Method for expanding and culturing nucleus pulposus cells derived from intervertebral discs and use thereof

Abstract

To provide methods for culturing and expanding nucleus pulposus cells derived from intervertebral discs and application therefor.SOLUTION: The present invention primarily relates to a method for in vitro culturing and expanding nucleus pulposus cells derived from an intervertebral disc. A secondary objective of the present invention is to provide a method for improving lower back pain by administering an effective amount of a pharmaceutical composition containing said nucleus pulposus cells obtained through the aforementioned method to subjects experiencing lower back pain.SELECTED DRAWING: Figure 6

Description

[Technical field]

[0001] The present invention relates to the technical field of a cell expansion culture method, and in particular, to a method for expanding and culturing nucleus pulposus cells derived from an intervertebral disc and use This relates to the technical field of: [Background technology]

[0002] Intervertebral disc tissue is located between the vertebrae, and is composed of the annulus fibrosus and nucleus pulpous, and can cushion the friction caused by the movement between the vertebrae. Degenerated disc disease often causes symptoms such as low back pain, and is prevalent in adults in their 30s to 50s, and in those with severe symptoms, the disc may collapse. According to statistics, 80% of adults experience low back pain in their lifetime, and about a quarter of them have difficulty working due to prolonged back pain.

[0003] In general, when the initial symptoms of disc degeneration appear, a clinician will give the patient a prescription for painkillers to temporarily relieve physiological pain. Currently, commonly used drugs include nonsteroidal anti-inflammatory drugs (NSAIDs), acetaminophen, steroid drugs, etc. These drugs may disrupt the balance of cell secretion and may cause side effects in the digestive system, such as the stomach and intestines. For people with severe symptoms of disc degeneration, disc replacement surgery is necessary, but the material of the artificial disc implant is a mixture of polymer and metal, and is not biocompatible, so it is prone to more serious side effects. None of the above methods can directly treat the degenerated disc, and no significant improvement in the quality of life of the patient has been seen.

[0004] When damage or degeneration occurs to the disc structure, the nucleus pulposus tissue can become unstable and, due to increased loads and pressures, can protrude outward or, in more severe cases, break through the annulus fibrosus. Such protruding nucleus pulposus tissue can press against surrounding neural structures, such as the spinal cord and nerve roots, causing symptoms such as pain, radiation, paralysis, and weakness. Thus, there is a close relationship between disc protrusion and the location and pressure of the nucleus pulposus tissue. Summary of the Invention [Problem to be solved by the invention]

[0005] In view of the limitations and shortcomings of the current treatment methods for degenerative disc disease, and the relationship between degenerative disc disease and nucleus pulposus tissue, the inventor of the present application has obtained nucleus pulposus cells from the nucleus pulposus tissue of the intervertebral disc of a patient at the early stage of degenerative disc disease, expanded and cultured in vitro, and then cryopreserved, and if requested by the patient, the cells can be thawed, recultured, and implanted in the affected area of ​​the patient to grow in the affected area, without causing immune rejection, and thus it is desired to achieve the objective of regenerating nucleus pulposus tissue and treating degenerative disc disease. For this reason, the inventor of the present application has researched and invented as much as possible, and has finally completed the research and development of the method for expanding and culturing nucleus pulposus cells derived from an intervertebral disc in vitro according to the present invention, and the use of the nucleus pulposus cells obtained by the above-mentioned method for producing a pharmaceutical composition for treating lower back pain. [Means for solving the problem]

[0006] A primary object of the present invention is to provide a method for expanding nucleus pulposus cells derived from intervertebral discs in vitro, which comprises: a) culturing nucleus pulposus tissue; The method includes the steps of: providing a nucleus pulposus (a primary culture medium); b) hydrolyzing the nucleus pulposus tissue with an enzyme and separating the unhydrolyzed nucleus pulposus tissue and primary nucleus pulposus cells that fall off from the tissue by sieving and centrifugation; c) performing primary culture of the primary nucleus pulposus cells obtained in step b); d) after the primary nucleus pulposus cells primary cultured in step c) reach a predetermined cell density, removing the primary nucleus pulposus cells and subculture them; e) selecting nucleus pulposus cells from the primary nucleus pulposus cells subcultured in step d) based on the expression levels of one or multiple genes; and f) performing expansion culture of the nucleus pulposus cells obtained in step e), wherein the one or multiple genes are genes selected from a gene module consisting of nucleus pulposus development genes, cartilage differentiation genes, nucleus pulposus-specific genes, nucleus pulposus degeneration genes, and annulus fibrosus-specific genes.

[0007] In the above method, the nucleus pulposus development gene is KDM4E, the cartilage differentiation genes include SOX9, COL2A1, and Agc1, the nucleus pulposus-specific gene is PAX1, the nucleus pulposus degeneration gene is SAA1, and the annulus fibrosus-specific gene is CD90.

[0008] Additionally, in the above method, the enzyme for hydrolyzing the nucleus pulposus tissue is collagenase, trypsin, or a combination thereof, and the primary nucleus pulposus cells primary cultured in step c) are extracted and subcultured after 0 to 14 days have passed and a cell density of 50 to 100%, more preferably after 5 to 7 days have passed and a cell density of 80 to 90% has passed.

[0009] Furthermore, in the above method, the expression level of the gene is detected and measured by quantitative polymerase chain reaction (qPCR), Northern blotting, Western blotting, or DNA microarray.

[0010] At the same time, a secondary object of the present invention is to provide a use of the nucleus pulposus cells obtained by the above method for producing a pharmaceutical composition for treating lower back pain, wherein the pharmaceutical composition includes a pharma- ceutical acceptable carrier. Effect of the Invention

[0011] According to the pharmaceutical composition, the nucleus pulposus cells obtained from the patient's nucleus pulposus tissue can be expanded and then re-transplanted into the affected area of ​​the patient (i.e., the site of degenerative intervertebral disc), where they can grow and achieve the therapeutic goal through regeneration of the nucleus pulposus tissue while avoiding the possibility of an immune rejection reaction in the patient receiving the transplant. [Brief description of the drawings]

[0012] [Figure 1] 1 is a bar graph showing the expression level of the nucleus pulposus development gene, KDM4E, in nucleus pulposus cells obtained by the method of the present invention for expanding and culturing nucleus pulposus cells derived from intervertebral discs in different age groups. [Figure 2A] 1 is a bar graph showing the expression level of the cartilage differentiation gene, SOX9, in nucleus pulposus cells obtained by the method of the present invention for expanding and culturing nucleus pulposus cells derived from intervertebral discs in different age groups. [Figure 2B] 1 is a bar graph showing the expression level of the cartilage differentiation gene, COL2A1, in nucleus pulposus cells obtained by the method of the present invention for expanding and culturing nucleus pulposus cells derived from intervertebral discs in different age groups. [Figure 2C] 1 is a bar graph showing the expression level of the cartilage differentiation gene, Agc1, in nucleus pulposus cells obtained by the method of the present invention for expanding and culturing nucleus pulposus cells derived from intervertebral discs in different age groups. [Diagram 3]1 is a bar graph showing the expression level of the nucleus pulposus-specific gene, PAX1, in nucleus pulposus cells obtained by the method of the present invention for expanding and culturing nucleus pulposus cells derived from intervertebral discs in different age groups. [Figure 4] 1 is a bar graph showing the expression level of the nucleus pulposus degeneration gene, SAA1, in nucleus pulposus cells obtained by the method of the present invention for expanding and culturing nucleus pulposus cells derived from intervertebral discs in different age groups. [Diagram 5] 1 is a bar graph showing the expression level of the annulus fibrosus-specific gene, CD90, in nucleus pulposus cells obtained by the method of the present invention for expanding and culturing nucleus pulposus cells derived from intervertebral discs in different age groups. [Figure 6] 1 is a flow diagram showing the method for expanding and culturing nucleus pulposus cells derived from an intervertebral disc according to the present invention and uses thereof. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0013] All technical and scientific terms described herein have the same meaning as commonly understood by those having ordinary technical knowledge in the technical field to which this disclosure belongs, unless otherwise defined. In the present invention, the following examples are described as exemplary examples, but are merely illustrative and are not necessarily limited thereto, and the present invention is not limited by the following examples. Unless otherwise stated, all materials used in the present invention are commercially available and easily available, and the following description is merely an example of available routes. EXAMPLES

[0014] <Processing of Nucleus pulposus tissue samples> 1. First, in a hospital operating room, nucleus pulposus tissue from a patient with degenerative disc disease or herniated disc is surgically removed in a sterile manner, placed in a sterile sealed carrier containing physiological saline containing an antibiotic composition, and then sent to a sterile workbench in a laboratory that complies with the Good Tissue Practice (GTP) standard for handling human cells and tissues for processing. 2. Record the weight of the nucleus pulposus tissue sample and basic information about the patient of said sample. 3. The specimen was placed in a 10 cm dish containing 2 mL of P0 medium (medium of passage number 0), and then the specimen was cut to an average size of 1 mm 3 The nucleus pulposus tissue is cut into small pieces smaller than 10 cm in diameter, and each 10 cm dish contains approximately 5 g of specimen. If the specimen is relatively large, the nucleus pulposus tissue is divided into several culture dishes based on a certain proportion, and the P0 medium contains components called DMEM (Dulbecco's modified Eagle's medium), human platelet lysate, and antibiotics. 4. Preparation of collagenase solution: 7 mL of P0 medium + 1 mL of type I collagenase, that is, the ratio of P0 medium to type I collagenase in the collagenase solution is 7:1 (volume ratio). 5. Add the prepared collagenase solution to the culture dishes containing the minced nucleus pulposus tissue, add 8 ml of collagenase solution to each culture dish, and culture overnight in an incubator at 37°C with 5% CO2. 6. After overnight culture, sieve the nucleus pulposus tissue specimens in each culture dish through a 100 μm cell strainer. During sieving, the tissue can be lightly polished using the piston of a 10 mL syringe. After polishing is complete, wash the cell strainer with 10 mL of DPBS (Dulbecco's phosphate-buffered saline). 7. The cell suspension collected after sieving is centrifuged at 200 xg for 5 minutes at room temperature and the supernatant is removed. 8. Next, wash twice with 20 mL of DPBS to obtain a cell pellet. 9. The resulting cell pellet is then resuspended in 2 mL of P0 medium, followed by a trypan blue cell count and calculation to obtain the cell number and its viability (80-99%). 10.5~10×10 5 Several types of cells are placed in a 10 cm dish containing 10 mL of P0 medium, and the cells at this stage are designated as P0 (cells of passage number 0), i.e., primary nucleus pulposus cells, and are cultured in an incubator at 37°C with 5% CO2 for primary culture. 11. The growth status of the primary nucleus pulposus cells is continuously observed, and on the third day, the old P0 medium is removed, followed by washing once with 6 mL of DPBS, and then 10 mL of new P0 medium is added and the cells are continuously cultured. 12. After the primary nucleus pulposus cells have been cultured for 0 to 14 days and a cell density of 50 to 100% has been reached, or more preferably after 5 to 7 days of culture, the cell density can reach 80 to 90%, at which point the primary nucleus pulposus cells can be extracted and subjected to a first subculture. EXAMPLES

[0015] <Subculture of primary nucleus pulposus cells> 1. When the cell density of the primary nucleus pulposus cells in Example 1 reaches 50 to 100%, or more preferably 80 to 90%, subculture is performed. 2. Remove the old P0 medium and wash the primary nucleus pulposus cells attached to the bottom of the 10 cm dish twice with 6 mL of DPBS. 3. Add 1 mL of 0.05% trypsin-EDTA (trypsin-ethylenediaminetetraacetic acid) to the 10 cm dish and allow it to react at 37° C. for 3 minutes in an incubator with 5% CO 2 at 37° C. 4. After completion of the action, the 10 cm dish is gently tapped with a hand to promote detachment of the primary nucleus pulposus cells from the bottom of the 10 cm dish. Trypsin is neutralized by adding 5.4 mL of P0 medium to the 10 cm dish, and then the primary nucleus pulposus cells suspended in P0 medium are collected, placed in a centrifuge tube, and centrifuged at room temperature at 200 x g for 5 minutes. 6. After removing the supernatant from the centrifuge tube, the primary nucleus pulposus cells are resuspended in an appropriate volume of culture medium. The volume of culture medium required for resuspending the primary nucleus pulposus cells is prepared based on a certain proportion, for example, the primary nucleus pulposus cells in one or two 10 cm dishes are resuspended in 1 mL of culture medium, in which the culture medium contains the following components, namely DMEM and Human Platelet Lysate. 7. Cell counting is performed using trypan blue to confirm the number and viability (80-99%) of the primary nucleus pulposus cells. 8. Resuspend the primary nucleus pulposus cells in a 10 cm dish at 1 x 10 6 Based on the proportion of the number of cells containing the above, subculture is carried out in 10 ml of culture medium, and the primary nucleus pulposus cells at this stage become P1 (ie, first passage cells, cells of passage number 1). 9. Subculture once every 3 to 4 days, i.e., repeat steps 1 to 8 above. EXAMPLES

[0016] <Evaluation of gene expression> 1. When subculture of P2 (cells of the second passage), P3 (cells of the third passage), and P4 (cells of the fourth passage) was performed using the subculture method in Example 2, approximately 5 × 10 5 The primary nucleus pulposus cells are collected and washed twice with 10 mL of DPBS. 2. The collected primary nucleus pulposus cells are resuspended in 1 mL of DPBS and transferred into a centrifuge tube, which is then centrifuged at 200xg for 5 minutes at room temperature. 3. Remove the supernatant in the centrifuge tube and add 1 mL of GENEzol to the centrifuge tube, then store the sample in the centrifuge tube at -80°C. 4. Extract RNA from the sample (primary nucleus pulposus cells) obtained in step 3 above, and detect and measure the expression level of each gene in the primary nucleus pulposus cells in the sample by qPCR (quantitative polymerase chain reaction), among which, each detected and measured gene is a gene selected from the following five types of gene modules: I. nucleus pulposus development genes, II. cartilage differentiation genes, III. nucleus pulposus-specific genes, IV. nucleus pulposus degeneration genes, and V. annulus fibrosus-specific genes. Furthermore, the nucleus pulposus development genes are KDM4E, the cartilage differentiation genes include SOX9, COL2A1, and Agc1, the nucleus pulposus-specific gene is PAX1, the nucleus pulposus degeneration gene is SAA1, and the annulus fibrosus-specific gene is CD90. EXAMPLES

[0017] <Cell cryopreservation> 1. When the primary nucleus pulposus cells are continuously expanded until they reach P2 or P3, the cells can be cryopreserved when the cell density reaches 80-90%. 2. Remove the old culture medium from the 10 cm dish and wash it twice with 6 mL of DPBS. 3. Add 1 mL of trypsin-EDTA to the 10 cm dish and incubate it in a 5% CO2 incubator at 37°C for 3 minutes. 4. After completion of the action, gently tap the 10 cm dish with your hand to promote detachment of the P2 or P3 primary nucleus pulposus cells from the bottom of the 10 cm dish. Trypsin is neutralized by adding 5.4 mL of culture medium to the 10 cm dish, and then the P2 or P3 primary nucleus pulposus cells suspended in the culture medium are collected, placed in a centrifuge tube, and centrifuged at room temperature at 200 x g for 5 minutes. 6. After removing the supernatant from the centrifuge tube, the P2 or P3 primary nucleus pulposus cells are resuspended in an appropriate volume of culture medium. The volume of culture medium required for resuspending the P2 or P3 primary nucleus pulposus cells is prepared based on a certain proportion, and for example, the P2 or P3 primary nucleus pulposus cells in one or two 10 cm dishes are resuspended in 1 mL of culture medium. 7. Cell counting is performed using trypan blue to confirm the number and viability (80-99%) of the P2 or P3 primary nucleus pulposus cells. 8. The P2 or P3 primary nucleus pulposus cells resuspended in culture medium in a centrifuge tube are centrifuged at room temperature at 200 xg for 5 minutes, and then the supernatant is removed. 9.2×10 6 The P2 or P3 primary nucleus pulposus cells are resuspended in an appropriate volume of cell cryopreservation medium at a cell concentration of 100 / ml. 10. Aliquot the P2 or P3 primary nucleus pulposus cell suspension into cryogenic storage vials based on a 1 mL / vial ratio. 11. Place the cryopreservation vial into a cell freezing container, then store the cell freezing container in a -80°C freezer overnight, and the next day, transfer the cryopreservation vial to liquid nitrogen and store in the vapor phase above the liquid nitrogen. In the above explanation, the gas phase storage is taken as an example, but the cryopreservation vial may be frozen and stored in the liquid phase of liquid nitrogen.

[0018] The sources of reagents used in each of the above examples are summarized in Table 1 below.

[0019] [Table 1] EXAMPLES

[0020] <Expression level of nucleus pulposus development gene: KDM4E in primary nucleus pulposus cells> 1 is a bar graph showing the results of the expression level of the nucleus pulposus development gene, KDM4E, obtained by analyzing primary nucleus pulposus cells obtained by the expansion culture methods of Examples 1 and 2 above in different age groups using the gene expression evaluation method of Example 3. As is clear from the bar graph in FIG. 1, the expression level of the nucleus pulposus development gene, KDM4E, in primary nucleus pulposus cells obtained in the age group of 35 or younger and the age group of 36 to 50 years old is found to be higher than that in the age group of 51 years old or older. Furthermore, the expression of the specific nucleus pulposus development gene, KDM4E, certainly proves that all of the multiple primary nucleus pulposus cells obtained by the expansion culture methods of Examples 1 and 2 of the present invention have been obtained through the developmental process of nucleus pulpous tissue (Nucleus pulpous). EXAMPLES

[0021] <Expression levels of SOX9, COL2A1, and Agc1, the chondrocyte differentiation genes in primary nucleus pulposus cells> 2A to 2C are bar graphs showing the results of the expression levels of cartilage differentiation genes: SOX9, COL2A1, and Agc1 obtained by analyzing primary nucleus pulposus cells obtained by the expansion culture methods of Examples 1 and 2 in different age groups using the gene expression evaluation method of Example 3. As is clear from the bar graph in Fig. 2A, the expression level of the cartilage differentiation gene: SOX9 in primary nucleus pulposus cells obtained in the age group of 35 years or younger was found to be significantly higher than that in the age groups of 36 to 50 years and 51 years or older, and among them, SOX9 is an important early transcription factor in cartilage differentiation. As is clear from the bar graph in FIG. 2B, the expression levels of the cartilage differentiation gene, COL2A1, in primary nucleus pulposus cells obtained in the age group of 35 or less and the age group of 36 to 50 years old were found to be higher than that in the age group of 51 years old or more, and the expression levels of the COL2A1 gene in the age group of 35 or less were also found to be significantly higher than that in the age group of 36 to 50 years old and the age group of 51 years old or more, of which, COL2A1 is a cartilage matrix gene important in cartilage differentiation. As is clear from the bar graph in FIG. 2C, the expression levels of the cartilage differentiation gene, Agc1, in different age groups of primary nucleus pulposus cells obtained by the expansion culture method of the present invention, when arranged in order from highest to lowest, were found to be the highest in the age group of 35 or less, the next highest in the age group of 36 to 50 years old, and the lowest in the age group of 51 years old or more, of which, Agc1 is a cartilage matrix gene important in cartilage differentiation. In addition, as can be seen from Figures 2A to 2C above, all of the primary nucleus pulposus cells obtained by the expansion culture methods of Examples 1 and 2 of the present invention express important cartilage differentiation genes involved in nucleus pulposus tissue, and the expression levels gradually decrease with increasing age. EXAMPLES

[0022] <Expression of nucleus pulposus-specific gene: PAX1 in primary nucleus pulposus cells> FIG. 3 is a bar graph showing the results of the expression level of the nucleus pulposus-specific gene: PAX1 obtained by analyzing the primary nucleus pulposus cells obtained by the expansion culture method of Examples 1 and 2 in different age groups by the gene expression evaluation method of Example 3. As is clear from the bar graph in FIG. 3, the expression level of the nucleus pulposus-specific gene: PAX1 in the primary nucleus pulposus cells obtained in the age group of 35 years or younger was found to be significantly higher than the age groups of 36 to 50 years and 51 years or older, and among them, the nucleus pulposus-specific gene: PAX1 is a precursor cell gene of the nucleus pulposus tissue, and the younger the donor, the higher the expression level of the nucleus pulposus tissue, and the higher the expression level, the higher the repair ability of the nucleus pulposus tissue. Therefore, as can be seen from FIG. 3, all of the primary nucleus pulposus cells obtained by the expansion culture method of Examples 1 and 2 of the present invention have expression of the PAX1 gene involved in the repair ability of the nucleus pulposus tissue, and the expression level is remarkably relatively high even among young people. EXAMPLES

[0023] <Expression of nucleus pulposus degeneration gene: SAA1 in primary nucleus pulposus cells> Fig. 4 is a bar graph showing the results of the expression level of the nucleus pulposus degeneration gene: SAA1 obtained by analyzing primary nucleus pulposus cells obtained by the expansion culture method of Examples 1 and 2 in different age groups using the gene expression evaluation method of Example 3. As is clear from the bar graph in Fig. 4, when the expression levels of the nucleus pulposus degeneration gene: SAA1 in different age groups of primary nucleus pulposus cells obtained by the expansion culture method of the present invention are arranged in order from highest to lowest, it was found that the expression level was highest in the age group of 51 years or older, the expression level was next highest in the age group of 36 to 50 years, and the expression level was lowest in the age group of 35 years or younger, and among them, when SAA1 is expressed during nucleus pulposus degeneration and apoptosis of nucleus pulposus cells, the expression level increases with increasing age. Therefore, as can be seen from Figure 4, the expression level of the nucleus pulposus degeneration gene, SAA1, in primary nucleus pulposus cells obtained by the expansion culture methods of Examples 1 and 2 of the present invention was shown to increase with age, that is, the primary nucleus pulposus cells expanded by the method of the present invention have the expression characteristics of cells in nucleus pulposus tissue. EXAMPLES

[0024] <Expression of CD90, a gene specific to the annulus fibrosus in primary nucleus pulposus cells> Fig. 5 is a bar graph showing the results of the expression level of the annulus fibrosus-specific gene, CD90, obtained in different age groups by analysis of primary nucleus pulposus cells obtained by the expansion culture methods of Examples 1 and 2 above, using the gene expression evaluation method of Example 3. As is clear from the bar graph in Fig. 5, the expression levels of the annulus fibrosus-specific gene, CD90, in different age groups of primary nucleus pulposus cells obtained by the expansion culture method of the present invention were all found to be very low, that is, the very low expression level of this annulus fibrosus-specific gene, CD90, can be inversely verified (explained) as being derived from a non-annulus fibrosus, primary nucleus pulposus cells obtained by the expansion culture method of the present invention.

[0025] The results of the expression levels of genes selected from five types of gene modules obtained by analyzing primary nucleus pulposus cells obtained by the expansion culture methods of Examples 1 and 2 above in different age groups using the gene expression evaluation method of Example 3: nucleus pulposus development genes, cartilage differentiation genes, nucleus pulposus-specific genes, nucleus pulposus degeneration genes, and annulus fibrosus-specific genes are summarized in Table 2 below.

[0026] [Table 2]

[0027] Table 2 shows the data representing the bar graphs of Figures 1 to 5, in which the data is the average value of gene expression data obtained in each age group. And, by taking together the data results shown in Figures 1 to 5 and Table 2, it is clear that, by the method of expanding and culturing primary nucleus pulposus cells and the method of evaluating gene expression in the method of expanding and culturing nucleus pulposus cells derived from intervertebral disc in vitro provided by the present invention, that is, by utilizing the expression levels of genes selected from five gene modules: nucleus pulposus development genes, cartilage differentiation genes, nucleus pulposus specific genes, nucleus pulposus degeneration genes, and annulus fibrosus specific genes, nucleus pulposus cells having the function of repairing nucleus pulposus tissue can be obtained, and then, the obtained nucleus pulposus cells can be used to manufacture a pharmaceutical composition for treating lower back pain, and the pharmaceutical composition containing the nucleus pulposus cells can be used to treat chronic lower back pain caused by intervertebral disc degeneration. EXAMPLES

[0028] <Flow of the method for expanding and culturing nucleus pulposus cells derived from intervertebral discs and its application> FIG. 6 is a flow diagram showing the method for expanding and culturing nucleus pulposus cells derived from an intervertebral disc of the present invention and its uses. First, as in the technical content described in Example 1, a nucleus pulposus tissue specimen is obtained from the spine of a patient with degenerative disc disease or herniated disc in a sterile manner after surgery, then the nucleus pulposus tissue specimen is transported in a sterile constant temperature transport manner to a sterile work bench in a laboratory that complies with the handling standards for human cells and tissues, where the nucleus pulposus tissue specimen is processed, and finally primary nucleus pulposus cells are obtained. Next, the primary nucleus pulposus cells obtained in Example 1 are subcultured by the method for subculture of primary nucleus pulposus cells described in Example 2, and the obtained primary nucleus pulposus cells are expanded, and then the primary nucleus pulposus cells are evaluated by the method for evaluating gene expression described in Example 3, and at the same time, a sterility safety test is performed on the primary nucleus pulposus cells to confirm whether the primary nucleus pulposus cells are not contaminated, for example, to confirm whether they are not contaminated with Mycoplasma, and then, as is clear from the results of the gene expression evaluation and sterility safety test described above, the primary nucleus pulposus cells obtained from the nucleus pulposus tissue specimen of the patient are cultured. A personalized report is generated regarding whether or not nucleus pulposus cells having regenerative ability and other characteristics of nucleus pulposus cells are present in the primary nucleus pulposus cells obtained, and according to this report, a pharmaceutical composition for treating lower back pain (disc degeneration or disc herniation) is customized for each patient using the nucleus pulposus cells having regenerative ability and other characteristics of nucleus pulposus cells. Finally, the pharmaceutical composition is implanted in the affected area of ​​the patient, causing the nucleus pulposus cells having regenerative ability to grow in the affected area of ​​the patient, thereby achieving the purpose of regenerating nucleus pulposus tissue and treating intervertebral disc degeneration or disc herniation.

[0029] As described above, the method for expanding and culturing nucleus pulposus cells derived from intervertebral discs according to the present invention and its uses have been fully and clearly described. It should be emphasized that the above detailed description specifically describes the feasible embodiments of the present invention, but the scope of the present invention is not limited to these embodiments, and equivalent implementations or modifications thereof are still included in the scope of the claims of the present application as long as they do not deviate from the technical spirit of the present invention.

Claims

1. A method for expanding nucleus pulposus cells derived from an intervertebral disc in vitro, comprising: a) providing nucleus pulposus tissue; b) hydrolyzing the nucleus pulposus tissue with an enzyme and separating the unhydrolyzed nucleus pulposus tissue and primary nucleus pulposus cells by sieving and centrifuging; c) performing primary culture of the primary nucleus pulposus cells obtained in step b); d) after the primary nucleus pulposus cells subjected to primary culture in step c) reach a predetermined cell density, the primary nucleus pulposus cells are extracted and subcultured; and e) detecting gene expression levels in the primary nucleus pulposus cells subcultured in step d), the detected genes being involved in nucleus pulposus development. A method for expanding and culturing nucleus pulposus cells derived from an intervertebral disc in vitro, comprising the steps of: selecting primary nucleus pulposus cells which contain the nucleus pulposus gene KDM4E, the cartilage differentiation genes SOX9, COL2A1, and Agc1, the nucleus pulposus-specific gene PAX1, the nucleus pulposus degeneration gene SAA1, and the annulus fibrosus-specific gene CD90, and which have high expression levels of KDM4E, SOX9, COL2A1, Agc1, and PAX1 and low expression levels of SAA1 and CD90, as nucleus pulposus cells having regenerative ability; and f) performing an expansion culture of the nucleus pulposus cells having regenerative ability obtained in step e).

2. 2. The method of claim 1, wherein the enzyme for hydrolyzing the nucleus pulposus tissue is collagenase, trypsin, or a combination thereof.

3. The method according to claim 1, characterized in that the cell density is 80-90% cell density.

4. The method according to claim 1, wherein the expression level of the gene is detected and measured by quantitative polymerase chain reaction (qPCR), Northern blotting, Western blotting, or DNA microarray.

5. 13. Use of the nucleus pulposus cells obtained by the method of claim 1 for producing a pharmaceutical composition for treating lower back pain.

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