阅读说明：本技术 将含有源自干细胞的外泌体的培养液作为有效成分包含的关节炎预防或治疗用组合物 (Composition for preventing or treating arthritis, comprising culture medium containing exosome derived from stem cell as active ingredient ) 是由 姜明姬 崔恩京 徐妲韵 于 2018-10-29 设计创作，主要内容包括：本发明涉及含有源自干细胞的外泌体的培养液作为有效成分的关节炎预防或治疗用组合物,具体地本发明涉及一种在干细胞培养液中接种TNF-α、在1-8％的氧浓度条件下培养获得的含有源自干细胞的外泌体的培养液作为有效成分包含的关节炎预防或者治疗用药物组合物。根据本发明的含有源自干细胞的外泌体的培养液包含大量含软骨再生促进因子等功能性有效成分的外泌体,从而确认与现有的用于治疗关节炎的干细胞移植方法及用通常的干细胞培养条件下获得的培养液处理相比,具有显著优异的关节炎治疗效果。从而根据本发明的含有源自干细胞的外泌体的培养液具有能够有效地用作关节炎治疗的新型治疗剂的效果。(The present invention relates to a composition for preventing or treating arthritis, which contains a culture solution containing exosomes derived from stem cells as an active ingredient, and more particularly, to a pharmaceutical composition for preventing or treating arthritis, which contains a culture solution containing exosomes derived from stem cells as an active ingredient, obtained by inoculating TNF- α in a culture solution of stem cells and culturing the culture solution under an oxygen concentration condition of 1 to 8%. The culture solution containing exosomes derived from stem cells according to the present invention contains a large amount of exosomes containing functional active ingredients such as cartilage regeneration-promoting factors, and thus has been confirmed to have a significantly excellent therapeutic effect on arthritis as compared with conventional stem cell transplantation methods for treating arthritis and treatment with culture solutions obtained under normal stem cell culture conditions. The culture solution containing the stem cell-derived exosomes according to the present invention thus has the effect of being effectively used as a novel therapeutic agent for the treatment of arthritis.)

1. A pharmaceutical composition for preventing or treating arthritis, which comprises a culture medium containing exosomes derived from stem cells as an active ingredient.

2. The pharmaceutical composition for the prevention or treatment of arthritis according to claim 1, wherein said culture solution containing exosomes derived from stem cells is obtained by seeding TNF- α in a stem cell culture solution, cultured under an oxygen concentration condition of 1-8%.

3. The pharmaceutical composition for the prevention or treatment of arthritis according to claim 2, wherein TNF- α is treated at a concentration of 5 to 500ng/ml and said incubation is performed for 12 to 72 hours.

4. The pharmaceutical composition for the prevention or treatment of arthritis according to claim 1, wherein said stem cells are selected from the group consisting of neural stem cells, oligodendrocyte precursor cells, adipose stem cells, amniotic membrane stem cells, amniotic fluid stem cells, placental stem cells, umbilical cord blood stem cells and bone marrow stem cells.

5. The pharmaceutical composition for the prophylaxis or treatment of arthritis according to claim 4, wherein said neural stem cells are immortalized neural stem cell CBNU-NSC cell line (accession number: KCTC12635BP), said amniotic fluid stem cells are immortalized amniotic fluid stem cell CBNU-AFSC cell line (accession number: KCTC12634BP), and said oligodendrocyte precursor cells are immortalized oligodendrocyte precursor cell CBNU-NSC.

6. The pharmaceutical composition for the prevention or treatment of arthritis according to claim 1, wherein the cell number of said stem cells is 1x10⁴～1x10⁶。

7. The pharmaceutical composition for the prevention or treatment of arthritis according to claim 1, wherein said arthritis is selected from the group consisting of osteoarthritis, rheumatoid arthritis, fibromyalgia, gout, and lupus.

Technical Field

The present invention relates to a composition for preventing or treating arthritis, which contains a culture solution containing exosomes secreted from stem cells as an active ingredient.

Background

Arthritis is one of the most common diseases that induce pain, stiffness, and motor insufficiency. Rheumatoid Arthritis (RA) is an inflammatory disease caused by autoimmune reaction to type II collagen (collagen II), with joint edema and pain. In contrast, Osteoarthritis (OA) is characterized by degenerative changes in the joints, inducing pain and joint deformity due to destruction of chondrocytes and tissues. Especially, as osteoarthritis ages and undergoes apoptosis, the decrease in the number and function of chondrocytes (chondrocytes) accelerates the deterioration of the composition and structure (architecture) of normal extracellular matrix (ECM), such as Peptidoglycan (PG), collagen type II (collagen II), and proteoglycan (aggrecan). In addition, analgesic and anti-inflammatory agents for relieving pain and inflammation have been mainly prescribed in the treatment of rheumatoid arthritis so far, and a radical therapeutic agent capable of fundamentally solving immune response has not been developed yet. And therapeutic agents that can arrest the progression and rehabilitate osteoarthritis have not been disclosed, relying only on temporary pain relief or surgical treatment.

In view of this, a novel therapeutic strategy capable of protecting and regenerating joints using Mesenchymal Stem Cells (MSCs) is recently attracting great attention. In particular, it has been reported that mesenchymal stem cells can be obtained from various tissues such as bone marrow, fat, umbilical cord blood, etc., have excellent self-division and proliferation abilities, and can be differentiated into cartilage tissues in vitro and in vivo, thereby being capable of replacing damaged joints. In fact, it has recently been demonstrated that Umbilical Cord Blood Stem Cells (UCBSC) and Adipose Stem Cells (ASC) improve joint damage through pain relief of joints and regeneration of chondrocytes in osteoarthritis animal models and clinical trials without side effects. However, in comparison with autologous stem cells (autologous stem cells), the problem of accumulation of exudate in the joint cavity often occurs when allogeneic stem cells (allogeneic stem cells) or xenogeneic stem cells (xenogeneic stem cells) are injected at a high dose, and technical studies for restoring cartilage damage by injecting only Culture Medium (CM) instead of directly transplanting stem cells are being conducted as a method for solving the problem. Stem cells are known to have an advantage of releasing various secreted factors (secretor factors) such as cytokines (cytokines), chemokines (chemokines), Growth Factors (GF), Neurotrophic Factors (NF) and exerting a paracrine (paracrine effect) effect of promoting the recovery of damaged tissues, and in particular, thrombospondin 2 (TSP 2) released from mesenchymal stem cells such as umbilical cord blood stem cells, adipose stem cells, Bone Marrow Stem Cells (BMSCs) is known to differentiate stem cells into chondrocytes and promote the proliferation of chondrocytes, thereby recovering osteoarthritis. However, the concentration of functional components such as cartilage regeneration-promoting factors in the stem cell culture solution is too low, and the administration of the stem cell culture solution alone has a problem that the actual arthritis therapeutic effect is lower than that of the stem cells directly transplanted. Accordingly, the present inventors have focused on secretion of effective functional components in the form of exosomes (exosomes) in lipid layers when they are secreted from stem cells, and established new culture conditions capable of increasing the secretion of exosomes by a culture method using normal (normal) stem cells, and have established new culture conditions for this purpose, in which Amniotic Fluid Stem Cells (AFSC), Amniotic Membrane Stem Cells (AMSC), Placental Stem Cells (PSC), Umbilical Cord Blood Stem Cells (UCBSC), Neural Stem Cells (NSC), oligodendrocyte precursor cells (OPC precursor cells, bmc), adipose stem cells (adipose tissue stem cells, ASC), and bone marrow stem cells (asbone marrow necrosis factor cells, TNF-tumor necrosis factor-containing cells (TNF-tumor necrosis factor-enriched sc) are cultured together under a culture condition containing TNF-enriched culture medium (msc-TNF-tumor cell) in the present invention conditioned medium, ERCM), and the excellent therapeutic effect on arthritis of the culture solution was confirmed, thereby completing the present invention.

Disclosure of Invention

Object of the Invention

An object of the present invention is to provide a pharmaceutical composition for preventing or treating arthritis, which contains a culture solution of exosomes derived from stem cells as an active ingredient.

Technical scheme

In order to achieve the above objects of the present invention, the present invention provides a pharmaceutical composition for preventing or treating arthritis, comprising a culture solution containing exosomes derived from stem cells as an active ingredient

Furthermore, according to an embodiment of the present invention, the culture solution containing exosomes derived from stem cells is cultured under an oxygen concentration condition of 1-8% inoculated with TNF- α.

Furthermore, according to an embodiment of the present invention, TNF- α is treated at a concentration of 5 to 500ng/ml, and the culturing is performed for 12 to 72 hours.

Furthermore, according to an embodiment of the present invention, the stem cell is selected from the group consisting of a neural stem cell, an oligodendrocyte precursor cell, an adipose stem cell, an amniotic membrane stem cell, an amniotic fluid stem cell, a placental stem cell, a cord blood stem cell and a bone marrow stem cell.

Furthermore, according to an embodiment of the present invention, the neural stem cell is an immortalized CBNU-NSC cell line (accession number: KCTC12635BP) of neural stem cell, the amniotic fluid stem cell is an immortalized CBNU-AFSC cell line (accession number: KCTC12634BP) of amniotic fluid stem cell, and the oligodendrocyte precursor cell is an immortalized CBNU-NSC. olig2 cell line (accession number: KCTC12636BP) of oligodendrocyte precursor cell.

In addition, according to an embodiment of the present invention, the number of the neural stem cells may be 1 × 10⁴～1x10⁶。

Furthermore, according to an embodiment of the present invention, the Arthritis is selected from the group consisting of Osteoarthritis (Osteoarthritis), Rheumatoid Arthritis (rhematoid Arthritis), Fibromyalgia (Fibromyalgia), gout (gout), and lupus (lupus).

Effects of the invention

The present invention relates to a composition for preventing or treating arthritis, which contains a culture solution containing exosomes derived from stem cells as an active ingredient, and more particularly, to a pharmaceutical composition for preventing or treating arthritis, which contains a culture solution containing exosomes derived from stem cells as an active ingredient, obtained by inoculating TNF- α in a culture solution of stem cells and culturing the culture solution under an oxygen concentration condition of 1 to 8%. The culture solution containing exosomes derived from stem cells according to the present invention contains a large amount of exosomes containing functional active ingredients such as cartilage regeneration-promoting factors, and thus has been confirmed to have a significantly excellent therapeutic effect on arthritis as compared with conventional stem cell transplantation methods for treating arthritis and treatment with culture solutions obtained under normal stem cell culture conditions. The culture solution containing the stem cell-derived exosomes according to the present invention thus has the effect of being effectively used as a novel therapeutic agent for the treatment of arthritis.

Drawings

FIG. 1 is a photograph of X-ray (X-ray) analysis of a group induced with osteoarthritis, a group treated with a culture solution rich in exosomes derived from stem cells of the present invention, and a group treated with a control stem cell culture solution; white arrows indicate medial femoral condylar osteophytes; white arrows indicate femoral or tibial deformity; the white arrow indicates that the tibial spur (spine) becomes sharp.

Fig. 2 is a photograph for visually observing the degree of osteoarthritis symptoms in the group induced osteoarthritis, the group treated with the culture solution rich in exosomes derived from stem cells of the present invention, and the group treated with the control stem cell culture solution. In fig. 3, black arrows indicate erosion and ulcer; white arrows indicate meniscal adhesions.

FIG. 3 is a result of observing a group induced osteoarthritis, a group treated with a culture solution rich in exosomes derived from stem cells of the present invention, and a group treated with a control stem cell culture solution with a microscope, and white arrows indicate a chondrocyte layer stained with hematoxylin (hematoxylin); black arrows indicate the Peptidoglycan (PG) layer stained red by safranin o (safranin o).

Detailed Description

In the course of research to use stem cells as a material for arthritis therapeutic agents more effectively, the present inventors have confirmed that a culture solution containing exosomes derived from stem cells, which is obtained by increasing the secretion of exosomes from stem cells, can effectively prevent or treat arthritis as a method for enhancing the availability and potency of stem cell-derived active ingredients.

Accordingly, the present invention is characterized by providing a pharmaceutical composition for preventing or treating arthritis, which comprises a culture medium containing a stem cell-derived extracellular medium as an active ingredient.

The method capable of increasing secretion of exosomes from stem cells according to the present invention may include the steps of seeding TNF- α in a stem cell culture solution, culturing under a low oxygen concentration condition; preferably, the method comprises the steps of treating TNF-alpha at a concentration of 5-500ng/mL and culturing the treated TNF-alpha for 12-72 hours under an oxygen concentration of 1-8%. More preferably, the method comprises the step of inoculating TNF-alpha in a stem cell culture solution at a concentration of 50ng/mL and culturing the solution under an oxygen concentration of 5% for 24 hours.

In the present invention, the term "stem cell" refers to a cell that has not been differentiated into a specific cell, and has the ability to differentiate into all kinds of cells constituting the body, such as nerve, blood, cartilage, and the like, if necessary.

In the present invention, the type of the stem cell capable of secreting exosome may be, but is not limited to, neural stem cell, oligodendrocyte precursor cell, adipose stem cell, amniotic membrane stem cell, amniotic fluid stem cell, placental stem cell, umbilical cord blood stem cell or bone marrow stem cell.

According to an embodiment of the present invention, the neural stem cell uses an immortalized neural stem cell CBNU-NSC cell line (accession number: KCTC12635BP), the amniotic fluid stem cell uses an immortalized amniotic fluid stem cell CBNU-AFSC cell line (accession number: KCTC12634BP), and the oligodendrocyte precursor cell uses an immortalized oligodendrocyte CBNU-nscc. olig2 cell line (accession number: KCTC12636 BP).

This is because the primary culture of amniotic fluid stem cells and neural stem cells (neural stem cells, oligodendrocyte precursor cells) is vulnerable to a hypoxic environment and thus has a problem of easy apoptosis, and in order for the stem cells to secrete a large amount of exosomes trapping functional components, it is necessary to culture the stem cells under a hypoxic condition.

The inventor of the present invention obtained a deposit number from a warehouse-popping mechanism before the filing date of the present application, for these immortalized cell lines, the inventor of the present invention obtained patent rights for patent application, and the production content of each immortalized cell line can refer to the following granted patent documents.

In addition, "exosomes" are small-sized (30-150nm) vesicles containing delicate RNA and protein-transporting substances, and are membrane-structured vesicles secreted from a variety of cell types. The exact molecular mechanisms and functions of secretion, absorption and composition of exosomes are not yet fully studied, but are known to function to bind membrane constituents of other cells and tissues, and to transport proteins and RNA.

It is thus understood that the present inventors have studied conditions for grafting liposomes composed of lipids to active ingredients, which are more effective than conventional techniques for secreting a large amount of exosomes from stem cells, in order to improve the in vivo absorption rate of stem cells or active ingredients contained in a stem cell culture solution, that is, conditions for inoculating TNF- α into a stem cell culture solution and culturing at an oxygen concentration of 1 to 8%, and that such culture conditions can solve problems such as an additional treatment process by a liposome grafting method, contamination of foreign substances, and formulation of culture solution components at the time of trapping liposomes.

Thus, when TNF-alpha is inoculated into a stem cell culture solution and the stem cells are cultured under an oxygen concentration condition of 1 to 8%, secretion of exosomes from the stem cells can be increased, and a culture solution rich in exosomes can be produced, and exosomes can be produced in large quantities from the stem cells.

In particular, the present inventors have found that TNF- α effectively promotes secretion of exosomes from many substances in the process of studying a promoter for promoting secretion of exosomes from stem cells, and have confirmed that an appropriate amount of TNF- α capable of maximally secreting exosomes is treated at a concentration of 5 to 500 ng/mL.

When the inoculation is carried out beyond the above range, stem cells may be damaged and the cells may not function normally; on the contrary, when the amount is less than the above range, there is a problem that the secretion amount of the exosome is insufficient.

Stem cells, like macrophages (macrophages), are dependent on TNF- α movement and activation. Compared with activated macrophages that secrete reactive oxygen radicals (active oxygen chemicals) to feed microbes and tumors that flow into the body, activated stem cells secrete Growth Factors (GF) and nerve growth factors (NF) to protect and regenerate tissues.

This GF/NF protein was found to be secreted by exosome encapsidation. Therefore, when TNF-alpha is inoculated in the stem cell culture process, the secretion of a large amount of exosomes containing GF/NF can be promoted, and the exosomes containing a large amount of GF/NF can be obtained.

Further, as a requirement for inducing a large amount of secretion of exosomes having an arthritis therapeutic effect from stem cells, it was confirmed that culture at an oxygen concentration significantly lower than the normal concentration (about 21% oxygen) is necessary, and culture at a low oxygen concentration of 1 to 8% for 12 to 72 hours after TNF- α inoculation is preferable. When the oxygen concentration condition is less than 1%, problems may occur to the normal politics and growth of cells; when the content exceeds 8%, secretion of exosome is insufficient. Thus, the culture is preferably carried out under a low oxygen condition of 1 to 8%, more preferably at an oxygen concentration of 5%.

In addition, according to the method of the present invention, any culture medium can be used as long as it is a medium for culturing stem cells, but in one embodiment of the present invention, Dulbecco's Modified Eagle's Medium (DMEM) is used.

The cell number of the TNF-alpha-seeded stem cell at a concentration of 5 to 500ng/mL is preferably 1X10⁴-1x10⁶。

As described above, when stem cells are cultured under the conditions of the present invention, exosomes containing physiologically active substances such as genes, proteins, growth factors, and the like, which are secreted by stem cells during the growth of stem cells, can be promoted and enhanced, and the secretion and production of exosomes can be significantly increased as compared with a method for obtaining a general stem cell culture solution (i.e., a method in which culturing is not performed under TNF- α and hypoxic conditions).

In addition, it was confirmed that the stem cell-derived exosomes obtained by the method of the present invention had an arthritis preventing or treating effect, and according to an embodiment of the present invention, a culture solution rich in stem cell-derived exosomes of the present invention and a culture solution containing stem cell-derived exosomes obtained by culturing under 21% of specimens (normal culture solution) were separately administered into joint cavities, with a rabbit animal model inducing osteoarthritis by anterior cruciate ligament resection, and the degree of improvement of arthritis symptoms was analyzed.

As a result, it was confirmed that the group enriched in culture fluid repulsive force derived from exosomes of stem cells of the present invention exhibited significantly superior arthritis-improving and therapeutic effects in different types of stem cells, could inhibit damage to the joint surface and the disappearance of chondrocytes, prevent joint deformation such as meniscus adhesion, femoral and tibial deformation, and osteophyte formation, and had the effect of regenerating damaged cartilage, compared to the normal culture fluid-treated group as the control group.

In addition, the inhibition of inflammatory cytokines that cause the induction of arthritis and the worsening of symptoms was significantly superior to the group treated with the culture solution containing exosomes derived from stem cells according to the present invention compared to the group treated with the normal culture solution used as the control group (see table 5).

Accordingly, the inventors of the present invention confirmed that the culture solution containing exosomes derived from stem cells according to the present invention can prevent or treat arthritis with more excellent effects than the existing stem cells themselves or the culture solution containing exosomes obtained under the ordinary stem cell culture conditions (culture under 21% oxygen conditions).

Accordingly, the present invention can provide a pharmaceutical composition for the prevention or treatment of Arthritis, which may be Osteoarthritis (Osteoarthritis), Rheumatoid Arthritis (rhematoid Arthritis), Fibromyalgia (Fibromyalgia), gout (gout), or lupus (lupus), comprising a culture solution of exosomes derived from stem cells as an effective ingredient, but is not limited thereto.

In addition, the pharmaceutical composition of the present invention may further comprise an appropriate carrier, excipient or diluent according to a general method. As the carrier, excipient and diluent that can be contained in the pharmaceutical composition of the present invention, lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, amorphous cellulose, polyvinylpyrrolidone, methylparaben, propylparaben, talc, magnesium stearate, mineral oil and the like can be used, but not limited thereto.

The pharmaceutical composition of the present invention can be prepared into oral dosage forms such as powder, granule, tablet, capsule, suspension, emulsion, syrup, and aerosol, external preparations, suppositories, and sterile injectable solutions by a conventional method. Specifically, the formulation can be prepared by using a diluent or excipient such as a generally used filler, extender, binder, wetting agent, disintegrant, or surfactant.

Solid preparations for oral administration include tablets, pills, powders, granules, capsules and the like, and such solid preparations can be prepared by mixing the above-mentioned pharmaceutical composition of the present invention with one or more excipients, for example, starch, calcium carbonate (calcium carbonate), sucrose (sucrose), lactose (lactose), gelatin and the like. Besides simple excipients, lubricants such as magnesium stearate and talc may be used.

Liquid preparations for oral administration include suspensions, solutions for internal use, emulsions, syrups and the like, and may contain various excipients such as wetting agents, sweeteners, aromatics, preservatives and the like, in addition to the simple diluents usually used, such as water and liquid paraffin.

Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, freeze-dried preparations and suppositories. As the nonaqueous solvent or suspension, propylene glycol (propylene glycol), polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, or the like can be used. Synthetic fatty acid esters (witepsol), polyethylene glycol, tween (tween)61, cocoa butter, glyceryl laurate, glycerogelatin, and the like can be used as a base for suppositories. Further, as the parenteral preparation, an injection such as ampoule for injection, which can be prepared by mixing with an injection solution immediately before use, an injection such as infusion bag, or a spray such as aerosol, and the like are preferable. In addition, the infusion bag may be made of polyvinyl chloride or polyethylene.

The compositions of the present invention may be administered to an individual by a variety of routes. All modes of administration are envisioned, e.g., administration may be by oral, rectal or intravenous, intramuscular, subcutaneous, endometrial or intra-articular injection, preferably directly to the site of the lesion in the individual in need of treatment.

The pharmaceutical composition of the present invention may be variously modified depending on various factors including the activity of the specific active ingredient used, age, body weight, general health, sex, diet, administration time, administration route, discharge rate, drug compatibility, and the severity of the specific disease to be prevented or treated, and the dosage of the pharmaceutical composition may vary depending on the state, body weight, disease degree, drug form, administration route and time of the patient, but may be appropriately selected by those skilled in the art, and may be administered at 0.0001 to 50mg/kg or 0.001 to 50mg/kg per day. Administration may be once a day or divided into several times. The above-mentioned dosage does not limit the scope of the present invention in any way. The pharmaceutical composition according to the present invention may be formulated as pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions.

Detailed Description

The present invention will be described in further detail below with reference to examples. These examples are intended to illustrate the present invention more specifically, and the scope of the present invention is not limited by these examples.

Preparation example: stem cells

In the following examples, the following cells were used as stem cells in order to obtain a stem cell culture solution rich in exosomes as active ingredients for confirming the effectiveness of arthritis treatment.

First, immortalized Amniotic Fluid Stem Cells (AFSC), immortalized Neural Stem Cells (NSC), and immortalized Oligodendrocyte Precursor Cells (OPC) are cell lines established by the present inventors as immortalized cell lines, each of which has been deposited from a microorganism depository, and immortalized amniotic fluid stem cell CBNU-NSC cell lines (deposition number: KCTC12635BP, korean patent publication No. 10-1719274), immortalized amniotic fluid stem cell CBNU-AFSC cell lines (deposition number: KCTC12634BP, korean patent publication No. 10-1719274), and immortalized oligodendrocyte precursor cell CBNU-NSC. olig2 cell lines (deposition number: KCTC12636BP, korean patent publication No. 10-1740357) are used, respectively, and the methods for producing these immortalized cell lines can refer to the contents of patent authorizations of each cell line. In the examples and experimental examples of the present invention, the amniotic fluid stem cells were analyzed using an immortalized amniotic fluid stem cell CBNU-AFSC cell line, the neural stem cells were using an immortalized neural stem cell CBNU-NSC cell line, and the oligodendrocyte precursor cells were using an immortalized oligodendrocyte precursor cell CBNU-NSC. In addition, amnion stem cells (AMSC), Placental Stem Cells (PSC) and Umbilical Cord Blood Stem Cells (UCBSC) are collected and used separately from stem cells that the parturient agrees to take during normal production; adipose-derived stem cells (ASC) and Bone Marrow Stem Cells (BMSC) were collected by abdominal liposuction and bone marrow aspiration, respectively, in normal women aged 53 and 50 years, and examined and approved by the Institutional Review Board (IRB), respectively, after obtaining his/her own consent.

< example 1>

1-1 Stem cell culture and Ex-corpore-enriched Medium

The prepared stem cells (1X 10)⁶cells/mL) were inoculated in Dulbecco's Modified Eagle's Medium (DMEM) in 5% carbon dioxide (CO)₂) The cells were cultured at 37 ℃ for 24 hours in a feed medium. Thereafter, in order to obtain an exosome-rich culture solution (ERCM) from stem cells, TNF- α was added to the above culture medium at a concentration of 50ng/mL, the oxygen concentration was reduced to 5%, and after culturing for 24 hours, the culture solution was collected and filtered with a filter, and then concentrated 10-fold at low temperature, to obtain a culture solution rich in exosomes derived from stem cells according to the present invention.

As a comparative example, the procedure of example 1 was conducted except for TNF-. alpha.treatment (50ng/mL) and oxygen (O) treatment in the course of obtaining an exosome-rich culture solution from stem cells, respectively₂) A normal culture solution (CM) of stem cells was obtained in the same manner as in example 1 except that the concentration was maintained at 21%, and the normal culture solution (CM) was used as a comparative example (control group) in a normal method.

1-2 analysis of effective secretion factor content in culture solution

After the exosomes were disrupted by sonication for 5 minutes in the stem cell culture solution, the contents of the major functional secretion factors, brain-derived neurotrophic factor (BDNF), Nerve Growth Factor (NGF), platelet-derived growth factor (PDGF), transforming growth factor- β (TGF- β), and Vascular Endothelial Growth Factor (VEGF), were analyzed using a protein liquid chromatograph (protein liquid chromatograph) equipped with a sephadex gel filtration column (sephadex) column. The analysis result confirms that the culture solution of the amniotic fluid stem cells contains a large amount of HGF, and also contains a considerable amount of BDNF, TGF and VEGF. Amniotic stem cells also secrete high amounts of HGF and considerable amounts of VEGF, placental stem cells secrete relatively small amounts of protein components in addition to HGF; in contrast, cord blood stem cells secrete high amounts of HGF and VEGF. Interestingly, neural stem cells and oligodendrocyte precursor cells secrete a certain amount of TGF in addition to BDNF, NGF and PDGF, and oligodendrocyte precursor cells secrete a considerable amount of HGF. In comparison, it was confirmed that bone marrow stem cells secreted moderate amounts of HGF and PDGF.

In addition, it was confirmed that all stem cells in the exosome-rich culture solution of the present invention obtained by treating stem cells with TNF- α and hypoxia contained the potent factor at a 2.5 to 3.5-fold higher content than that in the normal culture solution (control group), and it was found that the cells showed more excellent efficacy for the treatment of various diseases (see table 1 below).

[ TABLE 1 ]

Content of effective components in culture solution

< Experimental example 1>

Analysis of arthritis treatment Effect of the exosome culture solution rich in stem cells of the present invention

1-1 raising of rabbits for laboratory animals

Male New Zealand white rabbits (NZW) 8 months (3.5kg) after birth were used after 2 weeks of laboratory acclimation. Each rabbit was housed in a cage with 1 test animal, and the animals were raised in a laboratory at an environment adjusted to 23. + -. 2 ℃ with a relative humidity of 55. + -. 10%, a ventilation frequency of 12 times/hour, an illumination period of 12 hours (07:00-19:00), and a light level of 150-. Free feed intake of small particle type (pellet) solid feed Purina Rabbit supplied for experimental animalsAnd sterilized purified water, and the experiment was carried out in accordance with the Standard Operating Procedures (SOP) of the institute under the approval of the Institutional Animal experimental ethics Committee (IACUC) of the research support center for laboratory animals in north university.

1-2 by anterior cruciate ligament excision (ACLT) Induction of osteoarthritis and administration of exosome culture fluid rich in stem cells according to the invention

The rabbits bred in the above 1-1 were administered with intramuscular injection of ketamine (ketamine)50mg/kg and xylazine (xylazine)5mg/kg for general anesthesia, and after removing the hairs around the right knee, the skin was sterilized with povidone-iodine (Betadine) and 70% ethanol, and the operation was started under aseptic conditions.

After cutting the skin, incising (medial approach) the fascia and joint capsule, the patella (patella) is flexed as shown below to expose the anterior cruciate ligament. The medial parenchymal portion of the anterior cruciate ligament and the Medial Meniscal Tibial Ligament (MMTL) and the Lateral Meniscal Tibial Ligament (LMTL) are cut with a surgical knife and then sutured.

During a period of 3 days after surgery, the antibiotic Foxolin (manufactured by Korea's Sanyo pharmaceutical Co., Ltd., 10mg/kg) and the analgesic Maritrol (manufactured by Korea's first pharmaceutical Co., Ltd., 3mg/kg) were injected intramuscularly 2 times a day, after which the prescription Foxolin was continued once a day for 3 days. After 8 weeks of arthritis induction surgery, the exosome culture solution (ERCM) rich in stem cells derived from the above example 1 and the control normal culture solution (CM) were administered into the joint cavity in an amount of 0.1ml per rabbit at 2-day intervals every week for 8 weeks.

In the upper diagram, F: femur (femur), T: tibia (tibia), MM: medial menisci (media meniscus), ACL: anterior cruciate ligament (anterior), MMTL: medial meniscal tibial ligament (medial meniscotinial ligament), LMTL: : the lateral meniscal tibial ligament (lateral meniscotinial ligament).

1-3 radiologic evaluation of therapeutic efficacy

An osteoarthritis index (osteoarthritic diseases scales) was analyzed by X-ray (X-ray) photography according to Modified Kellgren-Lawrence scale (KL score) during eight weeks of osteoarthritis induction and after 8 weeks of administration of exosome culture fluid (ERCM) rich in stem cells and control culture fluid (CM). The KL score as an analysis index was evaluated by the sum (0-15 points) of 3 indexes of medial femoral condylar osteophytes (0-5 points), femoral or tibial deformities (0-5 points), and tibial spines (0-5 points).

After analysis, when a rabbit was used as an animal model to induce osteoarthritis by cutting the anterior cruciate ligament and medial and lateral meniscal tibial ligaments for 8 weeks, it was confirmed that the abnormal symptoms (lesions) of medial femoral condylar osteophyte, medial tibial deformity, and tibial bone tip were 10.1 out of 15 out of the total points, and severe arthritis was induced.

When a control group normal culture solution (CM, 0.1mL per rabbit) obtained by culturing stem cells at an oxygen concentration of 21% was administered into the articular cavity of the above-described osteoarthritis-induced rabbit, and continued for 8 weeks at intervals of 2 days, excellent therapeutic efficacy was shown in the order of neural stem cells (lesion mean 6.0 points) ≥ oligodendrocyte precursor cells (6.1 points) > adipose-derived stem cells (6.4 points) > amniotic fluid stem cells (6.8 points) > amniotic membrane stem cells (7.1 points) ≥ umbilical cord blood stem cells (7.3 points) > placental stem cells (8.5 points) ≥ bone marrow stem cells (8.5 points) in a radioactive ray analysis.

In addition, when the exosome-rich culture solution (ERCM) of the present invention obtained by adding TNF-alpha (50ng/mL) to a stem cell culture medium and culturing the cells at 5% hypoxia concentration was administered, all stem cells showed more excellent therapeutic efficacy than the administration of the control culture solution, and the therapeutic efficacy was shown in the order of neural stem cells (lesion mean 4.8 min) ≥ oligodendrocyte precursor cells (5.0 min) ≥ adipose stem cells (5.2 min) ≥ amniotic fluid stem cells (5.4 min) > amniotic membrane stem cells (5.7 min) > umbilical cord blood stem cells (6.2 min) > placental stem cells (6.8 min) ≥ bone marrow stem cells (6.9 min).

[ TABLE 2 ]

Results of radiology observations and evaluations

Significant difference compared to osteoarthritis (ACLT) induced group (P < 0.05); # was significantly different from normal medium (P <0.05).

1-4 visual evaluation of therapeutic efficacy

After the 8-week administration period of exosome culture solution (ERCM) and control culture solution (CM) rich in stem cells was completed, the animals were sacrificed, the experimental part of the knee joint was exposed by the medial joint arrival method, the soft tissue around the patella was removed without damaging the cartilage, and the upper part of the condyle of the femur including the experimental part was extracted. The edema, laceration, formation of bony spine, and synovial pachynsis of knee joint were visually observed, and oederick scoring (O' driscoring) analysis was performed to observe and photograph the change of cartilage defect site, the surface state of cartilage defect site, and the continuity of critical site between defect critical site and new tissue, and the treatment efficacy was evaluated by the sum of 4 indices (score 0-20) of surface roughness of surface (score 0-5), meniscus adhesion (score 0-5), hypertrophy of joint (score 0-5), and osteophyte (score 0-5).

After 8 weeks from the induction of osteoarthritis, the rabbits were dissected and subjected to a visual examination of the arthritic site in accordance with the above method, and as shown in table 3 and fig. 2, it was confirmed that the abnormal symptoms (lesions) of surface roughness, meniscus adhesion, joint hypertrophy and osteophyte formation were 18.1 points out of 20 points in total, and severe damage was confirmed. When a normal culture solution used as a control group was administered into the articular cavity of the above osteoarthritis-inducing rabbit, the therapeutic efficacy was visually analyzed in the order of neural stem cells (lesion mean 13.4 points) > oligodendrocyte precursor cells (13.8 points) > amniotic fluid stem cells (14.3 points) > adipose stem cells (14.7 points) > amniotic membrane stem cells (15.4 points) > umbilical cord blood stem cells (15.8 points) > placental stem cells (16.3 points) — bone marrow stem cells (16.3 points).

On the other hand, when the exosome culture solution rich in stem cells of the present invention was administered, all stem cells had more excellent therapeutic efficacy than the group administered as a normal culture solution used as a control group, and the degree of pathological symptoms was significantly improved in the order of neural stem cells (mean lesion 9.9 points) ≥ oligodendrocyte precursor cells (10.0 points) > adipose stem cells (10.7 points) > amniotic fluid stem cells (11.2 points) > amniotic membrane stem cells (12.6 points) > umbilical cord blood stem cells (13.1 points) > placental stem cells (13.4 points) ≥ bone marrow stem cells (13.5 points).

[ TABLE 3 ]

Evaluation results were observed with naked eyes

Significant differences compared to osteoarthritis (ACLT) induced group (P <0.05) # significant differences compared to normal broth group (P <0.05).

1-5 evaluation of the efficacy of the treatment by microscopic examination

The rabbit joint tissue obtained in the above example was fixed in 10% neutral formalin, and after 60 days of decalcification with EDTA, paraffin sections were prepared. Thereafter, the cells were stained with hematoxylin (hematoxylin) and eosin (H & E) and imaged, and the cartilage area was quantitatively analyzed by using microscopic analysis software ImageJ 5.1 at a ratio of cartilage regeneration site/cutting surface width.

In addition, the stability of hyaline cartilage (hyaline cartilage) was analyzed by safranin o (safranin o)/fast green (fast green) staining of intrachondrogenic peptidoglycals (pg). That is, the structure (0 to 11 minutes), the number of chondrocytes (0 to 4 minutes) and the number of chondrocytes were determined from the above-mentioned dyeing results,The sum (0-24 points) of 4 indexes (cluster, score 0-5) and chondrocyte component (PG, score 0-6) was used to evaluate the efficacy of treatment on the degree of cartilage damage.

[ TABLE 4 ]

Histopathological observation index of chondrocyte damage

After analysis, the arthritis-inducing tissues were stained with H & E and safranin o (safranin o) and the price difference was measured under a microscope, and the severity of abnormal symptoms such as cartilage structure, chondrocyte number, cell cluster (cluster) formation, and Peptidoglycan (PG) content was 17.2 points out of 24 points in total, indicating a serious level. When a normal culture medium was administered into the articular cavity of the osteoarthritis-inducing rabbit, the therapeutic effect of microscopic analysis was shown in the order of neural stem cells (mean lesion 13.1 points) > oligodendrocyte precursor cells (13.7 points) > adipose stem cells (14.2 points) > amniotic membrane stem cells (14.8 points) > umbilical cord blood stem cells (15.5 points) > amniotic fluid stem cells (15.6 points) > placental stem cells (16.3 points) > bone marrow stem cells (16.3 points).

On the other hand, when the exosome culture solution rich in stem cells of the present invention was administered, all stem cells showed more excellent improvement in lesion symptoms than the group administered as a normal culture solution used as a control group, and in particular, the exosome culture solution rich in stem cells of the present invention was found to be very effective in treating arthritis in the order of neural stem cells (lesion average 10.4 points) ≧ adipose stem cells (10.5 points) > oligodendrocyte precursor cells (10.9 points) > amniotic fluid stem cells (11.7 points) > amniotic fluid stem cells (11.9 points) > umbilical cord blood stem cells (12.8 points) > bone marrow stem cells (14.0 points) > placental stem cells (14.3 points) (see fig. 3 and table 5).

[ TABLE 5 ]

Analysis result observed by microscope

Significant differences compared to osteoarthritis (ACLT) induced group (P <0.05) # significant differences compared to normal culture broth (P <0.05).

1-6 analysis of inflammatory cytokines

For the analysis of inflammatory factors, 0.1mL of physiological saline was injected into the joint cavity immediately before the opening of the joint cavity, and 0.1 to 0.2mL of diluted joint cavity fluid was collected after about 5 piston movements.

Analysis shows that the content of cytokines TNF-alpha, IL-6 and IL-8 in the joint cavity for inducing arthritis is increased by more than 2 times compared with the concentration in normal cartilage, and the effect of reducing the content of inflammatory cytokines is shown when a control group normal culture solution is administrated into the joint cavity of a rabbit for inducing arthritis.

In terms of relative effects, the amniotic fluid stem cells, the umbilical cord blood stem cells, the neural stem cells and the adipose-derived stem cells show more excellent inflammatory cytokine-reducing effects than oligodendrocyte precursor cells, amniotic membrane stem cells, placental stem cells and bone marrow stem cells. On the other hand, when the exosome culture solution rich in stem cells of the present invention was administered, all stem cells showed significantly superior inflammatory cytokine-reducing effects compared to the control normal culture solution-administered group, and specifically, amniotic fluid stem cells, neural stem cells, oligodendrocyte precursor cells showed more superior inflammatory cytokine-inhibiting effects compared to umbilical cord blood stem cells, amniotic membrane stem cells, placental stem cells, and bone marrow stem cells (see table 6 below).

[ TABLE 6 ]

Concentration of inflammatory cytokines in joint cavity fluids

Significant differences compared to osteoarthritis (ACLT) induced group (P <0.05) # significant differences compared to normal broth group (P <0.05).

1-7 statistical analysis

All experimental results are expressed as mean ± SEM, and the inter-group differences were analyzed by oneway analysis of variance (ANOVA) (SPSS 12.0version), and when the P value was <0.05 deficiency, it was judged to have statistically significant differences.

The present invention has been described above by way of surrounding preferred embodiments thereof. However, it will be apparent to those skilled in the art that the present invention may be embodied in various modified forms without departing from the essential characteristics thereof. Thus, the illustrated embodiments are not to be considered in a limiting sense, but rather are to be understood as illustrative of the invention. The scope of the invention is indicated in the appended claims rather than in the foregoing description, and all differences within the scope and range of equivalents thereof will be construed as falling within the scope of the invention.

[ accession number ]

Name of depository Korean institute of Life engineering

Accession number KCTC12634BP

Storage date 20140725

Name of depository Korean institute of Life engineering

Accession number KCTC12635BP

Storage date 20140725

Name of depository Korean institute of Life engineering

Accession number KCTC12636BP

Storage date 20140725

Storage date 20140725