Pharmaceutical composition for preventing or treating cartilage disease or inflammatory joint disease

Abstract

The present invention relates to a pharmaceutical composition for preventing or treating cartilage disease or inflammatory joint disease and a method for preparing a chondrospheroid having chondrocyte differentiation ability. The present invention provides a pharmaceutical composition comprising a stem cell-derived spheroid as an active ingredient for preventing or treating cartilage disease or inflammatory joint disease, and a method for preparing a chondrospheroid having chondrocyte differentiation ability, the method comprising the steps of: Inoculating stem cells into a three-dimensional (3D) cell culture vessel made of a plastic material; treating the stem cells inoculated into the 3D cell culture vessel with a chondrocyte differentiation-inducing factor; culturing the stem cells treated with the chondrocyte differentiation-inducing factor to form chondrospheroids; and separating the formed chondrospheroids.

Description

Pharmaceutical composition for the prevention or treatment of cartilage disease or inflammatory joint disease

[0001] The present invention relates to a pharmaceutical composition for the prevention or treatment of cartilage diseases or inflammatory joint diseases comprising a stem cell-derived chondropheroid with chondrodifferentiation ability.

[0002] Osteoarthritis, also known as degenerative arthritis, is a joint disease that occurs as the cartilage, which protects the ends of bones, wears away. It is the most common type of arthritis and manifests symptoms such as joint pain, swelling, and stiffness, particularly in the morning. Cartilage is a fibrous connective tissue located on the surface of joints that connect two bones throughout the body, and it is composed of chondrocytes and chondroblasts. The core component of cartilage is collagen, specifically Type II collagen.

[0003] Currently, treatment for osteoarthritis focuses on relieving pain and maintaining joint flexibility. Medication, exercise, and physical therapy are commonly used, while surgeries such as artificial joint replacement are performed in limited cases where the joint is severely damaged.

[0004] Meanwhile, with the recent surge in interest in stem cell-based technologies, pluripotent stem cells—which possess the ability to form various organs through proliferation and differentiation—are recognized as capable of treating most diseases as well as fundamentally resolving organ damage.

[0005] Stem cells are cells capable of differentiating into various types of cells that constitute biological tissues; the term collectively refers to undifferentiated cells obtainable from the tissues of embryos, fetuses, and adults. Stem cells differentiate into specific cell types depending on differentiation stimuli (environment) and possess the characteristic of self-renewal, which allows them to produce identical cells through cell division. Furthermore, they possess plasticity, which enables them to differentiate into different cell types depending on the differentiation stimulus.

[0006] Stem cells can be classified into totipotency, pluripotency, multipotency, and unipotency stem cells based on their differentiation ability. Pluripotent stem cells possess the potential to differentiate into all cell types, whereas some stem cells possess the potential for multipotency or unipotency.

[0007] Stem cells exhibit a paracrine effect by expressing various bioactive factors that promote the growth and differentiation of surrounding cells. Due to these characteristics, stem cells are utilized as raw materials for cell therapy and are receiving significant attention in the field of regenerative medicine, including the treatment of intractable and degenerative diseases and organ regeneration.

[0008] The development of cell therapies utilizing the differentiation potential of these stem cells has been actively underway, and various studies have suggested the potential for application in conditions requiring the recovery and regeneration of lost cells, such as neurological disorders, heart disease, lung disease, liver disease, and tissue damage following cancer resection. Through this, it is expected that these therapies can be utilized to restore the functions of damaged cells and tissues.

[0009] The background description of the invention is provided to facilitate a better understanding of the present invention. The matters described in the background description should not be construed as an acknowledgment that they exist as prior art.

[0010] Meanwhile, regarding osteoarthritis, conservative treatments such as drug therapy aim to alleviate symptoms and restore limited function. Currently, while artificial joint replacement surgery is the standard treatment for advanced osteoarthritis, there are problems such as the short lifespan of artificial joints, which prevent lifetime use and require re-surgery. Consequently, there is a demand for the development of new cell therapies that can halt the progression of osteoarthritis and induce symptom improvement before joint destruction, and interest in stem cell-based cell therapies is particularly growing.

[0011] However, existing stem cell therapies have limitations, as the application method is highly invasive, involving the incision of the damaged area to apply cells to the lesion; the healing mechanism has been found to be based on secretory effects rather than the direct differentiation of transplanted cells, and structural improvement through the regeneration of hyaline cartilage cannot be achieved. Therefore, the need to develop a new therapeutic strategy to overcome these limitations has been recognized.

[0012] Furthermore, it was noted that, unlike bone marrow which has limitations in tissue harvesting, adipose tissue allows for the easy acquisition of large quantities of cells without donor site dysfunction using a relatively simple liposuction, and that, similar to bone marrow cells, adult stem cells isolated from adipose tissue originate from the embryonic mesoderm and can differentiate into bone or cartilage.

[0013] Accordingly, the inventors of the present invention aimed to develop a cell therapy agent capable of achieving cartilage regeneration through the direct differentiation of stem cells in lesions relatively non-invasively using adipose stem cells.

[0014] As a result, the inventors of the present invention manufactured an adipose stem cell-derived chondrosphere with excellent cartilage differentiation ability using a 3D cell culture vessel made of plastic material, and confirmed that injecting the adipose stem cell-derived chondrosphere into the joint cavity and transplanting it into a lesion has the effect of reducing inflammation levels within the lesion and regenerating cartilage, thereby completing the present invention.

[0015] In addition, it was discovered that when the above-mentioned adipose stem cell-derived chondrospheres are transplanted to the lesion site and a bioadhesive is applied to the transplant site to fix the cells to the lesion, the transplanted chondrospheres survive and engraft for a long period without being lost within the lesion, resulting in a significant increase in cartilage regeneration efficiency.

[0016] Accordingly, the problem that the present invention aims to solve is to provide a method for manufacturing a chondrosphere having the ability to differentiate into chondrocytes using stem cells.

[0017] In addition, another problem that the present invention aims to solve is to provide a chondrosphere produced by the method for producing a chondrosphere having the ability to differentiate into cartilage cells.

[0018] In addition, another problem that the present invention aims to solve is to provide a pharmaceutical composition for the prevention or treatment of cartilage disease or inflammatory joint disease for co-administration with a bioadhesive containing a stem cell-derived spheroid as an active ingredient.

[0019] In addition, another problem that the present invention aims to solve is to provide a cell therapy agent for the prevention or treatment of cartilage disease or inflammatory joint disease for co-administration with a bioadhesive containing a stem cell-derived spheroid as an active ingredient.

[0020] In addition, another problem that the present invention aims to solve is to provide a kit for the prevention or treatment of cartilage disease or inflammatory joint disease comprising a stem cell-derived spheroid and a bioadhesive.

[0021] In addition, another problem that the present invention aims to solve is to provide a method for preventing or treating cartilage disease or inflammatory joint disease, comprising the step of administering the pharmaceutical composition to an individual.

[0022] The problems of the present invention are not limited to those mentioned above, and other unmentioned problems will be clearly understood by those skilled in the art from the description below.

[0023] In order to solve the problem described above, a method for producing a chondrosphere having excellent cartilage cell differentiation ability according to one embodiment of the present invention is provided.

[0024] Specifically, the method for manufacturing a chondrosphere having excellent cartilage cell differentiation ability comprises the steps of inoculating stem cells into a 3D cell culture vessel made of plastic material, culturing the stem cells inoculated into the 3D cell culture vessel to form a chondrosphere, and separating the formed chondrosphere.

[0025] According to a feature of the present invention, the stem cell may be an adipose-derived stem cell.

[0026] According to another feature of the present invention, the stem cells are 1.0 × 10 5 cells / cm 2 Up to 5.0 × 10 6 cells / cm 2 It may be inoculated at a cell density.

[0027] According to another feature of the present invention, the 3D cell culture vessel may be composed of 300 to 450 concave microwells with a diameter of 550 μm to 650 μm, and the wells may be arranged in a hexagonal array.

[0028] According to another feature of the present invention, the culture may be carried out under culture conditions of a temperature of 30°C to 40°C and a CO2 concentration of 3% to 7%.

[0029] According to another feature of the present invention, the culture may be performed for at least 3 days.

[0030] In order to solve other problems as described above, a chondrosphere having cartilage cell differentiation ability is provided, which is manufactured by a method for manufacturing a chondrosphere according to another embodiment of the present invention.

[0031] According to a feature of the present invention, the chondrosphere may have a diameter of 150 to 250 μm.

[0032] In order to solve other problems as described above, a pharmaceutical composition for the prevention or treatment of cartilage disease or inflammatory joint disease is provided for co-administration with a bioadhesive containing a stem cell-derived spheroid as an active ingredient according to another embodiment of the present invention.

[0033] According to a feature of the present invention, the stem cell may be an adipose-derived stem cell.

[0034] According to another feature of the present invention, the spheroid may be a chondrospheroid having the ability to differentiate into chondrocytes.

[0035] According to another feature of the present invention, the bioadhesive may comprise at least one selected from the group consisting of fibrin, fibrinogen, thrombin, fibrin glue, alginate gel, hyaluronic acid gel, chitosan gel, collagen gel, gelatin gel, gelatin-resorcin-formalin adhesive, polysaccharide gel, polyethylene glycol-based hydrogel, polylactic acid / polyglycolic acid copolymer hydrogel, poly(vinyl alcohol) hydrogel, polyacrylamide gel, oxalaldehyde-based hydrogel, cyanoacrylate glue, synthetic acrylate-based adhesive, cellulose-based adhesive, basement membrane matrix, laminin, elastin, proteoglycan, and autoglue.

[0036] According to another feature of the present invention, the cartilage disease may be selected from the group consisting of osteoarthritis, chondromalacia patellae, cartilage defect, osteochondral injury, meniscus tear, avascular necrosis, and osteochondritis dissecans.

[0037] According to another feature of the present invention, the inflammatory joint disease may be selected from the group consisting of ankylosing spondylitis, psoriatic arthritis, rheumatoid arthritis, gouty arthritis, reactive arthritis, and infectious arthritis.

[0038] In order to solve other problems as described above, a cell therapy agent for the prevention or treatment of cartilage disease or inflammatory joint disease is provided for co-administration with a bioadhesive containing a stem cell-derived spheroid as an active ingredient according to another embodiment of the present invention.

[0039] According to a feature of the present invention, the stem cell may be an adipose-derived stem cell.

[0040] According to another feature of the present invention, the spheroid may be a chondrospheroid having the ability to differentiate into chondrocytes.

[0041] According to another feature of the present invention, the spheroids are 20 spheroids / mm 3 Up to 50 spheroids / mm 3 It may be administered at a spheroid density.

[0042] To solve other problems as described above, a kit for preventing or treating cartilage disease or inflammatory joint disease is provided, comprising a stem cell-derived spheroid and a bioadhesive according to another embodiment of the present invention.

[0043] According to the features of the present invention, the bioadhesive may be a gelling bioadhesive.

[0044] According to another feature of the present invention, the gelling bioadhesive may comprise at least one selected from the group consisting of fibrinogen, thrombin, alginate gel, hyaluronic acid gel, chitosan gel, collagen gel, gelatin gel, polyethylene glycol-based hydrogel, polylactic acid / polyglycolic acid copolymer hydrogel, poly(vinyl alcohol) hydrogel, polyacrylamide gel, oxalaldehyde-based hydrogel, and gelatin-resorcin-formalin adhesive.

[0045] In order to solve other problems as described above, a method for treating cartilage disease or inflammatory joint disease is provided, comprising the step of administering to an individual a pharmaceutical composition containing the chondrosphere as an active ingredient according to another embodiment of the present invention.

[0046] The adipose stem cell-derived chondrosphere according to the present invention, when administered to a lesion, reduces inflammation levels within the lesion and possesses excellent cartilage differentiation ability, thereby effectively regenerating damaged cartilage; thus, it can be usefully utilized as a pharmaceutical composition for the prevention or treatment of cartilage diseases or inflammatory joint diseases.

[0047] Furthermore, when the adipose stem cell-derived chondrosphere according to the present invention is administered in combination with a bioadhesive, it prevents the loss of transplanted cells and enables long-term survival by adhering and fixing the transplanted cells to the lesion site. This action has the effect of significantly increasing the efficiency of cartilage regeneration by inducing the differentiation and regeneration of the transplanted cells into chondrocytes.

[0048] Furthermore, the method for manufacturing chondrospheres according to the present invention has the advantage of being able to easily mass-culture using high-yield adipose stem cells, form high-quality spheroids with cartilage differentiation function with a short-term culture period of about 3 days, and significantly reduce manufacturing costs because there is no need to use expensive growth factors required for inducing cartilage differentiation.

[0049] The effects according to the present invention are not limited to those exemplified above, and various other effects are included in this specification.

[0050] FIG. 1a illustrates a flowchart of a method for producing a chondrosphere with cartilage cell differentiation ability according to one embodiment of the present invention.

[0051] FIG. 1b illustrates a schematic diagram of osteoarthritis treatment using a composition for the prevention or treatment of osteoarthritis containing chondrospheres according to one embodiment of the present invention.

[0052] FIG. 2a illustrates the results of observing the characteristics of chondrospheres produced in a polydimethylsiloxane (PDMS) spheroid culture vessel and a plastic spheroid culture vessel according to one embodiment of the present invention.

[0053] FIG. 2b illustrates the results of spheroid size analysis produced in a polydimethylsiloxane (PDMS) spheroid culture vessel and a plastic spheroid culture vessel according to one embodiment of the present invention.

[0054] FIG. 2c illustrates the results of confirming the chondrogenic differentiation ability of spheroids produced in polydimethylsiloxane (PDMS) spheroid culture vessels and plastic spheroid culture vessels according to one embodiment of the present invention through safranin O / fast green double staining.

[0055] FIG. 2d illustrates the results of evaluating the internal and external cell viability of spheroids produced in a polydimethylsiloxane (PDMS) spheroid culture vessel and a plastic spheroid culture vessel according to one embodiment of the present invention.

[0056] Figure 3a shows the results of an ELISA analysis for inflammatory cytokines TNF-α and IL-1B in the synovial fluid of an animal model of osteoarthritis according to one embodiment of the present invention.

[0057] FIG. 3b illustrates the results of observing bone regeneration according to whether or not chondrospheres are implanted in a cartilage defect area of ​​an animal model of osteoarthritis according to one embodiment of the present invention.

[0058] FIG. 3c shows an ICRS Macroscopic Score graph based on the results of observing bone regeneration according to whether or not chondrospheres were implanted in the cartilage defect area of ​​an animal model of osteoarthritis according to one embodiment of the present invention.

[0059] FIG. 3d shows the results of Safranin-O / Fast Green staining depending on whether chondrosphere implantation is performed in a femoral section of an animal model of osteoarthritis according to one embodiment of the present invention.

[0060] FIG. 3e shows a graph of the Histological Score based on the results of Safranin-O / Fast Green staining according to whether or not chondrospheres were implanted in femoral sections of an animal model of osteoarthritis according to one embodiment of the present invention.

[0061] Figure 4 shows the results of immunochemical staining using Human nuclear antigen (HN) after chondrophing and fibrin fixation in knee cartilage tissue of an animal model of osteoarthritis according to one embodiment of the present invention.

[0062] The advantages and features of the present invention and the methods for achieving them will become clear by referring to the embodiments described below in detail together with the accompanying drawings. However, the present invention is not limited to the embodiments disclosed below but may be implemented in various different forms. These embodiments are provided merely to ensure that the disclosure of the present invention is complete and to fully inform those skilled in the art of the scope of the invention, and the present invention is defined only by the scope of the claims.

[0063] In the following, terms used within this specification are explained for clarity of explanation.

[0064] In this document, expressions such as "have," "can have," "include," or "can include" refer to the existence of the relevant feature (e.g., numerical values, functions, actions, or components, etc.) and do not exclude the existence of additional features.

[0065] In this document, "or" means "and / or" unless otherwise noted. Expressions such as "A or B," "at least one of A or / and B," or "one or more of A or / and B" may include all possible combinations of items listed together. For example, "A or B," "at least one of A and B," or "at least one of A or B" may refer to cases including (1) at least one A, (2) at least one B, or (3) both at least one A and at least one B.

[0066] As used in the present invention, the term “stem cell” refers to an undifferentiated cell having the ability to differentiate into various cell types, and preferably refers to an adult stem cell.

[0067] As used in the present invention, the term “adult stem cell” refers to a stem cell derived from adult tissue that has the ability to differentiate into a specific cell type. For example, hematopoietic stem cells can differentiate into blood cells, and mesenchymal stem cells can differentiate into bone, cartilage, and fat cells.

[0068] The term “Adipose-derived Stem Cells (ADSCs)” as used in this invention refers to mesenchymal Stem Cells (MSCs) derived from adipose tissue. Adipose-derived stem cells have the ability to differentiate into various tissues, and in particular, can differentiate into cartilage, bone, and fat cells.

[0069] The term “spheroid” used in the present invention refers to a circular cell aggregate in which a plurality of single cells are cultured in three dimensions. The spheroid is characterized by the fact that the cells are arranged in three dimensions to provide an environment similar to actual tissue, and that active interaction between cells occurs, allowing it to better mimic the function of tissue.

[0070] The term “chondrosphere” used in the present invention refers to a three-dimensional (3D) cell aggregate composed of chondrocytes and chondrocytes.

[0071] The term “three-dimensional cell” or “3D cell” used in the present invention refers to a cell cultured in a three-dimensional environment. Three-dimensional cell culture is carried out in an artificially created environment where cells can grow in all directions and interact with the surrounding environment. It is characterized by high bio-likeness and active inter-cell interactions and is used in various fields such as cancer research, drug screening, and regenerative medicine.

[0072] The term “scaffold” as used in the present invention refers to a structure that serves to provide a physical support structure for cells to grow in 3D cell culture. Such a scaffold mimics the extracellular matrix (ECM) of biological tissues to help cells grow, differentiate, and migrate in a three-dimensional environment.

[0073] As used in the present invention, the term “bioadhesive” refers to a medical adhesive used to stably fix transplanted cells, promote tissue regeneration, and prevent infection. These adhesives play an important role in stably maintaining transplanted cells and promoting tissue regeneration after cell transplantation.

[0074] The term “gelling bioadhesive” as used in the present invention refers to an adhesive that changes from a liquid state to a gel state and fixes within the body or at the application site.

[0075] The term “autologous glue” as used in this invention refers to an adhesive derived from the patient’s own tissue or blood.

[0076] As used in the present invention, the term “concurrent administration” means administering two or more components or preparations simultaneously or sequentially.

[0077] As used in the present invention, the term "administration" encompasses all appropriate methods of providing the composition of the present invention to an individual, and is not necessarily limited to intramuscular injection, but also includes methods such as implantation, application, and spraying. Furthermore, the route of administration may be any general route as long as it can reach the target tissue. It may be administered intra-articularly, intraperitoneally, intravenously, intramuscularly, subcutaneously, intradermally, orally, locally, intranasally, intrapulmonaryly, or rectally, but is not limited thereto.

[0078] The term "prevention" above refers to any act that reduces the frequency or severity of pathological phenomena. Prevention may be complete or partial. In this case, it may refer to a phenomenon in which symptoms of cartilage disease or inflammatory joint disease within an individual are reduced compared to the case where the composition is not used.

[0079] The above "treatment" refers to any act of clinical intervention intended to alter the natural processes of the target or cell to be treated, and may be performed during the progression of a clinical pathological condition or to prevent it. The intended therapeutic effects may include preventing the onset or recurrence of the disease, alleviating symptoms, reducing all direct or indirect pathological consequences associated with the disease, preventing metastasis, slowing the rate of disease progression, alleviating or temporarily resolving the disease state, or improving the prognosis.

[0080] The term "pharmacologically effective amount" as used in the present invention refers to an amount sufficient to treat a disease with a reasonable benefit / risk ratio applicable to medical treatment and that does not cause side effects, and the effective dose level may be determined based on factors including the patient's health status, type and severity of the disease, drug activity, sensitivity to the drug, method of administration, time of administration, route of administration and elimination rate, duration of treatment, drugs used in combination or concurrently, and other factors well known in the medical field.

[0081] As used in the present invention, the term "individual" refers to an individual intended for the prevention or treatment of cartilage disease or inflammatory joint disease, and is not particularly limited to animals including humans, for example, non-primates (e.g., cattle, pigs, horses, cats, dogs, rats, and mice) and mammals including primates (e.g., monkeys, e.g., cynomolgous monkeys and chimpanzees). In some cases, it may be an individual excluding humans.

[0082] As used in the present invention, the term "pharmaceutical acceptable" refers to a composition that is physiologically acceptable and, when administered to humans, does not typically cause allergic reactions such as gastrointestinal disorders or dizziness, or similar reactions.

[0083] As used in this invention, the term "cell therapy agent" refers to a pharmaceutical product (under U.S. FDA regulations) used for the purposes of treatment, diagnosis, and prevention, consisting of cells and tissues produced through isolation, culture, and special manipulation from an individual. It refers to a pharmaceutical product used for treatment, diagnosis, and prevention through a series of actions, such as proliferating and selecting living autologous, allogeneic, or xenogeneic cells in vitro to restore the function of cells or tissues, or altering the biological characteristics of the cells by other methods.

[0084] In one aspect, the present invention relates to a method for producing a chondrosphere having the ability to differentiate into chondrocytes using stem cells.

[0085] FIG. 1a illustrates a flowchart of a method for producing a chondrosphere with cartilage cell differentiation ability according to one embodiment of the present invention.

[0086] Referring to FIG. 1a, the present invention includes the steps of inoculating stem cells into a 3D cell culture vessel made of plastic material (S110), culturing the stem cells inoculated into the 3D cell culture vessel to form chondrospheres (S120), and separating the formed chondrospheres (S130).

[0087] More specifically, in the step (S110) of inoculating stem cells into a 3D cell culture vessel made of plastic material, the stem cells may be adipose-derived stem cells, but are not limited thereto.

[0088] In addition, the above stem cells are 1.0 × 10 5 cells / cm 2 Up to 5.0 × 10 6 cells / cm 2 It may be inoculated at a cell density of , preferably 2.5 × 10 5 cells / cm 2 Up to 2.0 × 10 6 cells / cm 2 It may be inoculated at a cell density of 0.5 × 10⁶, and more preferably 0.5 × 10⁶ 6 cells / cm 2 Up to 1.5 × 10 6 cells / cm 2 It may be inoculated at a cell density, but is not limited to this.

[0089] In addition, the 3D cell culture vessel may be composed of 300 to 450 concave microwells with a diameter of 550 μm to 650 μm, and the wells may be arranged in a hexagonal array, but are not limited thereto.

[0090] In addition, the 3D cell culture vessel may further include a 3D cell culture scaffold to improve spheroid formation ability, but is not limited thereto.

[0091] Referring again to FIG. 1a, in the step (S120) of forming chondrospheres by culturing stem cells inoculated in a 3D cell culture vessel, the culture may be performed under culture conditions of a temperature of 30°C to 40°C and a CO2 concentration of 3% to 7%, and preferably under culture conditions of a temperature of 35°C to 39°C and a CO2 concentration of 4% to 6%, but is not limited thereto.

[0092] In addition, the above culture may be performed for at least 3 days, preferably for 3 to 14 days, but is not limited thereto and the culture period may be appropriately adjusted as needed.

[0093] At this time, the present invention can form chondrospheres within a relatively short period (e.g., 3 to 7 days) compared to conventional technology, thereby demonstrating superior effects in terms of time and cost. However, even if cultured for a longer period as needed, the quality or function of the chondrospheres is not affected.

[0094] Referring again to FIG. 1a, the step (S130) of separating the formed chondrospheres can be performed by a suitable method known in the art depending on the type of culture vessel and cell. Specifically, methods for separating and obtaining cells from a known culture medium may be used, although not specifically limited thereto, such as centrifugation, filtration, enzymatic separation using a trypsin-EDTA solution, or mechanical separation using a cell scraper or pipette.

[0095] In one aspect, the present invention relates to a chondrosphere having chondrocyte differentiation ability produced by the above-described method for producing a chondrosphere.

[0096] In one embodiment of the present invention, the chondrosphere may have a diameter of 150 to 250 μm, but is not limited thereto.

[0097] In one aspect, the present invention relates to a pharmaceutical composition for the prevention or treatment of cartilage disease or inflammatory joint disease for co-administration with a bioadhesive comprising a stem cell-derived spheroid as an active ingredient.

[0098] Figure 1b illustrates a schematic diagram of osteoarthritis cell therapy using chondrospheres and bioadhesives.

[0099] Referring to FIG. 1b, in one embodiment of the present invention, the composition may significantly increase the efficiency of cartilage regeneration by preventing the loss of cells transplanted to the lesion site and enabling long-term survival by administering it in combination with a bioadhesive, thereby inducing differentiation and regeneration of the transplanted cells into chondrocytes.

[0100] In one embodiment of the present invention, the stem cell may be an adipose-derived stem cell, but is not limited thereto.

[0101] In one embodiment of the present invention, the stem cells may be obtained not only from the patient themselves but also from another person or from other medical animals. The cells may also be used without limitation if they have been frozen.

[0102] In one embodiment of the present invention, the spheroid may be a chondrospheroid having the ability to differentiate into chondrocytes, but is not limited thereto.

[0103] In one embodiment of the present invention, the bioadhesive may comprise at least one selected from the group consisting of fibrin, fibrinogen, thrombin, fibrin glue, alginate gel, hyaluronic acid gel, chitosan gel, collagen gel, gelatin gel, gelatin-resorcin-formalin adhesive, polysaccharide gel, polyethylene glycol-based hydrogel, polylactic acid / polyglycolic acid copolymer hydrogel, poly(vinyl alcohol) hydrogel, polyacrylamide gel, oxalaldehyde-based hydrogel, cyanoacrylate glue, synthetic acrylate-based adhesive, cellulose-based adhesive, basement membrane matrix, laminin, elastin, proteoglycan, and autoglue, but is not limited thereto.

[0104] In one embodiment of the present invention, the cartilage disease refers to a disease caused by cartilage damage, and may be selected from the group consisting of, for example, osteoarthritis, chondromalacia patellae, cartilage defect, osteochondral injury, meniscus tear, avascular necrosis, and osteochondritis dissecans, but is not limited thereto.

[0105] In one embodiment of the present invention, the disease may be selected from the group consisting of ankylosing spondylitis, psoriatic arthritis, rheumatoid arthritis, gouty arthritis, reactive arthritis, and infectious arthritis, but is not limited thereto.

[0106] The pharmaceutical composition of the present invention is administered in an amount that is pharmaceutically effective. The composition of the present invention may be administered as an individual therapeutic agent or in combination with other therapeutic agents, may be administered sequentially or simultaneously with conventional therapeutic agents, and may be administered as a single or multiple doses. Taking all of the above factors into consideration, it is important to administer an amount that obtains maximum effect with a minimum amount without side effects, and this can be easily determined by a person skilled in the art.

[0107] The composition according to the present invention may comprise a pharmaceutically effective amount of stem cell-derived spheroids alone, or one or more pharmaceutically acceptable carriers, excipients, or diluents. In the above, a pharmaceutically effective amount refers to an amount sufficient to prevent, improve, and treat symptoms of cartilage disease or inflammatory joint disease.

[0108] Additionally, a composition comprising a pharmaceutically acceptable carrier may be one or more formulations selected from the group comprising oral formulations, topical preparations, suppositories, sterile injectable solutions, and sprays, such as various formulations including capsules, liquids, injections, soft capsules, granules, or tablets. When formulated, it may be prepared using diluents or excipients such as commonly used fillers, extenders, binders, wetting agents, disintegrants, and surfactants. The above carrier, excipient, and diluent may be one or more selected from the group consisting of lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinylpyrrolidone, physiological saline, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil, dextrin, calcium carbonate, propylene glycol and liquid paraffin, but are not limited thereto, and any conventional carrier, excipient, or diluent may be used. The above components may be added to the stem cell-derived spheroid, which is the active ingredient, either independently or in combination.

[0109] In addition, the pharmaceutical composition of the present invention may have any one formulation selected from the group consisting of tablets, pills, powders, granules, capsules, suspensions, liquid formulations, emulsions, syrups, sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized formulations, and suppositories. Witepsol, macrogol, Tween 61, cacao oil, laurin oil, glycerol, gelatin, etc. may be used as the base for suppositories.

[0110] The stem cell-derived spheroids of the present invention can be administered in various oral and parenteral formulations during clinical administration, and when formulated, they are manufactured using diluents or excipients such as commonly used fillers, extenders, binders, wetting agents, disintegrants, and surfactants.

[0111] Solid dosage forms for oral administration include tablets, pills, powders, granules, capsules, troches, etc., and these solid dosage forms are prepared by mixing at least one excipient, such as starch, calcium carbonate, sucrose, lactose, or gelatin, with one or more stem cell-derived spheroids of the present invention. In addition, lubricants such as magnesium styrate talc are also used in addition to simple excipients. Liquid dosage forms for oral administration include suspensions, liquid formulations, emulsions, or syrups, and may include various excipients, such as humectants, sweeteners, flavorings, and preservatives, in addition to commonly used simple diluents such as water and liquid paraffin.

[0112] Preparations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, suppositories, etc. As non-aqueous solvents and suspensions, propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate may be used. As bases for suppositories, witepsol, macrogol, tween 61, cocoa paste, laurin paste, glycerol, gelatin, etc. may be used.

[0113] The pharmaceutical composition of the present invention may be administered by any device capable of moving stem cell-derived spheroids to target cells. Preferred modes of administration and formulations include intravenous injections, subcutaneous injections, intradermal injections, intramuscular injections, drip injections, etc. The injection may be prepared using aqueous solvents such as physiological saline solution or Ringer's gel solution, non-aqueous solvents such as vegetable oils, higher fatty acid esters (e.g., ethyl oleate), and alcohols (e.g., ethanol, benzyl alcohol, propylene glycol, glycerin, etc.), and may include a pharmaceutical carrier such as a stabilizer to prevent deterioration (e.g., ascorbic acid, sodium bisulfite, sodium pyrosulfite, BHA, tocopherol, EDTA, etc.), an emulsifier, a buffer to adjust pH, and a preservative to inhibit microbial growth (e.g., phenylmercury nitrate, thimerosal, benzalkonium chloride, phenol, cresol, benzyl alcohol, etc.).

[0114] The pharmaceutical composition of the present invention can also be provided in the form of an external preparation containing a stem cell-derived spheroid as an active ingredient.

[0115] When the pharmaceutical composition of the present invention is used as a topical application for the skin, it may additionally contain adjuvants commonly used in the field of dermatology, such as fatty substances, organic solvents, solvents, thickeners and gelling agents, emollients, antioxidants, suspending agents, stabilizers, foaming agents, fragrances, surfactants, water, ionic emulsifiers, nonionic emulsifiers, fillers, metal ion chelating agents, chelating agents, preservatives, vitamins, blockers, humectants, essential oils, dyes, pigments, hydrophilic activators, lipophilic activators, or lipid vesicles, and any other ingredients commonly used in topical applications for the skin. Furthermore, said ingredients may be introduced in amounts commonly used in the field of dermatology. When the pharmaceutical composition of the present invention is provided as a topical application for the skin, it may be in the form of an ointment, patch, gel, cream, or spray, but is not limited thereto.

[0116] The pharmaceutical composition of the present invention may further include pharmaceutically acceptable additives, wherein the pharmaceutically acceptable additives may include starch, gelatinized starch, microcrystalline cellulose, lactose, povidone, colloidal silicon dioxide, calcium hydrogen phosphate, lactose, mannitol, malt syrup, gum arabic, pregelatinized starch, corn starch, powdered cellulose, hydroxypropyl cellulose, Opadry, sodium starch glycolate, carnauba wax, synthetic aluminum silicate, stearic acid, magnesium stearate, aluminum stearate, calcium stearate, sucrose, dextrose, sorbitol, and talc. The pharmaceutically acceptable additive according to the present invention is preferably included in an amount of 0.1 to 90 parts by weight with respect to the composition, but is not limited thereto.

[0117] In addition, the effective dosage of the stem cell-derived spheroid of the present invention to the human body may vary depending on the patient's age, body weight, gender, form of administration, health condition, and degree of disease, and is generally about 0.001-100 mg / kg / day, preferably 0.01-35 mg / kg / day. Based on an adult patient weighing 70 kg, it is generally 0.07-7000 mg / day, preferably 0.7-2500 mg / day, and may be administered in divided doses once or several times a day at regular intervals according to the judgment of a doctor or pharmacist.

[0118] The pharmaceutical composition of the present invention may be used alone or in combination with methods using surgery, radiation therapy, hormone therapy, chemotherapy, or biological response modifiers.

[0119] In one aspect, the present invention relates to a cell therapy agent for the prevention or treatment of cartilage disease or inflammatory joint disease for co-administration with a bioadhesive containing a stem cell-derived spheroid as an active ingredient.

[0120] In the cell therapy agent for the prevention or treatment of cartilage disease or inflammatory joint disease for co-administration with a bioadhesive according to the present invention, the description of the stem cell-derived spheroid is as described above.

[0121] In one embodiment of the present invention, the stem cell may be an adipose-derived stem cell, but is not limited thereto.

[0122] In one embodiment of the present invention, the spheroid may be a chondrospheroid having the ability to differentiate into chondrocytes, but is not limited thereto.

[0123] In one embodiment of the present invention, the cell therapy agent may be administered to a cartilage defect site or an inflamed joint site, but is not limited thereto.

[0124] The administration route of the above cell therapy composition may be parenteral as long as it can reach the target tissue. Parenteral administration may be, for example, intra-articular administration, intraperitoneal administration, intravenous administration, intramuscular administration, subcutaneous administration, intradermal administration, local administration, intranasal administration, intrapulmonary administration, or rectal administration, and preferably may be administered into the joint cavity, but is not limited thereto.

[0125] The above administration may be local or systemic. Local administration may be, for example, administered directly to the lesion or around the lesion. The above administration may involve administering an effective amount to prevent or treat the disease. Such an effective amount can be readily selected by a person skilled in the art depending on the conditions of the selected disease.

[0126] The cell therapy composition of the present invention may be used without freezing or may be frozen for future use. If freezing is required, a standard cryopreservation agent (e.g., DMSO, glycerol, Epilife (Epilife® cell freezing medium (Cascade Biologics)) may be added to the cell population before freezing.

[0127] In addition, the cell therapy composition may be formulated into a unit-dose pharmaceutical preparation suitable for administration into a patient's body according to conventional methods in the pharmaceutical field, and said preparation contains an effective dosage amount in one or several doses. Preferably, formulations suitable for this purpose include parenteral preparations such as injectable ampoules, injectable preparations such as infusion bags, and spray preparations such as aerosol preparations. The injectable ampoule may be prepared by mixing with an injection solution immediately before use, and physiological saline, glucose, mannitol, Ringer's solution, etc., may be used as the injection solution. In addition, the infusion bag may be made of polyvinyl chloride or polyethylene, and examples include infusion bags from Baxter, Becton Dickinson, Medcep, National Hospital Products, or Terumo.

[0128] In addition to the active ingredient, the above pharmaceutical formulation may further include one or more pharmaceutically acceptable conventional inert carriers, for example, in the case of an injectable formulation, a preservative, an analgesic, a solubilizing agent, or a stabilizer, and in the case of a formulation for topical administration, a base, an excipient, a lubricant, or a preservative.

[0129] In addition, the cell therapy composition may further include a support for accommodating the cells, preferably a biodegradable support. The biodegradable support may be a hydrogel such as fibrin glue, hyaluronic acid, gelatin, collagen, alginic acid, cellulose, pectin, chitin, polyglycolic acid, or polylactic acid, but is not limited thereto.

[0130] In addition, the cell therapy composition may further include a pharmaceutically acceptable carrier, and the pharmaceutically acceptable carrier may be used by mixing one or more of saline solution, sterile water, Ringer's solution, buffered saline solution, dextrose solution, maltodextrin solution, glycerol, ethanol, and other components thereof, and other conventional additives such as antioxidants, buffers, and bacteriostatic agents may be added as needed.

[0131] The above buffer may optionally be acetate, citrate, tartrate, lactate, succinate, or phosphate. The above stabilizer may be mannitol, histidine, lysine, glycine, sucrose, fructose, trehalose, lactose, or a mixture thereof. The above isotonic agent may be glycerin, lactose, mannitol, dextrose, sodium chloride, sodium sulfate, or sorbitol. The above antioxidant may be acetone, sodium bisulfite, butylated hydroxyanisole, butylated hydroxytoluene, cysteine, cysteineate HCl, sodium dithionite, gentisic acid, gentisate ethanolamine, glutamate monosodium, formaldehyde sulfoxylate sodium, potassium metabisulfite, sodium metabisulfite, monothioglycerol, propyl gallate, sodium sulfite, sodium thioglycolate, or ascorbic acid. The above bulking agent may be mannitol, glycine, lactose, sucrose, trehalose, dextrin, hydroxyethyl starch, picol, or gelatin.

[0132] The cell therapy composition or pharmaceutical preparation of the present invention prepared in this manner may be administered using administration methods commonly used in the art, together with other stem cells used for transplantation and other purposes, or in the form of a mixture with such stem cells. Preferably, it may be directly engrafted or transplanted into the diseased site of a patient requiring treatment, or directly transplanted or injected into the joint cavity, but is not limited thereto. Furthermore, the administration may be performed using both non-surgical methods such as injection or transplantation after incision of the diseased site, as well as surgical methods such as injection or transplantation; however, non-surgical methods using syringes or catheters are more preferable. In addition, in addition to parenteral administration according to conventional methods, such as direct administration to the lesion, transplantation by intravascular injection, which is a general method of cell transplantation, is also possible.

[0133] In addition, the dosage of stem cell-derived chondrospheres is 20 spheroids / mm³ 3 Up to 50 spheroids / mm 3 It may be administered at a spheroid density, preferably at an implantation site of 1 mm 3 22 to 36 spheroids may be administered in a single dose or in multiple doses. However, it should be understood that the actual dosage of the active ingredient should be determined in light of various relevant factors such as the disease to be treated, the severity of the disease, the route of administration, the patient's weight, age, and gender, and therefore, the above dosage does not limit the scope of the invention in any way.

[0134] In one aspect, the present invention relates to a kit for the prevention or treatment of cartilage disease or inflammatory joint disease, comprising a stem cell-derived spheroid and a bioadhesive.

[0135] In the kit for the prevention or treatment of cartilage disease or inflammatory joint disease according to the present invention, the description of the stem cell-derived spheroid is as described above.

[0136] In one embodiment of the present invention, the kit for preventing or treating cartilage disease or inflammatory joint disease is,

[0137] (i) stem cell-derived spheroids and,

[0138] (ii) By including a bioadhesive that can be attached to and fixed to tissue,

[0139] It enables the provision of a composition that can be effectively applied to cartilage defect sites or inflamed joint sites.

[0140] The above spheroid is a three-dimensional structure capable of promoting intercellular interaction and the autosecretion of the extracellular matrix (ECM), and has the advantage of a high survival rate after transplantation and easy induction of differentiation into cartilage tissue.

[0141] The above bioadhesive may be used by being prepared in advance in the form of a gel or solid adhesive and then directly injected or applied to a body part, or by forming a gel or exhibiting adhesive properties in the body environment immediately after or during application, but is not limited thereto.

[0142] In this case, when using a gelling bioadhesive, the spheroid can be stably fixed to the lesion site during the gelling process, and it can be implanted at the graft site without sutures or mechanical fixation.

[0143] In one embodiment of the present invention, the gelling bioadhesive may comprise at least one selected from the group consisting of fibrinogen, thrombin, alginate gel, hyaluronic acid gel, chitosan gel, collagen gel, gelatin gel, polyethylene glycol-based hydrogel, polylactic acid / polyglycolic acid copolymer hydrogel, poly(vinyl alcohol) hydrogel, polyacrylamide gel, oxalaldehyde-based hydrogel, and gelatin-resorcin-formalin adhesive, but is not limited thereto.

[0144] In one embodiment of the present invention, the kit may additionally include components necessary for the cultivation of spheroids.

[0145] In one embodiment of the present invention, the kit may further include a formulation or solvent for suspending a spheroid in a solid or gel state.

[0146] In one embodiment of the present invention, the kit may take the form of a bottle, tub, sachet, envelope, tube, ampoule, etc., and may be formed partially or wholly from plastic, glass, paper, foil, wax, etc. The container may include a cap that is initially part of the container or may be attached to the container by mechanical, adhesive, or other means and may be fitted with a stopper that allows access to the contents by a needle. Additionally, if the spheroid is provided in a solid or gel state, the kit may further include conventional delivery means such as a mixer or syringe. The kit may include an outer package, and the outer package may include instructions for the use of the components.

[0147] In one aspect, the present invention relates to a method for preventing or treating cartilage disease or inflammatory joint disease, comprising the step of administering to an individual a pharmaceutical composition containing the chondrosphere as an active ingredient.

[0148] In one embodiment of the present invention, the pharmaceutical composition may be a composition for co-administration with a bioadhesive, but is not limited thereto.

[0149] In the present invention, the term "individual" refers to a subject requiring a method for the prevention, control, or treatment of a disease, and may be used without limitation and may include humans, dogs, monkeys, cats, rodents, e.g., mice, genetically modified mice, etc. More specifically, it refers to mammals such as humans or non-human primates, mice, rats, dogs, cats, horses, and cattle.

[0150] The pharmaceutical composition of the present invention may be administered in a therapeutically effective amount or a pharmaceutically effective amount.

[0151] In the present invention, the term "therapeutically effective amount" refers to a pharmaceutically acceptable amount of salt of a composition effective for preventing or treating a target disease, and the therapeutically effective amount of the composition of the present invention may vary depending on various factors, such as the method of administration, the target site, the patient's condition, etc. Therefore, when used in humans, the dosage should be determined as an appropriate amount by considering both safety and efficacy. It is also possible to estimate the amount used in humans from the effective amount determined through animal experiments. Such matters to be considered when determining the effective amount are described, for example, in Hardman and Limbird, eds., Goodman and Gilman's The Pharmacological Basis of Therapeutics, 10th ed. (2001), Pergamon Press; and EW Martin ed., Remington's Pharmaceutical Sciences, 18th ed. (1990), Mack Publishing Co.

[0152] In the present invention, the term “pharmacologically effective amount” refers to an amount sufficient to treat a disease with a reasonable benefit / risk ratio applicable to medical treatment and that does not cause side effects. The effective dose level may be determined based on factors including the patient’s health status, type and severity of the disease, drug activity, sensitivity to the drug, method of administration, time of administration, route of administration and elimination rate, duration of treatment, drugs used in combination or concurrently, and other factors well known in the medical field. The composition of the present invention may be administered as an individual therapeutic agent or in combination with other therapeutic agents, may be administered sequentially or simultaneously with conventional therapeutic agents, and may be administered as a single or multiple doses. Taking all of the above factors into consideration, it is important to administer an amount that obtains maximum effect with a minimum amount without side effects, and this can be easily determined by a person skilled in the art.

[0153] The present invention will be explained in more detail below through examples. However, since these examples are merely illustrative of the present invention, the scope of the present invention should not be interpreted as being limited by these examples.

[0154] Example 1: Preparation of Adipose Stem Cell-Derived Chondropheroids with Chondrodifferentiation Potential

[0155] To develop an effective cell therapy for osteoarthritis, adipose stem cells were cultured in PDMS spheroid culture vessels or plastic spheroid culture vessels to produce chondrospheres with cartilage differentiation ability.

[0156] First, human adipose-derived stem cells (hASCs) were isolated from lipoaspirates obtained from three patients (average age: 45, range: 37–49) who underwent liposuction at Dongguk University Ilsan Hospital. Informed consent was obtained from all donors, and all experiments were conducted in compliance with relevant guidelines and regulations and approved by the Institutional Review Board (IRB) of Dongguk University Ilsan Hospital (IRB file number: DUIH 2012-34). hASCs (1 × 10⁶ 6 hASCs (100 mm) were cultured in Alpha MEM medium (Gibco, Grand Island, New York, USA) supplemented with 10% FBS (Gibco) and 1% penicillin-streptomycin (100×) (Welgene, Daegu, Korea) under conditions of 5% CO₂ and 37°C. During the proliferation period, the medium was replaced every 3 days. All cell lines were tested for Mycoplasma contamination. hASCs were confirmed to be mesenchymal stem cells by marker analysis using a Flow cytometer (CytoFLEX, Beckman Coulter, Indianapolis, IN, USA) with CD34 (hematopoietic stem cell and endothelial cell positive), CD45 (leukocyte positive), and CD73, CD90, and CD105 (mesenchymal stem cell positive, respectively).

[0157] Separate cultured adipose stem cells from the dish into single cells: 1 × 10 6 Adjusted to a cell / ml (culture medium) concentration, approximately 1.0 × 10⁻⁶ in a washed and prepared PDMS spheroid culture vessel (StemFIT 3D®H389600H, Microfit Co., Ltd., Korea) for the production of chondrosphere spheroids 6 cells / cm 2 To achieve a cell density of 1 × 10 61 ml of a cell suspension at a concentration of cells / ml was injected, and approximately 0.5 × 10⁻⁶ were placed in a plastic spheroid culture vessel (StemFIT 3D-Plastic® H389600H, Microfit Co., Ltd., Korea). 6 cells / cm 2 or about 1.0 × 10⁻⁶ 6 cells / cm 2 To achieve a cell density of 1 × 10 6 1 ml or 2 ml of a cell suspension at a concentration of cells / ml was injected. The spheroid culture vessels used consisted of 389 concave microwells with a diameter of 600 μm, regardless of the material (PDMS or plastic), and the wells were arranged in a hexagonal array. Five minutes after cell injection, cells settled at the rim of the spheroid culture vessel, excluding the wells, were removed by washing with a buffer solution (PBS). The spheroid culture vessels injected with adipose stem cells were incubated for 72 hours in an incubator maintained at 37°C and 5% CO2 after adding 3 ml of DMEM / F12 medium (Dulbecco's Modified Eagle Medium and Ham's F12 nutrient mixture, 1:1 mixture; Gibco) supplemented with 1% FBS and 1% Antimycotic.

[0158] Chondrospheres prepared by the method described above were observed for their characteristics and size using an optical microscope on days 0, 1, and 3 after cell injection into a culture vessel. Chondrogenic differentiation ability was confirmed on day 3 of cell injection using the Safranin O / Fast Green double staining method, and internal and external cell viability was evaluated on day 3 of cell injection using Live / Dead fluorescence staining.

[0159] As a result, as shown in Figs. 2a to 2d, it was found that spheroids formed well even when using plastic culture vessels compared to the PDMS culture vessels previously used for spheroid production (Fig. 2a). On the third day after cell injection, the average size of spheroids cultured in PDMS culture vessels was 184±10μm, and the average size of spheroids cultured in plastic culture vessels was 167±17μm (1 × 10⁻⁶). 6 ) and 214±16 μm(2 × 10⁻⁶ 6 As shown in ), it was confirmed that the size of the spheroids increased with the number of introduced cells when using plastic culture vessels (Fig. 2b). Safranin O / Fast Green staining results, which allow for the verification of proteoglycan expression levels produced by precursor chondrocytes and chondrocytes, showed a deep red color in all experimental groups, confirming that the adipose stem cell-derived spheroids cultured in the PDMS or plastic spheroid culture vessels were chondrospheres possessing excellent chondrogenic differentiation ability; in particular, in the plastic culture vessel, approximately 1.0 × 10 6 cells / cm 2 Inoculated adipose stem cells at a cell density (2 × 10 6 It was confirmed that the spheroids prepared by culturing and culturing showed significantly improved chondrogenic differentiation ability (Fig. 2c). In addition, Live / Dead fluorescence staining results confirmed that there were almost no dead cells even after 3 days had passed since the preparation of the spheroids (Fig. 2d).

[0160] These results show that 2 × 10⁶ in plastic spheroid culture vessels 6 It was confirmed that cells produced by injecting dog adipose stem cells and culturing them for 3 days under conditions of 37°C and 5% CO2 had a large spheroid shape, excellent chondrogenic differentiation ability, and excellent spheroid stability and spheroid viability.

[0161] Therefore, in subsequent experiments, 2 × 10⁶ in plastic spheroid culture containers 6 Dog adipose stem cells (approx. 1.0 × 10⁶ 6 cells / cm 2 Chondrospheres prepared by injecting cell density were used.

[0162] Example 2: Confirmation of cartilage regeneration effect after chondrosphere implantation and bioadhesive administration in an osteoarthritis animal model

[0163] To confirm the cartilage regeneration effect of the chondrosphere prepared in Example 1 above, an osteoarthritis animal model (rabbit) was prepared, the chondrosphere was implanted into the cartilage damage site, a bioadhesive was administered, and ELISA analysis for inflammatory cytokines, visual observation of the cartilage damage site through a light microscope, and Safranin-O / Fast Green double staining were performed. As a control, only fibrin glue was applied to the cartilage defect site as a bioadhesive without implanting the chondrosphere.

[0164] Osteoarthritis induction in rabbits was performed by removing the ligament connected to the medial meniscus. Eight weeks after the ligament removal, cartilage defect induction was performed by checking the exact defect size in the rabbit patellofemoral groove using a 4 mm diameter punch and scraping out the cartilage using a dental scale. Chondrospheres were then implanted into the cartilage defect site and fixed by applying fibrin glue. For this purpose, Greenplast Q from GC Green Cross, which contains fibrinogen and thrombin separately, was used as the fibrin glue. The fibrinogen solution and thrombin solution were each thawed and placed in 1 mL syringes.

[0165] Specifically, the maximum cartilage defect size that can be induced in the patellofemoral groove of a rabbit (2mm × 2mm × 2mm × 3.14 = 25.12mm) 3 Considering this, the spheroid density was set to 28 spheroids / mm³ to 30 spheroids / mm³ to fill the area, and accordingly, 700 to 750 chondrospheres of Example 1 were implanted into the cartilage defect area.

[0166] Next, a fibrin adhesive was applied to the site where the chondrosphere was implanted. First, 1 to 2 drops of fibrinogen solution were applied, followed by the subsequent application of an equal volume (1:1, v / v) of thrombin solution. A reaction time of approximately 3 minutes was then allowed to form a fibrin gel directly on the cartilage defect site through a reaction immediately after application. After confirming the fixation of the chondrosphere, the patella was restored to its proper position and fixed, and final suturing was performed.

[0167] After 12 weeks following the induction of osteoarthritis and the transplantation of chondrospheres, articular cartilage tissue was harvested, and the International Cartilage Repair Society (ICRS) macroscopic score was performed using macroscopic images of the cartilage tissue. The ICRS macroscopic score was analyzed by assigning scores as defined in Table 1 below, based on the paper (M. Van den Borne, N. Raijmakers, J. Vanlauwe, J. Victor, S. De Jong, J. Bellemans, D. Saris, International Cartilage Repair Society (ICRS) and Oswestry macroscopic cartilage evaluation scores validated for use in Autologous Chondrocyte Implantation (ACI) and microfracture, Osteoarthr. Cartil. 15(12)(2007) 1397-1402.).

[0168] [Table 1]

[0169]

[0170] Subsequently, Safranin-O / Fast Green staining was performed to evaluate GAG ​​expression in the cartilage. Specifically, the recovered cartilage tissue was fixed in 10% formalin for 24 hours, subjected to an acidic decalcification process, prepared into paraffin blocks, and sectioned to a thickness of 8 μm for Safranin-O / Fast Green staining. Safranin-O was stained with a 4% solution for 10 minutes, and Fast Green with a 0.01% solution for 1 hour. The stained tissue slides were examined under a microscope. The Histological Score was calculated by referring to Table 2 below. Surface regularity was evaluated as the ratio of the smooth portion of the repaired cartilage to the total defect area, and cartilage thickness was evaluated as the average value obtained by comparing the thickness of the repaired cartilage with the thickness of the surrounding cartilage.

[0171] [Table 2]

[0172]

[0173] As a result, as shown in Fig. 3, it was confirmed that the levels of inflammation caused by osteoarthritis in the synovial fluid of rabbits with osteoarthritis induced by meniscus resection were reduced in the experimental group administered chondrosphere and fibrin in combination compared to the control group (fibrin alone) (Fig. 3a). In addition, when observing bone regeneration in the cartilage-damaged area following chondrosphere implantation, effective bone regeneration was observed in the experimental group administered chondrosphere and fibrin in combination compared to the control group (Fig. 3b), and the ICRS Macroscopic Score was found to increase significantly (Fig. 3c). Safranin-O / Fast Green staining results of femoral sections also showed that the red area indicating healthy cartilage significantly increased in the experimental group administered chondrosphere and fibrin in combination compared to the control group (Fig. 3d), and the Histological Score was found to decrease significantly (Fig. 3e).

[0174] Through these results, it was confirmed that implanting chondrospheres and applying fibrin to the cartilage damage site significantly increases the bone regeneration effect of adipose stem cell-derived chondrospheres by adhesively fixing the chondrospheres to the cartilage damage site.

[0175] Example 3: Confirmation of the mechanism of action of cartilage regeneration using a chondrosphere adhesive-fixed cell therapy

[0176] To analyze the mechanism of action following the injection of chondrospheres into animal models of osteoarthritis-induced and cartilage defects, immunohistochemical staining using Human Nuclear Antigen (HN) was performed on selected tissues. As a control group, only fibrin was applied to the cartilage-damaged area as a bioadhesive without the implantation of chondrospheres.

[0177] Immunohistochemical staining was performed by freezing cartilage tissue that had undergone acid demineralization into 10 μm thick sections, reacting them with anti-HN antibodies, and detecting them using a secondary antibody labeled with a fluorescent substance.

[0178] As a result, as shown in Figure 4, it was confirmed that Human nuclear antigen (HN) was expressed in the cartilage regeneration induced in the case where chondrospheres were transplanted, unlike in the control group where chondrospheres were not transplanted.

[0179] Through these results, it was confirmed that the cartilage regeneration effect of the chondrosphere adhesive-fixed cell therapy is achieved through the direct engraftment of the transplanted chondrospheres.

[0180] Although embodiments of the present invention have been described in more detail with reference to the attached drawings, the present invention is not necessarily limited to these embodiments and may be modified in various ways within the scope of the technical spirit of the present invention. Accordingly, the embodiments disclosed in the present invention are intended to explain, not limit, the technical spirit of the present invention, and the scope of the technical spirit of the present invention is not limited by these embodiments. Therefore, the embodiments described above should be understood as illustrative in all respects and not restrictive. The scope of protection of the present invention shall be interpreted by the claims below, and all technical spirits within an equivalent scope shall be interpreted as being included within the scope of rights of the present invention.

[0181]

[0182] [National R&D projects that supported this invention]

[0183] [Project ID] 2460000017

[0184] [Assignment No.] 00070271

[0185] [Ministry Name] Ministry of Health and Welfare

[0186] [Project Management (Specialized) Agency Name] Pan-Governmental Regenerative Medicine Technology Development Agency

[0187] [Research Project Name] Inter-Ministerial Regenerative Medicine Technology Development Project

[0188] [Project Title] Development of Chondrosphere-Adhesive Cell Therapy for Osteoarthritis

[0189] [Name of Project Performing Organization] Dongguk University Industry-Academic Cooperation Foundation

[0190] [Research Period] April 1, 2022 ~ December 31, 2024

Claims

1. A step of inoculating stem cells into a plastic 3D cell culture container, A step of forming chondrospheres by culturing stem cells inoculated into a 3D cell culture vessel, and A step comprising separating the formed chondrosphere, Method for manufacturing chondrospheres having excellent cartilage cell differentiation ability.

2. In Paragraph 1, The above stem cells are, A method for producing chondrospheres having excellent cartilage cell differentiation ability, characterized by being adipose-derived stem cells.

3. In Paragraph 1, The above stem cells are, 1.0 × 10 5 cells / cm 2 Up to 5.0 × 10 6 cells / cm 2 A method for producing a chondrosphere having excellent cartilage cell differentiation ability, characterized by being inoculated at a cell density.

4. In Paragraph 1, The above 3D cell culture vessel is, It is composed of 300 to 450 concave microwells with a diameter of 550 μm to 650 μm, and A method for producing a chondrosphere having excellent cartilage cell differentiation ability, characterized in that the wells are arranged in a hexagonal array.

5. In Paragraph 1, The above culture is, A method for producing a chondrosphere having excellent cartilage cell differentiation ability, performed under culture conditions of a temperature of 30℃ to 40℃ and a CO2 concentration of 3% to 7%.

6. In Paragraph 1, The above culture is, A method for producing a chondrosphere having excellent cartilage cell differentiation ability, characterized by being performed for at least 3 days.

7. A chondrosphere having chondrocyte differentiation ability, manufactured by the method of any one of claims 1 to 6.

8. In Paragraph 7, The above chondrosphere is, A chondrosphere having the ability to differentiate into chondrocytes, having a diameter of 150 to 250 μm.

9. A pharmaceutical composition for the prevention or treatment of cartilage disease or inflammatory joint disease for co-administration with a bioadhesive, comprising a stem cell-derived spheroid as an active ingredient.

10. In Paragraph 9, The above stem cells are, A pharmaceutical composition for the prevention or treatment of cartilage disease or inflammatory joint disease for co-administration with a bioadhesive, characterized by being an adipose-derived stem cell.

11. In Paragraph 9, The above spheroid is, A pharmaceutical composition for the prevention or treatment of cartilage disease or inflammatory joint disease for co-administration with a bioadhesive, characterized by being a chondrospheroid having the ability to differentiate into chondrocytes.

12. In Paragraph 9, The above bioadhesive is, A pharmaceutical composition for the prevention or treatment of cartilage disease or inflammatory joint disease for concomitant administration with a bioadhesive, comprising at least one selected from the group consisting of fibrin, fibrinogen, thrombin, fibrin glue, alginate gel, hyaluronic acid gel, chitosan gel, collagen gel, gelatin gel, gelatin-resorcin-formalin adhesive, polysaccharide gel, polyethylene glycol-based hydrogel, polylactic acid / polyglycolic acid copolymer hydrogel, poly(vinyl alcohol) hydrogel, polyacrylamide gel, oxalaldehyde-based hydrogel, cyanoacrylate glue, synthetic acrylate-based adhesive, cellulose-based adhesive, basement membrane matrix, laminin, elastin, proteoglycan, and autoglue.

13. In Paragraph 9, The above cartilage disease is, A pharmaceutical composition for the prevention or treatment of cartilage disease or inflammatory joint disease for co-administration with a bioadhesive, selected from the group consisting of osteoarthritis, chondromalacia patellae, cartilage defect, osteochondral injury, meniscus tear, avascular necrosis, and osteochondritis dissecans.

14. In Paragraph 9, The above-mentioned inflammatory joint disease is, A pharmaceutical composition for the prevention or treatment of cartilage disease or inflammatory joint disease for co-administration with a bioadhesive, selected from the group consisting of ankylosing spondylitis, psoriatic arthritis, rheumatoid arthritis, gouty arthritis, reactive arthritis, and infectious arthritis.

15. A cell therapy product for the prevention or treatment of cartilage disease or inflammatory joint disease for co-administration with a bioadhesive, comprising a stem cell-derived spheroid as an active ingredient.

16. In Paragraph 15, The above stem cells are, A cell therapy agent for the prevention or treatment of cartilage disease or inflammatory joint disease for co-administration with a bioadhesive, characterized by being an adipose-derived stem cell.

17. In Paragraph 15, The above spheroid is, A cell therapy agent for the prevention or treatment of cartilage disease or inflammatory joint disease for co-administration with a bioadhesive, characterized by being a chondrospheroid having the ability to differentiate into chondrocytes.

18. In Paragraph 15, The above spheroid is, 20 spheroids / mm 3 Up to 50 spheroids / mm 3 A cell therapy agent for the prevention or treatment of cartilage disease or inflammatory joint disease for co-administration with a bioadhesive, administered at a spheroid density.

19. A kit for the prevention or treatment of cartilage disease or inflammatory joint disease, comprising stem cell-derived spheroids and a bioadhesive.

20. In Paragraph 19, The above bioadhesive is, A kit for the prevention or treatment of cartilage disease or inflammatory joint disease, characterized by being a gelling bioadhesive.

21. In Paragraph 20, The above-mentioned gelling bioadhesive is, A kit for the prevention or treatment of cartilage disease or inflammatory joint disease, comprising at least one selected from the group consisting of fibrinogen, thrombin, alginate gel, hyaluronic acid gel, chitosan gel, collagen gel, gelatin gel, polyethylene glycol-based hydrogel, polylactic acid / polyglycolic acid copolymer hydrogel, poly(vinyl alcohol) hydrogel, polyacrylamide gel, oxalaldehyde-based hydrogel, and gelatin-resorcin-formalin adhesive.

22. A method for preventing or treating cartilage disease or inflammatory joint disease, comprising the step of administering the pharmaceutical composition of claim 9 to an individual.

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