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] This invention relates to a method for producing mature chondrocytes.

Background Art

[0002] In surgical fields, reconstruction by bone and cartilage transplantation has been performed frequently. Sites used as donors have included the skull, ribs, costal cartilage, ilium, etc. However, it cannot be denied that if a large amount of cartilage is harvested, there is a possibility of functional problems and deformities after donor bone harvesting. In cases requiring multiple reconstruction surgeries, many donor sites are often sacrificed. Also, there is a limit to the amount that can be harvested.

[0003] Therefore, Vacanti et al. invented a tissue engineering method for regenerating cartilage from a limited number of chondrocytes obtained by enzymatic treatment of animal cartilage tissue. This method is an invention in which chondrocytes are seeded on a mesh-structured scaffold made of an artificially synthesized degradable polymer, and the cells adhere and proliferate on the scaffold to regenerate cartilage tissue [1, 2]. However, since cartilage was not uniformly generated in the scaffold, clinically applicable results have not yet been obtained. Furthermore, it has been found that the synthetic polymer of the scaffold induces inflammation when absorbed in the body and causes absorption of the regenerated cartilage [3 - 4]. As later reported by Vacanti et al. , An artificial cartilage structure containing a large number of cells usually requires a high-oxygen environment for its growth and proliferation. Therefore, when cartilage is not formed to the center of the structure, the perichondrium is not formed [5]. Therefore, their blood and nutrient supply is quite limited. This has been found to cause cell death and inevitable necrosis of the artificial construct, and then the shape and function are lost.

Prior Art Documents

Non-Patent Documents

[0004]

Non-Patent Document 1

Non-Patent Document 7

Summary of the Invention

Problems to be Solved by the Invention

[0005] Previously, even when cultured chondrocytes were transplanted, it was difficult to obtain a sufficient amount 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 Problems

[0006] The first invention described in this specification relates to a method for producing mature chondrocytes. These mature chondrocytes can be used, for example, in cartilage regeneration surgery 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. And the first culture medium is a medium containing hydrocortisone and FGF-2.

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

[0008] The cartilage culture step includes, for example, a step of culturing cartilage in an environment of 2% or more and 15% or less of carbon dioxide gas and 1% or more and 10% or less of oxygen gas. In the cartilage culture step, different media may be used for primary culture and subculture. For example, for primary culture, a medium containing autologous serum and FGF-2 may be used. Also, for example, when culturing cartilage or mature chondrocytes after cryopreservation for a certain period, 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 includes a cartilage culture step of culturing cartilage using a first culture medium to obtain mature chondrocytes, and a mature chondrocyte-containing composition obtaining step of obtaining a composition containing mature chondrocytes, the mature chondrocytes, and a conditioned medium obtained through the cartilage culture step. And the first culture medium is a medium containing hydrocortisone and FGF-2.

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

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

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

[0013] The composition containing mature chondrocytes is a composition used for transplantation for cartilage regeneration. The composition containing mature chondrocytes is a composition used for auricle formation, external auditory canal formation, nose formation, mandible formation, hard tissue depression formation surgery of the cheekbone, hard tissue depression formation surgery of the skull, trachea, funnel chest, etc.

[0014] Another invention described in this specification relates to a method for producing an angiogenesis promoter. The angiogenesis promoter includes a composition containing mature chondrocytes, which includes a tube-inducing factor and VEGFR2-positive cells. And this method includes a step of producing a composition containing mature chondrocytes by the method for producing the composition containing mature chondrocytes described above.

Advantages of the Invention

[0015] According to this invention, a method for producing mature chondrocytes suitable for transplantation can be provided. For example, by adding cultured chondrocytes and a conditioned medium (for example, the supernatant of mature chondrocytes) simultaneously at the time of transplantation, angiogenesis around the cartilage can be increased, the engraftment rate of chondrocyte transplantation can be improved, the size of the transplanted cartilage can be enlarged, and long-term survival can be made possible. As a result, the transplanted regenerated cartilage can be sufficiently supplied with blood and nutrients, suppressing cell death and necrosis, and then maintaining its shape and function. In addition, since nutrients are supplied to the cartilage, a large number of chondrocytes can engraft, cartilage absorption is suppressed, and large regenerated cartilage that has not been obtained so far can be obtained.

[0016] Cultured chondrocytes and the supernatant produced by these cells can be added simultaneously during transplantation to promote chondrogenesis, greatly increase the regenerated cartilage, and enable successful transplantation. When transplanted together with chondrocytes and the supernatant (conditioned medium), angiogenesis occurred around the cartilage and a high transplantation rate was achieved. Therefore, when we confirmed what factors were present in the cultured cells, several cytokines / chemokines that cause high-concentration angiogenesis were specifically found. Among mature chondrocytes and the specific cytokines / chemokines produced by these mature chondrocytes that promote angiogenesis, the supernatant (CM: conditioned medium) containing particularly GRO, IL8, MCP-1, and VEGF can be added to chondrocytes to suppress absorption after transplantation and improve the engraftment rate.

Brief Description of the Drawings

[0017]

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

Figure 8

Figure 9

Figure 10

BEST MODE FOR CARRYING OUT THE INVENTION

[0018] Hereinafter, embodiments for carrying out the present invention will be described with reference to the drawings. The present invention is not limited to the embodiments described below, and includes those appropriately modified by those skilled in the art within the obvious scope from the following embodiments.

[0019] The first invention described in this specification relates to a method for producing mature chondrocytes. These mature chondrocytes can be used, for example, in cartilage regeneration surgery 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 of autologous serum and FBS.

[0020] An example of the cartilage is auricular cartilage. The cartilage may be minced cartilage (microchondrocytes). The cartilage may be minced, washed, and then have its blood components removed.

[0021] The cartilage culture step includes, for example, a step of culturing cartilage in an environment of 2% or more and 15% or less of carbon dioxide gas and 1% or more and 10% or less of oxygen gas. Different media may be used for the primary culture and the subculture in the cartilage culture step. For example, a medium containing autologous serum and FGF-2 may be used for the primary culture. Also, for example, when culturing cartilage or mature chondrocytes after cryopreservation for a certain period, 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 auricle formation, external auditory canal formation, nose formation, mandible formation, hard tissue depression formation surgery of the zygomatic bone, hard tissue depression formation surgery of the skull, trachea, funnel chest, etc.

[0023] Cartilage culture step The cartilage culture step is a step for culturing cartilage using a first culture medium to obtain mature chondrocytes. In the cartilage culture step, the collected cartilage may be cultured as it is (after washing). Also, the cartilage culture step may be a step of culturing transected micro-cartilage, or may be a step of culturing minced and filtered micro-cartilage. Hereinafter, an explanation will be given based on the step of culturing minced and filtered micro-cartilage.

[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 preferably is a medium containing autologous serum, hydrocortisone and FGF-2, or a medium containing FBS, hydrocortisone and FGF-2.

[0025] For the medium, necessary elements may be appropriately added to the basal medium. Examples of the basal medium are α-MEM medium, Eagle's basal medium, and DMEM. For the reagents, known ones used for the medium may be appropriately added. Examples of the 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), TGFβ, BMP, steroid, chondroitin sulfate, soybean trypsin inhibitor, ascorbic acid, hyaluronic acid, proline, dexamethasone, insulin, transferrin, and selenous acid. When adding ascorbic acid or the like to the medium, those in the form of salts such as 2-phosphate may be added. They may be added so as to be 0.1 ng / mL or more and 20 μg / mL or less (or 0.2 ng / mL or more and 10 μg / mL or less) in the medium respectively. These may be appropriately adjusted and added according to the degree of purification and the required amount. Instead of FBS, autologous serum may be added. Also, a serum-free medium may be used. In any case, it is preferable to add HC (hydrocortisone) and FGF-2 to the medium.

[0026] An example of the culture medium is one obtained by adding 1 to 10% fetal bovine serum (FBS), hydrocortisone at 20 ng / ml or more and 100 ng / ml or less, and FGF2 (Fibroblast Growth Factor 2) at 5 ng / ml or more and 20 ng / ml (or 50 ng / ml) to α-MEM medium. Instead of or together with 1 to 10% fetal bovine serum (FBS), 1 to 10% autologous serum may be added. These amounts may be appropriately adjusted. For example, FBS and autologous serum may be contained in the medium at 0.1% to 20%.

[0027] The minute cartilage may be cultured and proliferated under normal culture conditions. The cell amount can range from around 10% to a 100% confluent state, and it is also possible to culture in a high-density, multilayered state exceeding 100% confluence. Immediately after transplantation or after standing for a while, it may be transferred to a hypoxic state. For hypoxic culture, for example, a hypoxic incubator of the type that mixes commercially available nitrogen gas or the like to lower the oxygen partial pressure can be used, or it may be cultured by blowing nitrogen gas or the like into an appropriate space to lower the oxygen partial pressure.

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

[0029] Step for obtaining a composition containing mature chondrocytes The step for obtaining a composition containing mature chondrocytes is a step for obtaining a composition containing mature chondrocytes, which includes mature chondrocytes and a conditioned medium obtained through the cartilage culture step. The composition containing mature chondrocytes may contain mature chondrocytes and the culture supernatant.

[0030] Preferred examples of mature chondrocytes are cells that express chemokine / cytokines interleukin 8 (IL-8), growth-related gene (GRO), monocyte chemoattractant factor-1 (MCP-1), and VEGF at least 4-fold more than IL-6. Any one or two or more of IL-8, GRO, and MCP-1 may be expressed at least 10-fold more than IL-6, or at least 20-fold more than IL-6, or at least 20-fold more than IL-6. By including such cytokines / chemokines with high angiogenesis-promoting ability and low inflammation, the mature chondrocyte-containing composition exhibits high angiogenesis-promoting ability without causing inflammation when transplanted, making it suitable for transplantation. This mature chondrocyte-containing composition preferably contains cartilage collected from a patient to be transplanted, in particular.

[0031] Preferred examples of mature chondrocytes are those that express any one or two or more of interleukin 8 (IL-8), growth-related gene (GRO), monocyte chemoattractant factor-1 (MCP-1), and VEGF at least 10-fold more than TNF-α, preferably at least 50-fold more than TNF-α, more preferably at least 100-fold more than TNF-α, and most preferably at least 500-fold more than TNF-α.

[0032] Preferred examples of mature chondrocytes are those that express any one or two or more of interleukin 8 (IL-8), growth-related gene (GRO), monocyte chemoattractant factor-1 (MCP-1), and VEGF at least 10-fold more than IL-1-β, preferably at least 50-fold more than IL-1-β, more preferably at least 100-fold more than IL-1-β, and most preferably at least 500-fold more than IL-1-β.

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

[0034] The mature chondrocyte-containing composition may contain an appropriate amount of mature chondrocytes and conditioned medium (or culture supernatant) according to the purpose. Further, this composition may be contained in a necessary container in the same manner as a normal composition and made available as needed. The amount of mature chondrocytes may be, for example, 1×10 4 cells or more to 1×10 10 cells per single use, or 1×10 5 cells or more to 1×10 8 cells per single use.

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

[0036] Examples of the culture supernatant of mature chondrocytes include a processed product obtained by removing moisture from the culture supernatant, which is the supernatant component obtained by solid-liquid separation of the culture supernatant by centrifugation, 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, or a processed product obtained by solid-liquid separation of the culture supernatant using a filter, or the stock solution of the culture supernatant before performing the above-described treatment. Further, for example, the supernatant obtained by culturing the mature chondrocytes of the present invention is centrifuged (for example, 1,000×g, 10 minutes), fractionated with ammonium sulfate (for example, 65% saturated ammonium sulfate), the precipitate is suspended in an appropriate buffer, followed by dialysis treatment, filtered through a syringe filter (for example, 0.2 μm), and a sterile culture supernatant may be obtained. The collected culture supernatant may be used as it is, or stored frozen and thawed at the time of use. Further, a pharmaceutically acceptable carrier may be added and dispensed into a sterile container so that the volume is easy to handle. Furthermore, as a measure against the risk of infectious pathogens, the culture supernatant may be treated by a virus clearance filter or ultraviolet irradiation. The mature chondrocyte-containing composition preferably contains 1 mL or more and 1,000 mL or less of the culture supernatant as one administration unit, and more preferably 30 mL or more and 300 mL or less.

[0037] By simultaneously adding and transplanting cultured chondrocytes and a conditioned medium (for example, the supernatant of mature chondrocytes) at the time of transplantation, angiogenesis around the cartilage can be increased, the engraftment rate of chondrocyte transplantation can be improved, the regenerated cartilage can be greatly increased, and the transplantation can be made successful. This composition is used, for example, in auricle formation, nose formation, mandible formation, hard tissue concave formation of the cheekbone, hard tissue concave formation of the skull, and hard tissue concave formation of pectus excavatum.

[0038] Another invention described in this specification relates to an angiogenesis promoter. This angiogenesis promoter includes, for example, a mature chondrocyte-containing composition obtained by the method for producing any of the above-described mature chondrocyte-containing compositions. That is, this angiogenesis promoter is the same as the above-described mature chondrocyte-containing composition. The angiogenesis promoter may be, for example, an injection. The above-described mature chondrocyte-containing composition may be contained in a syringe, mixed with a patient's tissue as necessary, and transplanted to the required part of the patient. The angiogenesis promoter includes a mature chondrocyte-containing composition containing a tube-inducing factor and VEGFR2-positive cells. And this method includes a step of producing a mature chondrocyte-containing composition by the method for producing the above-described mature chondrocyte-containing composition.

Example

[0039] 1 square centimeter of auricular cartilage to was collected from the remains of a patient's ear. The fragments were minced and rinsed with phosphate-buffered saline (Ca-) supplemented with antibiotics. Next, they were treated with 0.3% collagenase (Worthington Biochemical, Freehold, NJ) / PBS and rotated at 37 °C for 4 to 6 hours with a stirrer, and then chondrocytes were isolated. In the primary culture, chondrocytes were seeded at a cell density of 1×10 3 cells / cm 2 and primary cultured in DMEM medium supplemented with 10% autologous serum and FGF-2 (5 - 10 ng / ml, FIBRAST (registered trademark), Kaken Pharmaceuticals, Tokyo). In the subculture, chondrocytes were seeded at 1 2 per 175 cm × 10 3 cells / cm 2 . Next, the subcultured cells were seeded, and a graft material containing chondrocytes and CM was used as the final product (1 × 10 7cells / 1 cc CM) and obtained. 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 was carried out under 5 - 10% carbon dioxide 。 On the 12th day of primary culture, 2 - 3 × 10 6 cultured cells of cells were collected. A part of these cells was frozen, and the other cells were seeded at a density of 4 - 5x10 4 / cm 2 and cultured for 14 days. After 15 days, they were washed 3 times with PBS(-) and cultured for 2 days in a medium obtained by removing FBS from the above medium components 。 This medium was used for analysis as conditioned medium 。

[0040] Medium composition 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 was carried out under 10% carbon dioxide and 5% oxygen 。 Figure 1 is a photograph replacing the drawing showing the phase contrast micrograph of chondrocytes in Example 1

Example

[0041] Cytokine / chemokine analysis of the conditioned medium of cultured mature chondrocytes was performed by the Antibody-Immobilized Magnetic Beads method 。The supernatant obtained by centrifuging the conditioned medium sample at 13,000 G for 5 minutes at 4°C was used for measurement. The concentrations of 40 target proteins in the conditioned medium were measured using the Luminex® system. The pretreated samples were used at 25 μL per well, and the measurement was performed 3 times. One additional standard solution was added manually, and a 5-fold dilution series was prepared at 7 points and measured 3 times. The 40 target proteins were EGF, FGF-2, eotaxin, TGF-α, G-CSF, Flt-3L, GM-CSF, fractalkine, IFNα2, IFN γ , 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-1α, IL-9, IL-1 β , IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IP-10, MCP-1, MIP-1α, MIP-1 β , RANTES, TNFα, TNF β , and VEGF.

[0042] Cytokine / chemokine analysis of the conditioned medium of cultured mature chondrocytes was performed by the Antibody-Immobilized Magnetic Beads method. The results are shown in Figure 2. Figure 2 is a graph in place of a drawing showing the analysis results of cytokines and chemokines contained in the conditioned medium obtained when culturing mature chondrocytes. As shown in Figure 2, it was proved that a large amount (ng / ml) of IL-8, GRO, and MCP-1, which are cytokines with high angiogenesis ability, were produced. On the other hand, TNF-α that causes inflammation, IL-1-β with strong inflammatory induction ability, and INF-γ were hardly produced in these cells, indicating that it is safe.

Example

[0043] Cartilage cell transplantation into humans (chondrogenesis for auricular reconstruction) [Case 1]: A 22-year-old male with bilateral microtia (absence of the outer ear) and hypoplasia of the cheekbones underwent reconstructive treatment with cultured cartilage. Cartilage tissue measuring 15 mm × 15 mm was collected from the right residual auricular cartilage, sterilized, and seeded in a culture medium prepared by adding autologous serum, 5% FBS, hydrocortisone, and FGF2 to High glucose-DME medium. On the 11th day of primary culture, 4.3×10 6 cells were obtained, and on the 13th day of primary culture, 2.2×10^7 cells of cultured cells were recovered. These cultured cells were cryopreserved once. Before transplantation, the thawed cells were cultured in 30 flasks with a bottom area of 150 cm^2 for 40 days. A total of 230 cc (1 X 10 7 cells / 1 cc CM) of a composition containing cultured mature cartilage cells and conditioned medium was prepared. The composition containing cultured mature cartilage cells and conditioned medium was injected and transplanted into both lower abdominal regions, and the regenerated cartilage collected 1 year after transplantation was used to reconstruct both auricles and cheekbones. Figure 3 is a photograph replacing the drawing showing the regenerated cartilage collected from the abdomen 1 year after transplantation. In Figure 3, the size of the regenerated cartilage was 80x200x8 mm. The cross-section of the cartilage was a white cartilage tissue throughout the layer.

[0044] Next, histological analysis was performed to prove that cartilage had regenerated. The results are shown in Figure 4. Figure 4 is a photograph replacing the drawing showing the results of histological 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 elastic van Gieson staining. As shown in Figure 4A, a perichondrium was formed around the cartilage, and numerous vascular lumens were observed therein. This indicates that nutrition is supplied from the perichondrium to the cartilage. Also, from Figures 4B to 4D, it can be seen that cartilage has been formed because it is stained with dyes that specifically stain cartilage. 。As shown in Fig. 4, it can be seen that mature regenerated cartilage and regenerated perichondrium are formed after transplantation, and the traits and characteristics of elastic cartilage tissue derived from auricular origin are maintained.

Example

[0045] Cartilage cell transplantation into humans: Reconstruction of pectus excavatum chest depression In an 18-year-old male, residual chest depression (around the midsternal notch to the costal margin) was observed after pectus excavatum surgery. Cartilage tissue measuring 10 mm × 7 mm was collected from the left auricle, sterilized, and seeded for culture. The culture medium used was high glucose-DMEM medium supplemented with autologous serum, hydrocortisone, and fibroblast growth factor 2. On the 21st day of primary culture, 4.2×10 6 cells were collected, and on the 23rd day of primary culture, 3.3×10 6 cells of cultured cells were collected. These cultured cells were cryopreserved once. Before transplantation, the thawed cells were cultured in 15 flasks with a bottom area of 150 cm 2 for 40 days. The total amount of transplanted cultured mature cartilage cells and conditioned medium was 84 mL (1X10 7 cells / 1 cc CM). Two years after transplantation, the cartilage was not absorbed, and the external appearance of the chest was good.

Example

[0046] Cartilage cell transplantation into humans: Nasal deformation, cranial concave deformation A 20-year-old female with saddle nose, short nose, and concave deformation of the forehead underwent reconstruction treatment with cultured cartilage on the forehead and nose. Cartilage tissue measuring 10 mm × 15 mm was collected from the left auricle, sterilized, and seeded for culture. The culture medium used was high glucose-DMEM medium supplemented with autologous serum, hydrocortisone, and fibroblast growth factor 2. On the 18th day of primary culture, 1.1×10 7 cells of cultured cells were collected. These cultured cells were cryopreserved once. Before transplantation, the thawed cells were cultured in flasks with a bottom area of 150 cm 2Seven flasks were cultured for 40 days. The total amount of transplanted cultured mature chondrocytes and conditioned medium was 27.3 mL (1X10 7 cells / 1 cc CM). Three years after transplantation, the cartilage was not absorbed and the external appearance was good.

Example

[0047] Changes in VEGFR2 of cultured chondrocytes during passage VEGFR2 is only expressed in vascular endothelial cells and their progenitor cells. Therefore, if VEGFR2 appears at the beginning, it can be seen that the composition containing cultured mature chondrocytes and conditioned medium can form blood vessels. The cells used for transplantation were from P2 to P4. VEGFR2-expressing cells were present from P1 to P4. In this chondrocyte culture system, it was revealed that endothelial cells were included from P1 to P4. Therefore, if this cell line is transplanted, vascular endothelial cells will appear together with the cartilage tissue, and as a result, a vascular network will be constructed around the cartilage tissue, and the maintenance of the transplanted cartilage tissue can be expected. Figure 5 is a photograph instead of a drawing showing the changes in VEGFR2 of cultured chondrocytes during passage.

Example

[0048] Next, for the purpose of examining the number of passages possible for cultured chondrocytes, the number of passages possible and PDL of chondrocytes were measured. Tumorigenic cells have the characteristic of continuous growth without growth arrest. For the purpose of confirming the safety of the cultured chondrocytes prepared this time, an examination of chondrocyte aging was conducted. This is an examination to culture the cells and see if growth stops when continuous passage is carried out. The PDL (CPD) until growth arrest was measured by culturing auricular chondrocytes from 4 subjects for 3 months. (It means how many times the cells have divided). The results are shown in Figure 6. Figure 6 is a graph instead of a drawing showing the number of passages possible and PDL of chondrocytes. As shown in Figure 6, it was confirmed that growth arrest due to aging has occurred. 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] Since cells show positive staining for senescence-associated β-galactosidase (SA-β-gal) when they age, the senescence-associated enzyme Senescence-associate galactosidae activity staining of auricular cartilage cells at passages P13 and P14 from 4 individuals was performed to examine cell aging. As a result, at each stage, the enzyme activity appeared before growth arrest after performing Senescence-associate galactosidae activity staining, confirming that growth arrest due to Aging was occurring. For Senescence-associate galactosidae activity staining, the SA X-Gal staining Cellular Senescence Kit: OZBIOSCIENCES (Catalog Number: GXS0003) was used

[0050] Figure 7 is a photograph replacing the drawing showing cultured mature cartilage cells (P = 13) of a 20-year-old female A. Cartilage cells of 20-year-old female (growth arrest 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] Figure 8 is a photograph replacing the drawing showing cultured mature cartilage cells (P = 13) of a 63-year-old female D. Cartilage cells of 63-year-old female (growth arrest 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] Figure 9 is a photograph replacing the drawing showing cultured mature cartilage cells (P = 14) of a 37-year-old female B. Cartilage cells of 37-year-old female PDL = 0.5109 (P - 14: 1 x 10^5 ⇒ 1.425 x 10^5 / dish) Total PDL: 44.0962 SA X - Gal: 100% positive

[0053] Figure 10 is a photograph replacing the drawing showing cultured mature chondrocytes (P = 14) of a 29 - year - old female. C. Chondrocytes of 29 - year - old female PDL = 0.2013 (P - 14: 1 x 10^5 ⇒ 1.15 x 10^5 / dish) Total PDL: 47.1421 SA X - Gal: 100% positive

[0054] Discussion To suppress the formation and absorption of regenerated cartilage, the formation of perichondrium is necessary. An artificial cartilage structure containing a large number of cells usually requires a high - oxygen environment for its growth and proliferation. Therefore, if cartilage is not formed to the center of the structure, the perichondrium will not be formed [5]. Consequently, their blood and nutrient supply is quite limited. This has been found to cause cell death and inevitable necrosis of the artificial construct, and then its shape and function are lost [6 - 8]. On the other hand, a composition containing mature chondrocytes is suitable for the transplantation of regenerated cartilage because it contains cytokines / chemokines with high angiogenesis - promoting ability and low inflammation, and can exert high angiogenesis - promoting ability without causing inflammation when transplanted, so a regenerated perichondrium is formed around the regenerated cartilage. The chondrocyte - conditioned medium contains VEGF 7 times that of IL6, GRO 20 times that of IL6, IL8 80 times that of IL6, and MCP - 1 100 times that of IL6. It has also been shown to be safe because TNF - α, IL - 1 - β with strong inflammation - inducing ability, and INF - γ are hardly produced in these cells. In addition, in the Aging test, it was confirmed that growth arrest occurred, so it was confirmed that the cells were not tumorigenic and was also shown to be safe.

[0055] The angiogenic ability of IL-8 has also been shown in the literature (Mikula-Pietrasik J, Kuczmarska A, Kucinska 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 the above Keglowich1 et al.).

[0056] MCP-1 has been reported as an angiogenic chemokine, and its angiogenic ability has also been shown in another piece of literature (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). Additionally, it has been shown in the literature (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) that MCP-1 mediates the angiogenic ability of VEGF. 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 ability.

Industrial Applicability

[0058] This invention can be used in the field of medical devices and the like.

Claims

1. A cartilage culture step of culturing cartilage using a first culture medium to obtain mature chondrocytes, and A step of obtaining a composition containing mature chondrocytes, comprising the mature chondrocytes and a conditioned medium obtained through the cartilage culture step. A method for producing a composition containing mature chondrocytes, The first culture medium is A medium containing hydrocortisone and FGF-2 A method for producing a composition containing mature chondrocytes, The composition containing mature chondrocytes is a composition used for transplantation for cartilage regeneration, or A method that is a composition used for auricle formation, external auditory canal formation, nose formation, mandible formation, hard tissue depression formation surgery of the cheekbone, hard tissue depression formation surgery of the skull, hard tissue depression formation surgery such as trachea and funnel chest.

2. A method for producing a composition containing mature chondrocytes according to Claim 1, The cartilage culture step includes a step of culturing the cartilage in an environment of 2% or more and 15% or less of carbon dioxide gas and 1% or more and 10% or less of oxygen gas. A method for producing a composition containing mature chondrocytes.

3. A method for producing a composition containing mature chondrocytes according to Claim 1, The conditioned medium contains interleukin 8 (IL-8), growth-related gene (GRO), monocyte chemoattractant protein-1 (MCP-1), and VEGF.

4. A method for producing an angiogenesis promoter, A cartilage culture step of culturing cartilage using a first culture medium to obtain mature chondrocytes, and A step of obtaining a composition containing mature chondrocytes, comprising the mature chondrocytes and a conditioned medium obtained through the cartilage culture step. A method for producing a composition containing mature chondrocytes, The first culture medium is A medium containing hydrocortisone and FGF-2 By the method for producing a composition containing mature chondrocytes, A step of producing a composition containing mature chondrocytes, comprising an angiogenesis factor and VEGFR2-positive cells, The angiogenesis promoter contains the composition containing mature chondrocytes. Method.

5. The method according to Claim 4, The cartilage culture step includes a step of culturing the cartilage in an environment of 2% or more and 15% or less of carbon dioxide gas and 1% or more and 10% or less of oxygen gas. Method.

6. The method according to Claim 4, The method wherein the conditioning medium contains interleukin 8 (IL-8), growth-related gene (GRO), monocyte chemoattractant protein-1 (MCP-1), and VEGF.

Images