KR100522457B1 - Cultivation method of Osteoblast and The composition - Google Patents

Abstract

The present invention relates to an artificial culture of bone cells that can be clinically implanted into bone defect sites such as avascular necrosis, non-adhesive fractures, and complex fractures. Method and composition for culturing bone cells according to the present invention is a step of separating the bone tissue of the animal, the step of fixing the bone cells of the separated bone tissue in the culture vessel, the process of propagating bone cells settled in the culture vessel and expanded Selectively apply Ossol I, Ossol II, Ossol III, Oscot I, Oscot II, Odia I, and Odia II to the four basic culture processes of differentiating bone cells in a short time. It is possible to cultivate bone cells so that bone damage can be transplanted quickly.

Description

Cultivation method of Osteoblast and The composition

The present invention relates to artificial cultures of bone cells that can be clinically implanted in bone defect sites such as avascular necrosis, non-adhesive fractures, and complex fractures, and various compositions used in the method for producing the same.

In the United States, about 5.5 million fractures are received each year, and 3.1 million are operated on, according to the 1995 Medical Data International (MDI) statistics, about 426,000 bone grafts were performed. The cost of bone graft is around $ 800 million annually worldwide, and in 1995, 58% of autologous bone grafts, 34% of allografts and 8% of composite grafts were performed.

In the case of simple fractures, a few weeks of gypsum fixation is sufficient, but in cases of severe fractures or bone defects, bone grafts have been performed to date for better results. However, in the case of autograft, the pain at the bone extraction site is severe, the recovery period is long, and there is a difficulty in securing a donor for bone transplantation. In case of allogenic graft, bone strength is weakened and there is a fatal disadvantage that it may be rejection, possibility of infection such as hepatitis or AIDS.

Avascular necrosis of the bone is caused by patients with high intakes of steroids or alcohol, or when the blood supply to the bone is not known correctly. There is no proper treatment other than the surgical method, and the surgical treatment can be divided into the method of maintaining the original joint and replacing the artificial joint. The former method of removing the bones of the necrotic part and transplanting their fibula with blood vessels attached or extracting bones from the long bone is used. However, problems resulting from eliminating transplant donations are hard to overlook. Rotational osteotomy, which used to be the former method, is difficult to predict after surgery. Surgery is also difficult and has a high incidence of complications such as depression of the head and osteoarthritis in the future, and is only used as a temporary measure to delay the arthroplasty in younger patients.

When osteoporosis is a fracture, especially in the case of a fracture of the vertebral region, bed stabilization, assistive attachment, and injection of bone cement into the broken vertebrae with recent treatments are preferred. However, these treatments are not only a temporary treatment that is less effective than bone grafting to a broken vertebral body, but may also cause peripheral nerve damage or paralysis due to the thermal reaction of the polymer, and the injection may flow through the neural tube or blood vessel. It has a risk.

One of the surgical treatments for traumatic bone defects or lower limb immobilization is an angioplasty of the normal bone and an extension of the bone length gradually using an external fixator. However, the bone formation of the distraction portion during distraction surgery is slower than the rate of bone extension, so there is an inconvenience of waiting for a long period of external fixation to achieve bone union.

In the case of isolated bone cysts, which frequently develop pathological fractures and recur after treatment, surgeries such as bone graft, corticosteroid injection, and autologous bone marrow injection have been performed after complete curettage. If you don't get enough bone grafts, the procedure can be difficult.

The present invention has been made to improve the problems of the prior art as described above, to provide a method for culturing bone cells in the amount necessary in as short a time as possible to the bone cells clinically rarely rejection in transplantation. have.

When autologous cells cultured according to the present invention are used for bone transplantation, donor site morbidity that may occur during general autograft and immunological problems that may occur during allogenic graft, Problems such as implant loosening can be solved.

Bone is composed of cells and a large amount of bone matrix between them, and most of bone matrix is composed of organic components composed of collagen fibers (35%) and minerals composed of calcium and phosphoric acid (45%). Is done. Looking at the cross-section of a typical bone, there is a cortical and compact bone, which is a hard layer on the outside, and there is a relatively porous spongy bone inside, and the innermost part of the bone is made of bone cells and blood cells. It is composed of bone marrow.

In bone, osteoblasts, which maintain bone function, are trapped in a space called bone lacuna inside the bone matrix, leading to bone formation. Is present on the surface of the bone matrix and bone marrow. In addition, osteoclasts, which are essential for bone remodeling, are also present in bones, promote the absorption of bone tissue, allow for continuous turn-over of osteoblasts, and broken bones. Help restore normal organization. Of these bone cells, osteoclasts originate from progenitor cells of monocytes in the bone marrow, and the remaining cells are formed by differentiation of osteoprogenitors derived from mesenchymal stem cells in the bone marrow. do. Therefore, osteoblasts can be directly isolated from tissues such as bone marrow, spongy bone, and cortical bone, and cultured by inducing differentiation from primitive osteoblasts.

In order to achieve the above object, the method for culturing bone cells according to the present invention comprises the steps of: (a) separating bone tissue from an animal; (B) dispensing the bone cells of the separated bone tissue in a first culture vessel to settle the bone cells in the first culture vessel while exchanging the cell culture fluid at regular cycles; (C) propagating the bone cells in the culture medium for bone cell growth in the first culture vessel in which the bone cells are settled; (D) harvesting the proliferated bone cells from the first culture vessel and dispensing the bone cells into a second culture vessel and inserting a culture medium for bone cell differentiation to differentiate the bone cells; Method.

In the present invention, the (A) step, (A-1) separating the bone tissue; (A-2) sequentially or selectively using Ossol I, II, or III to remove red blood cells and to separate bone cells.

In the present invention, 'Oszol I' refers to a mixed solution containing ammonium chloride, sodium bicarbonate and EDTA in HBSS solution (Hank's Balanced Salt Solution), preferably 100 to 200mM in HBSS solution Ammonium chloride, 5-20 mM sodium bicarbonate, 0.1-0.5 mM EDTA. Such 'Oszol I' is used for the purpose of dissolving and removing red blood cells mixed in bone tissue in the present invention.

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Bone cells are relatively weakly attached to the surface of bone tissue or are contained within bone tissue. Therefore, in the step (a), the primary bone cell separation can release the bone cells, which are weakly attached to the bone surface, from the surface by shaking the solution containing the bone tissues. It can be liberated using chelating compounds such as enzymes and EDTA. Specifically, the bone tissue of the animal is immersed in a physiological buffer solution and transferred to a centrifuge tube and shaken several times, and then centrifuged to sink the bone tissue and then remove the supernatant. This procedure is repeated several times to wash the bone tissue, and then the bone tissue is suspended in Ossol I solution to dissolve the red blood cells. Centrifugation removes lysed red blood cells, and the precipitated bone tissue is selectively used in Ossol I and Ossol II to break down bone matrix components to release bone cells from the bone. The collected osteogenic cells are collected by centrifugation, and the collected cells are suspended in DMEM or alpha-MEM medium and used for the bone cell settlement step.

In the present invention, the step (b) comprises (b-1) calculating the number of living bone cells after floating the cell culture solution in the bone cells separated in the step (a); (B-2) dividing the living bone cells into the first culture vessels coated with the Oscot I.

In the present invention, 'Oscoat I' refers to a solution for cell culture container coating containing insulin (Insulin) in collagen type 1 (collagen type 1), preferably 5 to 20 mg / L collagen type 1 (collagen type 1) contains 5 to 20 mg / L insulin (Insulin). Such 'Oscoat I' is used in the present invention for the purpose of growing well attached bone cells on the surface of the cell culture vessel.

The cell culture medium is preferably DMEM medium or alpha-MEM medium containing FBS (fetal bovine serum).

In the present invention, the step (c) includes a step of proliferating bone cells inoculated into the first culture vessel using an Odia I culture medium.

In the present invention, 'Odia I' is IGF-1, (insulin-like growth factor type 1), TGF-β (transforming growth factor-β), b-FGF (basic fibroblastgrowth factor), EGF ( epidermal growth factor (PDGF), platelet derived growth factor (PDGF), acidic fibroblast growth factor (a-FGF), bone morphogeniic protein (BMP), keratinocyte growth factor (KGF), hepatocyte growth factor (HGF), etc. Dexamethasone, the growth factor, means a culture medium for bone cell proliferation mixed with insulin, preferably 5 to 20 ug / L IGF-1, 5 to 20 ng / L TGF-β, 5 to 20 mg / L b-FGF, 5 to 20 mg / L EGF, 5 to 20 ug / L PDGF, 5 to 20 ug / L a-FGF, 5 to 20 ug / L BMP, 5 to 20 mg / L KGF, 5 to Growth factors selected from growth factors consisting of 20 mg / L HGF and the like contain 0.5-20 ug / L dexamethasone, 5-20 mg / L insulin. Such 'Odia I' is used for the purpose of promoting bone cell growth in the present invention.

In the present invention, the step (d) may include (d) dividing the bone cells into a second culture vessel coated with Oscot II by collecting the proliferated bone cells from the culture vessels; (D-2) proliferating and differentiating the divided bone cells using an Odia II culture medium.

In the present invention 'Oscoat II' is composed of collagen type 1 (collagen type 1) TGF-β (transforming growth factor-β), BMP (bone morphogeniic protein), dexamethasone (Dexamethasone) and the like Means a cell culture vessel coating solution containing selected growth factors of growth factors, preferably 5 to 20 mg / L collagen type I 5 to 20 ng / L TGF-β, 5 to 20 ug / L BMP, 5-20 ug / L dexamethasone and the like. Such 'Oscoat II' is used for the purpose of increasing bone mineralization ability of bone cells at the same time grow well attached to the surface of the cell culture vessel.

In the present invention, 'Odia II' refers to a culture medium for bone cell differentiation in which BMP (bone morphogeniic protein), dexamethasone (Dexamethasone), bGP (beta-glycerol phosphate), and insulin are mixed. 5-20 ng / L BMP, 5-20 ug / L dexamethasone, 1-5 g / L bGP, 5-20 mg / L insulin. Such 'Odia II' is used for the purpose of increasing bone formation ability of bone cells in the present invention.

Another method for culturing bone cells according to the present invention in order to achieve the above object, (A) the bone tissue of the animals using Ossol I containing ammonium chloride, sodium bicarbonate, EDTA in HBSS Separating from red blood cells and separating bone cells using Ossol II containing collagenase in HBSS; (B) dispensing the separated bone cells into a first culture vessel to fix the bone cells into the first culture vessel while exchanging the culture solution at a predetermined cycle; (C) propagating the bone cells in the culture medium for bone cell growth in the first culture vessel in which the bone cells are settled; And (d) harvesting the proliferated bone cells from the first culture vessel to divide the bone cells into a second culture vessel and insert the culture medium for bone cell differentiation to differentiate the bone cells. Characterized in the culture method.

In the present invention, the step (a) comprises the steps of (a-1) separating the bone marrow tissue including the sponges; (A-2) removing red blood cells; (A-3) separating bone cells from cavernous bone fracture and cavernous bone;

Another method for culturing bone cells according to the present invention in order to achieve the above object, (A) the bone tissue of the animals using Ossol I containing ammonium chloride, sodium bicarbonate, EDTA in HBSS Separating from red blood cells and separating bone cells using Ossol II containing collagenase in HBSS; (B) dispensing the separated bone cells into a first culture vessel to fix the bone cells into the first culture vessel while exchanging the culture solution at a predetermined cycle; (C) propagating the bone cells in the culture medium for bone cell growth in the first culture vessel in which the bone cells are settled; And (d) harvesting the proliferated bone cells from the first culture vessel to divide the bone cells into a second culture vessel and insert the culture medium for bone cell differentiation to differentiate the bone cells. Characterized in that it is produced by the culture method.

(A) step in the present invention, (A-1) the process of collecting the sponge bone tissue; (A-2) removing red blood cells; (A-3) separating bone cells from cavernous bone fracture and cavernous bone;

Another method of culturing bone cells according to the present invention to achieve the above object, (A) after washing the bone tissue of the animal with HBSS (Hanks Balanced Salt Solution) solution to the collagenase (collagenase) in HBSS Separating bone cells using Ossol II containing; (B) dispensing the separated bone cells into a first culture vessel to fix the bone cells into the first culture vessel while exchanging the culture solution at a predetermined cycle; (C) propagating the bone cells in the culture medium for bone cell growth in the first culture vessel in which the bone cells are settled; And (d) harvesting the proliferated bone cells from the first culture vessel to divide the bone cells into a second culture vessel and insert the culture medium for bone cell differentiation to differentiate the bone cells. Characterized in the culture method.

In the present invention, the step (a) may include (a-1) collecting cortical bone tissue; (A-2) washing the cortical bone tissue; (A-3) separating bone cells from cortical bone fractures and cortical bones. In the case of cortical bone tissue, Ossol I does not need to be used because of less red blood cell contamination, and HBSS solution is used for cortical bone cleaning.

Hereinafter, the present invention will be described in more detail with reference to Examples. Since these examples are only for illustrating the present invention, the scope of the present invention is not to be construed as being limited by these examples.

Example 1

Example 1 removes erythrocytes from animal bone tissue using Ossol I solution and dispenses into Oscot I-coated culture vessel to periodically exchange Odia I cell culture media to separate and transplant bone cells. It was characterized by multiplying to a sufficient number and proceeded in the following order.

First, 2.0 ml of animal bone tissue was centrifuged at 1200 rpm for 10 minutes to remove supernatant, thereby obtaining a precipitate. The precipitate was suspended in 30 ml of Ossol I solution to dissolve erythrocytes and centrifuged to obtain cell precipitates from which erythrocytes were removed. The cell precipitate was observed under a microscope and the erythrocyte lysis process using Ossol I solution was repeated until the erythrocytes were completely lysed. The cell precipitate isolated in the above process is suspended in alpha-MEM medium to which FBS is added, and then divided into a culture vessel coated with Oscot I, and placed in a culture vessel at 37 ° C. supplied with 5% carbon dioxide to culture the cells. Settled in. Settled cells were exchanged with the Odia I cell culture medium every three days to continue to grow cells forming an adherent cell colony. When cells have grown more than 90% of the surface of the culture vessel, cells are removed from the surface of the vessel using 0.5% Trypsin- 5.3 mM EDTA solution and suspended in fresh Odia I cell culture medium. The aliquots were placed in an incubator at 37 ° C. supplied with 5% carbon dioxide, followed by cultivation to continue growing. When cells were isolated and proliferated from about 2.0 ml of bone marrow tissue, the cells proliferated to about 100 million bone cells after 3 weeks, and the total expression of basic alkaline phosphatase was about 9 mU per million cells. It was.

Example 2

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First, the bone tissue of the animal was chopped to a size of about 2-3 mm using a surgical blade, soaked in 50 ml of HBSS solution, gently shaken 10 times, allowed to stand for 10 minutes, and washed three times by removing the supernatant. . Suspended in DMEM or alpha-MEM medium and shaken 10 times or more to separate the cells attached to the surface of spongy bone. The remaining bone tissue was transferred to 10 ml of 5 mM EDTA solution and left to stand for 24 hours, exchanged with 10 ml of Ossol II solution, and left standing for 24 hours to separate bone cells in spongy bone tissue. The isolated bone cells were suspended in alpha-MEM medium to which FBS was added, and then aliquoted and fixed in culture coats coated with Oscot I. The settled cells were periodically exchanged with the Odia I cell culture medium to continue to grow cells forming an adherent cell colony. When cells have grown more than 90% of the surface of the culture vessel, cells are removed from the surface of the vessel using 0.5% Trypsin- 5.3 mM EDTA solution and suspended in fresh Odia I cell culture medium. The aliquots were placed in an incubator at 37 ° C. supplied with 5% carbon dioxide, followed by cultivation to continue growing. When cells were isolated and proliferated from about 100 mg of spongy bone tissue, they proliferated to more than 100 million bone cells after 3 weeks, and the total expression of basic alkaline phosphatase was about 12 mU per million cells. It was.

Example 3

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First, the bone tissue of the animal was centrifuged at 1200 rpm for 10 minutes to remove the supernatant to obtain a precipitate. The precipitate was suspended in 30 ml of Ossol I solution to dissolve erythrocytes and centrifuged to obtain cell precipitates from which erythrocytes were removed. The cell precipitate was observed under a microscope and the erythrocyte lysis process using Ossol I solution was repeated until the erythrocytes were completely lysed. The cell precipitate isolated in the above process is suspended in alpha-MEM medium to which FBS is added, and then divided into a culture vessel coated with Oscot I, and placed in a culture vessel at 37 ° C. supplied with 5% carbon dioxide to culture the cells. Settled in. Isolate the spongy bone tissue from the sediment from which the supernatant was removed, and cut the spongy bone tissue into a size of about 2-3 mm using a surgical blade, soak it in 50 ml of HBSS solution, shake it gently 10 times, settle for 10 minutes, and then remove the supernatant. It was washed three times. The spongy bone was suspended in alpha-MEM medium and shaken 10 times or more to separate the cells attached to the surface of the spongy bone. The bone tissue remaining in the above process was left in 10 ml of 5 mM EDTA solution for 24 hours, and then exchanged with 10 ml of Ossol II solution for 24 hours to separate bone cells in spongy bone tissue. The isolated bone cells were suspended in alpha-MEM medium to which FBS was added, and then aliquoted and fixed in culture coats coated with Oscot I. The settled cells were periodically exchanged with the Odia I cell culture medium to continue to grow cells forming an adherent cell colony. When cells have grown more than 90% of the surface of the culture vessel, cells are removed from the surface of the vessel using 0.5% Trypsin- 5.3 mM EDTA solution and suspended in fresh Odia I cell culture medium. The aliquots were placed in an incubator at 37 ° C. supplied with 5% carbon dioxide, followed by cultivation to continue growing. When cells were isolated and proliferated from 2.0 ml of bone marrow tissue containing about 100 mg of spongy bone, the cells proliferated to more than 100 million bone cells after 3 weeks, and the total expression of basic alkaline phosphatase was It was about 11 mU per million cells.

Example 4

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First, the bone tissue of the animal was chopped to a size of about 2-3 mm using a surgical blade, and then washed three times with HBSS solution. Bone cells in cortical bone tissues were allowed to stand in 10 ml of 5 mM EDTA solution for 24 hours and exchanged with 10 ml of Ossol II solution for 24 hours to separate bone cells in cortical tissues. The isolated bone cells are suspended in alpha-MEM medium, and then aliquoted and cultured in a culture vessel coated with Oscot I. Settled cells are periodically exchanged with the Odia I cell culture medium to continue proliferating cells forming an adherent cell colony. When cells have grown more than 90% of the surface of the culture vessel, cells are removed from the surface of the vessel using 0.5% Trypsin- 5.3 mM EDTA solution and suspended in fresh Odia I cell culture medium. The aliquots were placed in an incubator at 37 ° C. supplied with 5% carbon dioxide, followed by cultivation to continue growing. When cells were isolated and propagated from about 100 mg of cortical bone tissue by the above method, they proliferated to more than 10 million bone cells after 3 weeks, and the total expression of basic alkaline phosphatase was about 15 mU per million cells. .

Example 5

Example 5 was characterized by differentiating the expanded bone cells to express the phenotype as bone cells in a short time using Oscot II and Odia II and proceeded in the following order.

The cells proliferated in Example 3 were suspended in Odia II and then fixed in Oscot II coated cell culture vessels. The fixed cells were periodically exchanged with the Odia II cell culture medium to induce the expression of high concentration of alkaline phosphatase. In Example 3, the expression of alkaline dephosphatase (alkaline phosphatase) was 11 mU per million cells, and the expression of basic dephosphatase (alkaline phosphatase) was about 30 per million cells after 3-4 days. increased to mU.

(Measurement of bone cell count)

In the above embodiments, new culture medium and apparatus were introduced in all processes of separation, fixation, proliferation, and differentiation of bone cells, resulting in a significant time saving in each process. As a result, high quality bone cells were obtained. there was. Thus differentiated and expressed phenotype bone cells were released from the culture vessel and packaged in an airtight container. That is, after centrifugation of the bone cells expressing the phenotype, the supernatant was discarded and the cells were collected and counted as follows. The cells to be counted were collected using Trypsin-EDTA solution and then suspended in 10 mL of liquid medium (DMEM or alpha-MEM). Enter the conditions for dilution, measurement, and total cell count in the Coulter Counter, mix 10 mL of the cell suspension with a pipette, and then add 9.8 mL of counter buffer (ISOTON II). 0.2 mL of cell suspension was placed in a 15 mL tube. After shaking to prevent the cells from sinking, the cells were counted using a Coulter counter (total number of cells = number of cells (pieces / 0.5ml) x 2 x dilution factor (500)). The cells counted by the above method were mixed in a cryotube by mixing 1 ml of alpha-MEM (medium solution for cell survival) per 50 million cells, and then completely sealing the lid of the freezing tube. Packaging was completed in a Styropol mobile container maintained at 8 ℃.

(Measurement of bone cell activity)

The activity of bone cells was measured using the following method, and the amount of basic dephosphatase expressed by 100,000 bone cells collected was converted to the total amount of 1 million bone cells. More than one million cells incubated in 100 ul of liquid medium (DMEM or alpha-MEM) were prepared as samples. 25mM pH9.6 Glycine-NaOH buffer, 0.553 mg / ml pH9.6 para-Nitrophenyl phosphate (pNP) solution was prepared, and 120 ml of 0.553 mg / ml pNP solution was used. To the solution was added 1.25 ml of 1 M magnesium chloride (MgCl 2) solution and 15 ml of purified water, adjusted to pH 9.6 with 5N sodium hydroxide (NaOH) solution, and the final volume was 145 ml. The enzyme dilution buffer was prepared by mixing 500 ml of 25 mM glycine-sodium hydroxide buffer solution, 0.5 ml of 1 M magnesium chloride solution, 0.5 ml of 0.1 M zinc chloride (ZnCl 2) solution, and 50 ml of glycerol. The used substrate solution was added to each well of a 96-well plate (90-ul) and placed at 20 ° C. and left at room temperature for 20 minutes on the day of the experiment. Repeat the freeze / thaw process of the cultured bone cells three times and add 100ul of 0.2% Triton X-100 while shaking to mix and add 20ul of lysed cell solution to the wells (standard enzyme). In the case of the solution was diluted in advance by each concentration (0-400mU / ml) and the same amount of the medium was added to the control) was reacted for 5 minutes at 37C and 50N of 1N sodium hydroxide solution was added to stop the reaction. After 10 seconds of stirring, the absorbance was measured at 405 nm using a 96-well plate reader. The 96-well plate reader is a device that quantitatively and qualitatively analyzes the absorbance of a material by irradiating a specific wavelength (visible light or ultraviolet ray) by placing a sample subjected to color reaction in a 96 well plate. A standard curve was prepared using the absorbance measured from the dilution of the basic dephosphatase standard by the above method (FIG. 1). The standard curve equation was y = 0.0019x + 0.0969, where y is the absorbance read at 405 nm and x is the ALP enzymatic activity (mU / ml). To calculate the expression level of basic alkaline phosphatase in the final cultured cells, one million bone cells were dissolved in a volume of 200 ul and 20 ul of cell lysate samples corresponding to 100,000 cells were removed. The absorbance at 405 nm was measured by the phosphatase activity measuring method, and the absorbance of the measured medium was substituted into the standard curve equation to obtain the ALP enzyme activity (mU / ml) of the sample, and this was ALP enzyme activity per million cells. Converted to the total amount of (mU) and the results are shown in Table 1 below.

TABLE 1

Example O.D. (450 nm) ALP activity (mU / ml) Real ALP activity (mU) / 10 ⁶ cells One 0.182 45.0 9 2 0.211 60.1 12 3 0.201 54.8 11 4 0.239 75.0 15 5 0.382 150.1 30

As described above, the bone cell culture method according to the present invention comprises the steps of separating the bone tissue of the animal, the step of fixing the bone cells of the separated bone tissue in the culture vessel, the process of proliferating bone cells fixed in the culture vessel and bone Selectively apply Ossol Solution I, Ossol Solution II, Ossol Solution III, Oscot I, Odia I, Oscot II, and Odia II to each of the four basic processes to differentiate cells. Bone cells can be cultured so that bone damage can be implanted quickly.

Figure 1 is a standard curve of basic dephosphatase (alkaline phosphatase) activity of bone cells according to the present invention.

Claims (14)

(A) Bone tissues of animals were removed from red blood cells using Ossol Solution I containing 100-200 mM ammonium chloride, 5-20 mM sodium bicarbonate, 0.1-0.5 mM EDTA in HBSS solution, and then 1-20 g in HBSS solution. Separating bone cells from bone tissue by removing tissue components other than cells using Ossol Solution II containing / L collagenase; (B) The bone cells isolated above are dispensed into a first culture vessel coated with insulin (Insulin) in collagen type 1 and coated with a first culture vessel coated with insulin (Insulin), and the cell culture fluid is exchanged at regular intervals. Fixing to the first culture vessel; (C) growth factors selected from growth factors consisting of IGF-1, TGF-β, b-FGF, EGF, PDGF, a-FGF, BMP, KGF, HGF in the first culture vessel in which the bone cells are fixed Propagating the bone cells by putting a culture medium for bone cell proliferation containing Odia I mixed with dexamethasone and insulin; (D) a second coat of Oscot II coated with growth factors selected from the growth factors consisting of TGF-β, BMP, and dexamethasone in collagen fiber type I from the proliferated bone cells; And dividing the bone cells into a culture vessel and inserting a culture medium for bone cell differentiation to differentiate the bone cells. According to claim 1, wherein in the step (a) the bone tissue is bone marrow tissue; Cavernous bone tissue; Cortical bone tissue; Or a method of culturing bone cells, characterized in that the mixed tissue. delete delete delete delete delete delete delete delete 5-20 mg / L collagen fiber type I (collagen type 1) containing 5 to 20 mg / L insulin (Insulin) cell culture container coating composition (oscot I). 5 to 20 ng / L transforming growth factor-β (TGF-β), 5 to 20 ug / L bone morphogeniic protein (BMP), 5 to 20 to 5 to 20 mg / L collagen type 1 A composition for coating a cell culture container containing a growth factor selected from growth factors consisting of ug / L dexamethasone (Oscot II). 5-20 ug / L insulin-like growth factor type 1 (IGF-1), 5-20 ng / L transforming growth factor-β (TGF-β), 5-20 mg / L basic fibroblastgrowth factor (FGF) , 5 to 20 mg / L epidermal growth factor (EGF), 5 to 20 ug / L platelet derived growth factor (PDGF), 5 to 20 ug / L acidic fibroblastgrowth factor (5 to 20 ug / L BMP) 0.5-20 ug / L dexamethasone to growth factors selected from growth factors consisting of (bone morphogeniic protein), 5-20 mg / L keratinocyte growth factor (KGF), and 5-20 mg / L hepatocyte growth factor (HGF). (Dexamethasone), a composition for proliferating bone cells containing 5-20 mg / L insulin (Odia I). Bone cells containing 5-20 ng / L bone morphogeniic protein (BMP), 5-20 ug / L dexamethasone, 1-5 g / L bGP (beta-glycerol phosphate), 5-20 mg / L insulin Differentiation composition (Odia II).