A medium containing steroids and FGF-2 Method for producing mature chondrocytes.

2. 2. The method for producing mature chondrocytes according to claim 1, wherein the first culture medium further comprises an insulin-like growth factor.

3. 3. The method for producing mature chondrocytes according to claim 1 or 2, wherein the steroid is dexamethasone.

4. The method for producing mature cartilage cells according to claim 1, The method, wherein the cartilage is auricular cartilage.

5. The method for producing mature cartilage cells according to claim 1, The cartilage culturing step includes culturing the cartilage in an environment of 2% to 15% carbon dioxide gas and 1% to 10% oxygen gas. method.

6. a cartilage culturing step of culturing cartilage using the first culture medium to obtain mature chondrocytes; and a mature chondrocyte-containing composition obtaining step of obtaining a composition containing mature chondrocytes, the composition comprising the mature chondrocytes and a conditioned medium obtained through the cartilage culturing step. A method for producing a mature chondrocyte-containing composition, comprising: The first culture medium comprises: A medium containing steroids and FGF-2 A method for producing a composition containing mature chondrocytes.

7. 7. The method for producing a mature chondrocyte-containing composition according to claim 6, wherein the first culture medium further contains an insulin-like growth factor.

8. 8. A method for producing a mature chondrocyte-containing composition according to claim 6 or 7, wherein the steroid is dexamethasone.

9. A method for producing a mature chondrocyte-containing composition according to claim 6, comprising: The cartilage culturing step includes culturing the cartilage in an environment of 2% to 15% carbon dioxide gas and 1% to 10% oxygen gas. A method for producing a composition containing mature chondrocytes.

10. A method for producing a mature chondrocyte-containing composition according to claim 6, comprising: The conditioned medium comprises interleukin 8 (IL-8), growth-related gene (GRO), monocyte chemoattractant protein-1 (MCP-1), and VEGF.

11. A method for producing a mature chondrocyte-containing composition according to claim 6, comprising: A method wherein the mature chondrocyte-containing composition is a composition used for transplantation for cartilage regeneration.

12. A method for producing a mature chondrocyte-containing composition according to claim 6, comprising: The mature chondrocyte-containing composition comprises: A method for forming a composition used in ear formation, ear canal formation, nose formation, mandibular formation, hard tissue depression formation of the cheekbone, hard tissue depression formation of the skull, hard tissue depression formation of the trachea, pectus excavatum, etc.

13. A method for producing a vascular regeneration promoter, The vascular regeneration promoter comprises a mature chondrocyte-containing composition containing an angiogenic factor and VEGFR2-positive cells, A method comprising the step of producing a mature chondrocyte-containing composition by the method for producing a mature chondrocyte-containing composition according to claim 6.

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