Method for culturing nucleus pulposus cell

Abstract

To provide a method for culturing nucleus pulposus cells useful as regenerative medical preparations for treating intervertebral disc degeneration and the like.SOLUTION: When culturing nucleus pulposus cells, the use of a culture vessel coated with laminin enables mass culture of nucleus pulposus cells while maintaining expression of the cell surface marker Tie2, which is useful as a regenerative medical preparation for intervertebral disc degeneration and the like. As a result, it is possible to obtain highly functional nucleus pulposus cells that exhibit high expression of type II collagen and excellent colony-forming ability.SELECTED DRAWING: Figure 2

Description

[Technical field]

[0001] The present invention relates to a method for culturing nucleus pulposus cells for use in regenerative medicine, and more particularly to a culture method that enables mass culture of nucleus pulposus cells while maintaining the expression of the cell surface marker Tie2 (tyrosine kinase with Ig and EGF homology domain-2) for efficient production of a regenerative medicine preparation (intervertebral disc cell preparation) effective for the treatment of intervertebral disc degeneration and the like, and ultimately produces highly functional nucleus pulposus cells with high expression of type II collagen and excellent colony formation ability. [Background technology]

[0002] In Japan, lower back pain is a disease that many people suffer from, and has become a social problem, especially in modern times. Although the cause of lower back pain is unknown in many cases, the cause can be identified in about 20% of cases, and the most common cause is intervertebral disc degeneration. Examples of causative diseases include herniated disc, degenerative spondylosis, spinal canal stenosis, spondylolisthesis, and osteoporosis. These are degenerations caused by irreversible changes in the intervertebral disc tissue, and it is necessary to restore the function of the intervertebral disc itself through regenerative medicine, etc.

[0003] The intervertebral disc is a doughnut-shaped cartilage tissue, consisting of the nucleus pulposus (NP) at the center, the annulus fibrosus (AF) which is a fibrous cartilage that surrounds it, and the cartilage endplates (EP) which connect the adjacent vertebrae above and below. The gelatinous nucleus pulposus is an avascular tissue, and contains a large amount of extracellular matrix (ECM) consisting of large proteoglycans and collagen secreted by the notochord-derived nucleus pulposus cells contained in the nucleus pulposus.

[0004] Currently, various conservative therapies are used to treat degenerative disc disease, but the reality is that there is no definitive cure. When surgical treatment is performed, the symptomatic area is fixed with medical screws, and over time, there is a risk that similar degenerative disease will develop in the adjacent area. Therefore, there is a need for a completely different type of treatment, a regenerative therapy for intervertebral discs, that is, a treatment that returns the disc to its pre-degenerative state.

[0005] For intervertebral disc regeneration therapy, it is desirable to select and extract cells with high regenerative potential, but there is a problem in that only a small amount of nucleus pulposus cells can be extracted from the intervertebral disc tissue from which they are extracted. Therefore, a method for culturing and increasing nucleus pulposus cells with high regenerative potential is required. In reality, it is desirable to reduce the cost of culturing as much as possible and to implement industrialization at low cost.

[0006] Currently, research and development of intervertebral disc cell preparations containing a large amount of ECM and the like is being conducted. To produce such cell preparations, a certain amount of nucleus pulposus cells is required. For example, intervertebral disc nucleus pulposus tissue excised by surgery from a patient with herniated disc can be used as a source of nucleus pulposus cells to be used in such cell preparations, but the amount of nucleus pulposus tissue that can be collected, i.e., the number of nucleus pulposus cells contained therein, is limited. In addition, it is desirable to avoid mixing nucleus pulposus cells derived from multiple patients (donors) with herniated discs in order to collect the amount, taking into consideration the risk of viral infections and the like. Therefore, it is important to establish a technology for preparing a cell population containing a sufficient number of nucleus pulposus cells for treatment by culturing rare stem cells or progenitor cells that can be induced to differentiate into mature nucleus pulposus cells contained in a small amount of intervertebral disc tissue (nucleus pulposus, annulus fibrosus, etc.) derived from a single donor.

[0007] Patent Document 1 discloses an invention that provides a means for efficiently preparing a cell population rich in cells having a specific trait according to the application (e.g., type II collagen-positive nucleus pulposus cells) from a cell population containing stem cells and / or progenitor cells (e.g., nucleus pulposus stem / progenitor cells) positive for the expression of the cell surface marker Tie2. Specifically, the invention discloses a culture method in which a cell population containing Tie2-positive stem / progenitor cells is cultured (1) in a state in which the cells are present in undigested tissue, (2) in a medium containing at least one Tie2 expression enhancer other than growth factors, (3) in a culture vessel having a culture surface treated to enhance cell adhesion, and / or (4) in a medium containing an extracellular matrix degrading agent while suppressing the formation of spherical colonies.

[0008] The culture method described in Patent Document 1 (above (3)) uses a culture vessel that has been subjected to a surface treatment to enhance cell adhesion, and discloses a treatment in which a coating agent containing an extracellular matrix (ECM) or other bio-related substance is applied to the culture surface as a coating agent for cell adhesion treatment. Examples of the ECM include collagen (type I, type II, type IV, etc.), gelatin, which is a heat-treated product thereof, chondroitin sulfate A, fibronectin, laminin, thrombospondin, vitronectin, and proteoglycan (aggrecan, heparin sulfate proteoglycan, etc.), and examples of bio-related molecules other than ECM include polyamino acids such as polylysine (poly-L-lysine or poly-D-lysine). Furthermore, other coating agents for cell attachment treatment include polyglycolic acid, PLGA (polylactic acid-glycolic acid copolymer), polyhydroxyalkanoic acid (PHA), poly-ε-caprolactone, polyorthoester, polyanhydride, polyphosphazene, polydimethylsiloxane, polyurethane, polytetrafluoroethylene, polyethylene, polysulfone, poly-methyl methacrylate, poly-2-hydroxyethyl methacrylate, polyamide, polypropylene, polyvinyl chloride, polystyrene, polyvinylpyrrolidone, polyornithine, etc.

[0009] Specifically, in the examples of Patent Document 1, a test example of a culture process (differentiation culture stage) is disclosed in which poly-L-lysine (PLL), gelatin, collagen (type I, type IV), and fibronectin are used as coating agents for cell attachment treatment. In the test examples, PLL is used most frequently, and laminin, a type of ECM, is not used at all. As described above, in Patent Document 1, laminin is a type of ECM, and is listed as one example of many examples of coating agents, but the effects are not demonstrated in the examples, so it can be said that the usefulness of laminin as a coating agent in the culture method of the present application is neither described nor suggested. [Prior art documents] [Patent documents]

[0010] [Patent Document 1] International Publication WO2020 / 196615 Summary of the Invention [Problem to be solved by the invention]

[0011] As described above, in order to produce an intervertebral disc cell preparation for the treatment of intervertebral disc degeneration and the like, a certain amount of functional nucleus pulposus cells that produce extracellular matrix (ECM) such as type II collagen and proteoglycan is required. For this purpose, for example, it is necessary to efficiently amplify and differentiate Tie2-positive cells, which are contained in the intervertebral disc tissue (nucleus pulposus, etc.) excised from the affected area of ​​a patient with herniated disc and are considered to be stem cells and / or precursor cells of nucleus pulposus cells, to produce a large amount of functional nucleus pulposus cells. In particular, in order to enhance the therapeutic effect when an intervertebral disc cell preparation is administered to a patient with intervertebral disc degeneration and the like, when culturing and inducing differentiation of Tie2-positive cells (nucleus pulposus stem / progenitor cells) contained in the intervertebral disc, it is important not to simply differentiate into nucleus pulposus cells, but to differentiate into functional nucleus pulposus cells that produce a large amount of extracellular matrix such as type II collagen as efficiently as possible.

[0012] The objective of the present invention is to provide a culture method that enables mass culture of nucleus pulposus cells that maintain the expression of the cell surface marker Tie2, which is useful as a regenerative medicine preparation, and ultimately results in highly functional nucleus pulposus cells that have high type II collagen expression and excellent colony formation ability. [Means for solving the problem]

[0013] The present inventors conducted research focusing on the coating agent of the container (dish) used for culturing nucleus pulposus cells that maintain Tie2 expression, and found that the use of laminin as a coating agent can contribute to solving the above problems and provide better results than the previously reported methods. In the previously reported method described in Patent Document 1, for example, the results shown in Figures 6 (the figure numbers in this paragraph are those of Patent Document 1) and 15 for the type II collagen positivity rate, the number of type II collagen positive cells in Figure 7, the Tie2 positivity rate in Figure 13, and the total number of Tie2 positive cells in Figure 14 are better when PLL coating is used than when no coating agent is used. Furthermore, Figures 10 and 11 show the results that PLL is the most excellent coating agent in terms of type II collagen and proteoglycan positivity rate (production ability).

[0014] Laminin is a major protein that constitutes the basement membrane of the extracellular matrix. It promotes multicellular organization, tissue construction and maintenance, cell adhesion, cell migration, cell proliferation, etc. It is known to be closely related to cancer cells and to be expressed early in embryonic development (2-cell stage).

[0015] Coating of culture dishes and the like with laminin has already been performed, but there have been no reports to date showing that laminin, when used as a coating agent on the culture surface of a culture vessel in the culture of nucleus pulposus cells, is more effective than other coating agents. In addition, although Patent Document 1 shows that PLL is the most excellent coating agent in the culture of nucleus pulposus cells, it has been surprisingly found that coating with laminin is superior. That is, the present inventors have found that by adopting laminin rather than PLL as a coating agent for a culture vessel in the culture of nucleus pulposus cells, nucleus pulposus cells with high expression of type II collagen and excellent functionality in terms of colony formation ability and the like can be obtained more efficiently than expected while maintaining Tie2 expression, and have completed the present invention.

[0016] That is, the present invention includes the following aspects: [1] A method for culturing nucleus pulposus cells using laminin as a coating agent for the culture surface of a culture vessel. [2] The culture method according to the above [1], wherein the nucleus pulposus cells are collected from a human. [3] A culture vessel for culturing nucleus pulposus cells, the culture surface of which is coated with laminin. [4] The culture vessel according to the above [3], wherein the nucleus pulposus cells are collected from a human. [5] Use of laminin as a coating agent for the culture surface of a container for culturing nucleus pulposus cells. [6] The use according to [5] above, wherein the nucleus pulposus cells are obtained from a human. [Brief description of the drawings]

[0017] [Figure 1] The results were obtained by observing the cells with a phase contrast microscope after six days of culture using three types of dishes (non-coated [NON], poly-L-lysine-coated [PLL], and laminin-coated [Laminin]). Figures 2 to 7 also show the test results using the above three types of dishes. [Diagram 2] The results are the results of comparing the proliferation promoting effect of nucleus pulposus cells using the three types of dishes. [Diagram 3]This shows the results of flow cytometry detection of the cell surface marker Tie2 expressed on the surface of nucleus pulposus cells after culture. The gray cell population indicates Tie2-positive cells. [Figure 4] The percentage of Tie2-positive cells on the surface of the nucleus pulposus cells after culture was calculated based on the results detected by flow cytometry. [Diagram 5] The number of Tie2-positive cells on the surface of the nucleus pulposus cells after culture was calculated based on the results detected by flow cytometry. [Figure 6] The expression rate of type II collagen (Col2) on the surface of the nucleus pulposus cells after culture was calculated based on the results detected by flow cytometry. [Figure 7] This shows the results of comparing the spherical colony-forming ability of nucleus pulposus cells. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0018] In the present specification, the nucleus pulposus cells in the intervertebral disc cell preparation used for the treatment of degenerative disc disease and the like are those contained in a cell population (primary cultured cell population) contained in intervertebral disc tissue collected from the body of a human or other animal (donor), or a cell population (subcultured cell population) obtained by subculturing the primary cultured cell population. The nucleus pulposus cells are preferably collected from a human, and the cells obtained by amplifying and differentiating the nucleus pulposus cells of the patient himself or another person can be made into a preparation and used for the treatment of degenerative disc disease and the like. The method for amplifying and culturing the nucleus pulposus cells can be performed according to the conventional method described in the prior art such as Patent Document 1, and the present invention is characterized in that laminin is used as a coating agent for the culture vessel during the culture, and other culture conditions (culture medium, culture time, additives, etc.) are not particularly limited. In addition, the method for measuring the expression level of proteins such as Tie2 and type II collagen and mRNA, and the analysis and measurement using a microscope can be performed according to the usual method.

[0019] Coating of culture vessels such as culture dishes with laminin can be carried out according to standard methods. Commercially available laminins (e.g., iMatrix-511 manufactured by Nippi Corporation) or laminins extracted from biological tissues can be used. A laminin solution prepared to give 0.5 μg / cm2 for the above-mentioned products can be placed in a cell culture vessel (dish), incubated at a temperature close to that in the body for about an hour, and then the solution can be removed to obtain a dish coated with laminin.

[0020] An intervertebral disc cell preparation for the treatment of degenerative disc disease and the like, which contains nucleus pulposus cells obtained by the culture method of the present invention, can be produced by combining, as necessary, various additives such as other pharma- ceutically acceptable bases, solvents, stabilizers, plasticizers, fluidizing agents, buffers, antioxidants, humectants, fillers, lubricants, isotonicity agents, pH regulators, antiseptics, preservatives, soothing agents, etc. Furthermore, other medicinal active ingredients used in the treatment of degenerative disc disease and the like or considered to be useful for intervertebral disc regeneration can also be added to produce a formulation.

[0021] The above-mentioned intervertebral disc cell preparation may be in a form that can be transplanted or delivered to a target site (e.g., nucleus pulposus of an intervertebral disc), and examples of such preparations include vial or ampoule preparations for injection that dissolve when used, kit preparations filled in an injection syringe, etc. Targets for the application of the intervertebral disc cell preparation, that is, diseases that can be treated by administering the preparation, include diseases whose symptoms include damage or degeneration, herniation, etc. of the intervertebral disc (nucleus pulposus), such as lumbar or cervical discopathy, intervertebral disc herniation, cervical spondylotic myelopathy, radiculopathy, spondylolysis / spondylolisthesis, lumbar spinal canal stenosis, lumbar degenerative spondylolisthesis, and lumbar degenerative scoliosis.

[0022] The above-mentioned intervertebral disc cell preparation may be administered in an amount effective for achieving the desired therapeutic or preventive effect. Such an effective amount may be appropriately adjusted depending on the dosage per administration, the number of administrations, and the administration interval (number of administrations within a certain period of time), etc., while taking into consideration the ingredients contained in the preparation, the dosage form, the administration subject, the administration route, and other embodiments. The treatment using the intervertebral disc cell preparation may be performed on humans and vertebrates other than humans. EXAMPLES

[0023] [Test Method] 1. Materials and Methods Cells were isolated from human nucleus pulposus tissue (n=5, mean age: 19.1±3 years) collected from patients who had undergone surgery for lumbar disc herniation, with their informed consent. Cell isolation and culture methods were performed according to the recommended method established by the Spine Section of the Orthopaedic Research Society (ORS). The isolated cells were cultured for 6 days (20% FBS [fetal bovine serum], 37°C, O2: 5%, CO2: 21%, 3×10 4 Three types of dishes (non-coated, poly-L-lysine-coated, and laminin-coated) were used for culture and compared. Cell proliferation rates were calculated from the cell counts after culture, and phosphorylation of the extracellular signal-regulated kinase (Erk) pathway and serine-threonine kinase (Akt; protein kinase B) pathway was confirmed by Western blotting. In addition, the expression of specific cell surface markers of nucleus pulposus cells, Tie2, GD2, and GD24, as well as precursor matrix proteins type II collagen (Col2) and proteoglycan (PG), was evaluated by flow cytometry (n=5). Nucleus pulposus cells were further cultured in methylcellulose medium for 10 (or 14) days, and their spherical colony formation ability was evaluated by colony assay (n=5).

[0024] 2. Nucleus Pulposus Cell Isolation The collected nucleus pulposus tissue was washed with saline and chopped into small pieces. The chopped nucleus pulposus tissue was centrifuged with fetal bovine serum (FBS) and MEMα (containing L-glutamine and phenol red). The centrifuged tissue was then incubated with TrypLE. TM The cells were resuspended in Express Enzyme (1X) and phenol red. The suspension was incubated (37°C, 30 min) while gently swirling. Then, the tissue was observed for dissolution and centrifuged. The precipitate was resuspended in a solution of 10% FBS-MEMα and 0.25 mg / mL collagenase P and incubated (37°C, 2 hr). After treatment, the tissue was centrifuged again and resuspended in 10% FBS-MEMα solution, and the cell suspension was transferred to a 40 μm syringe filter and filtered to obtain nucleus pulposus cells.

[0025] 3. Treatment with coating agent Laminin (Nippi, iMatrix-511, Version 010.) was added to a 100 mm tissue culture dish (IWAKI 3020-100) and incubated in a humidified chamber (37°C, 1 hour). As a control without the use of coating agent, the same type of culture dish was treated in the same manner with an equal volume of phosphate buffered saline (PBS). In addition, a PLL-coated dish was used as a comparative control for the coating agent (IWAKI 4020-040100 mm Poly-L-Lysine-Coated 100mmDish). After incubation, the coating agent was removed, and culture medium was added to the culture dishes without washing, and nucleus pulposus cells were directly seeded onto each of the non-coated, PLL-coated, and laminin-coated culture dishes.

[0026] 3. Measurement of Cell Viability On day 6, the supernatant was collected from each culture dish and washed with 1% PBS. After washing, cells were harvested using TrypLE Express, and cell count and cell viability were evaluated. Cell viability was measured using a phase contrast microscope with 0.4 w / v% trypan blue and a hemocytometer plate.

[0027] 4. Protein Extraction and Western Blotting The expression level of type II collagen (Col2) on the 5th day of culture was detected by Western blot analysis. The supernatant was collected from each culture dish and washed with Tris-buffered saline. After washing, the cells were lysed in lysis buffer using TrypLE Express, MilliQ and RIPA Buffer, and 200 mM PMSF, and incubated on ice for 1 hour. After centrifugation, the proteins extracted in the supernatant were collected. The cells were incubated in a microplate reader and quantified by measuring the absorbance at a wavelength of 700 nm using DC protein assay reagent.

[0028] The extracted samples were pulverized using an ultrasonic pulverizer. The extracted samples were mixed with 4x Buffer and mercaptoethanol, heated, and denatured with SDS. Protein lysates were separated using polyacrylamide gels and MES running buffer. After electrophoresis, proteins were transferred onto an electrophoresis system using a PVDF membrane. To detect immunoreactive bands, the membrane was washed and blocked with 3% bovine serum albumin, then washed with buffer G (0.1% gelatin, 0.1% BSA, 0.1% NaN3 in PBS). Then, the primary antibodies Phospho-Akt Ser473 Antibody (Cell Signaling Technology 1:1000), Phospho-44 / 42 Erk1 / 2 (Cell Signaling Technology 1:1000), and monoclonal antibody GAPDH clone (Rabbit anti-GAPDH Polyclonal antibody 1:1000) were diluted and reacted at 4°C overnight. After reacting with the primary antibody, the sections were reacted with the secondary antibody at room temperature for 1 hour, and the immunoreacted bands were detected with a detection reagent (Millipore Immobileon [registered trademark]), and the protein signals were quantified using a multiimaging system.

[0029] 5. Flow Cytometry Nucleus pulposus cells were analyzed using a flow cytometer (BD FACSCalibur). The cells were treated with primary antibodies IgG1 mouse-FITC, IgG1 mouse-APC, IgG1 mouse-PE, anti-hTie-2 APC-conjugated mouse IgG1, PE mouse anti-human disialoganglioside GD2, and FITC mouse anti-human CD24 (10 μg / mL each) and incubated on ice for 1 hour. The cells were then washed with PBS and evaluated. Propidium iodide was used to target only live cells during analysis. In addition, this study was performed by fixing the cells and evaluating the ECM expressed within the cells. That is, the nucleus pulposus cells were fixed, cell membrane permeabilization was performed using IntraPrep Permeabilization Reagent, and flow cytometry was performed.

[0030] 6. Colony Assay Method The cells were cultured in methylcellulose medium (MethoCult H4230) for 10 days (37°C, O2:5%), and the number of spherical colonies formed per mL of medium was counted using an inverted phase contrast microscope.

[0031] 7. Statistics and Visualization Statistics for this study were performed using GraphPad Prism v9.0.1(128), (GraphPad Software Inc., USA). Normality of the data sets was confirmed by one-way or two-way analysis of variance followed by Dunnett's multiple comparison or Tukey's comparison test. P values ​​less than 0.05 were considered statistically significant (*: P<0.05, **: P<0.01). All data are shown as mean values, and error bars for variability are the standard deviation of the mean values.

[0032] 〔result〕 1. Evaluation of cell proliferation ability of nucleus pulposus cells As shown in Figure 1, the shape of the cells immediately before harvesting was spindle-shaped, typical of nucleus pulposus, and the size of the cells increased in the following order: non-coated [NON], PLL-coated [PLL], and laminin-coated [Laminin] (Figure 1). The cell proliferation rate was significantly higher in the PLL-coated compared to the non-coated, and furthermore in the laminin-coated compared to the PLL-coated (Figure 2). Furthermore, although not shown in the figure, analysis of cell activity signals showed a tendency for laminin-coated cells to increase phosphorylation of Erk and Akt, which are important signaling molecules in the cell proliferation and survival pathways.

[0033] 2. Evaluation of progenitor cell phenotypes using cell surface markers Using flow cytometry, the expression rates of Tie2, GD2, and CD24 on the surface of nucleus pulposus cells were detected. The results of flow cytometry for Tie2 are shown (Figure 3). The expression rate of Tie2 was promoted more in the laminin coating than in the PLL coating (Figure 4), and the number of Tie2-positive cells was calculated to be significantly higher (Figure 5). In addition, the production of intracellular type II collagen (Col2) was also significantly higher in the laminin coating than in the PLL coating (Figure 6). There was no difference in intracellular proteoglycan (PG) due to the difference in coating agent (not shown). In addition, although not shown in the figure, it was confirmed that the expression of GD2 and CD24, which are specific markers of nucleus pulposus cells, did not differ depending on the culture conditions. These results demonstrated that it is possible to expand and culture on laminin coating while maintaining the precursor cell phenotype.

[0034] The self-renewal function of nucleus pulposus cells was evaluated by colony assay. The number of spherical colonies formed on methylcellulose by cells seeded on methylcellulose was evaluated. As a result, the number of spherical colonies formed on the laminin coat was greater than that on the PLL coat (Figure 7). [Industrial Applicability]

[0035] From the above results, it was revealed that in the culture of nucleus pulposus cells for use in regenerative medicine for intervertebral disc degeneration, etc., when laminin is used as the coating agent, nucleus pulposus cells with extremely high usefulness can be obtained, compared with poly-L-lysine (PLL), which is the coating agent during culture that was considered optimal in the prior art. That is, by using a laminin-coated dish in the culture of nucleus pulposus cells, nucleus pulposus cells with significantly superior functionality in terms of the proliferation promotion effect, Tie2 expression effect, and type II collagen production effect of nucleus pulposus cells compared to PLL-coated dishes were efficiently obtained, and excellent effects were also shown in terms of spherical colony formation ability. Therefore, the present invention is very useful for producing a therapeutic preparation for regenerative medicine for intervertebral disc degeneration, etc.

Claims

1. A method for culturing nucleus pulposus cells, which uses laminin as a coating agent for the culture surface of a culture vessel.

2. 2. The method according to claim 1, wherein the nucleus pulposus cells are collected from a human.

3. A culture vessel for culturing nucleus pulposus cells, the culture surface of which is coated with laminin.

4. 4. The culture vessel according to claim 3, wherein the nucleus pulposus cells are collected from a human.

5. Use of laminin as a coating agent for the culture surface of a container for culturing nucleus pulposus cells.

6. 6. The use according to claim 5, wherein the nucleus pulposus cells are obtained from a human.

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