Method for selecting stem cells having enhanced cartilage differentiation capability and cell therapy product comprising same

Abstract

The present invention relates to a method for selecting stem cells having enhanced ability to differentiate cartilage, a cell therapy product comprising stem cells selected by said selection method, and a method for producing the same.

Description

Technical Field

[0001] The present invention relates to a method for selecting stem cells having enhanced chondrogenic differentiation ability, a cell therapy product comprising stem cells selected by the selection method, and a method for manufacturing the cell therapy product. Background Art

[0002] A joint is a site where bones converge and is composed of cartilage, joint capsule, synovium, ligaments, tendons, muscles, and others. Articular cartilage is vulnerable to injury and prone to pathological degeneration. After being damaged, articular cartilage generally does not heal or only partially heals under specific biological conditions.

[0003] Arthritis is an inflammation of joints caused by various factors and is often accompanied by fever, pain, stiffness, and swelling. There are more than 100 causes of arthritis, such as degenerative changes, immune system disorders, infections, trauma, and metabolic disorders.

[0004] Regarding existing arthritis treatments, there are no effective drugs, and non-drug treatments currently dominate over drug treatments. Drugs include NSAID-based pain relievers and anti-inflammatory agents - for relieving symptoms such as inflammation and pain, steroid preparations, glucosamine, etc., and treatments using hyaluronic acid or analogs are mainly used to protect joints through lubrication by intra-articular injection.

[0005] Surgical treatments, such as artificial joint replacement corresponding to non-drug treatments, are performed for severe arthritis, but this requires rehabilitation, the lifespan of the replaced joint is limited (within 10 years), it incurs costs, and causes side effects, such as inflammation caused by implant corrosion and secondary infections. Therefore, clinically, a therapy that can delay surgery for as long as possible is needed, and considering the efficacy and side effects of existing treatments, new drugs that are safe and effective in preventing joint wear need to be developed. Cell therapy is emerging as a promising tissue generation method, but quality issues need to be addressed to obtain a satisfactory solution.

[0006] Prior Art Documents

[0007] (Patent Document 1) Korean Patent No. 10-1669423 Summary of the Invention

[0008] Technical Problem

[0009] The present invention relates to a method for selecting stem cells having enhanced chondrogenic differentiation ability, a cell therapy product comprising stem cells selected by the selection method, and a method for manufacturing the cell therapy product.

[0010] Technical Solution

[0011] One aspect of the present invention is to provide a method for manufacturing a cell therapy product.

[0012] Another aspect of the present invention is to provide a pharmaceutical composition for preventing or treating arthritis.

[0013] Still another aspect of the present invention is to provide a method for selecting stem cells for preventing or treating arthritis.

[0014] Advantageous Effects

[0015] The method of the present invention can produce a cell therapy product with enhanced efficacy by effectively selecting stem cells with enhanced chondrogenic differentiation ability. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 Shows the results of analyzing the concentration of TGF-β1 secreted per 1 x 10 5 cells in eight batches of umbilical cord blood-derived mesenchymal stem cells at passage 2.

[0017] Figure 2 Shows the results of identifying the chondrogenic differentiation ability of eight batches of umbilical cord blood-derived mesenchymal stem cells at passage 6 by Safranin-O staining.

[0018] Figure 3 Shows the results of identifying the chondrogenic differentiation ability of eight batches of umbilical cord blood-derived mesenchymal stem cells at passage 6 by Safranin-O staining and quantitatively evaluating using a program.

[0019] Figure 4 Shows the results of analyzing the correlation coefficient between the result values of TGF-β1 secretion ability at passage 2 and the result values of Safranin O-positive area (%) at passage 6 of eight batches of umbilical cord blood-derived mesenchymal stem cells.

[0020] Figure 5 Shows the results of evaluating the cartilage tissue regeneration ability after in vivo administration in each of the high and low groups classified by the TGF-β1 secretion ability of stem cells at passage 2. DETAILED DESCRIPTION

[0021] One aspect of the present invention relates to a method for manufacturing a cell therapy product.

[0022] In one embodiment, the method for manufacturing a cell therapy product may include: measuring the concentration of TGF-β1 in the culture of stem cells subcultured for 2 passages or fewer; and isolating the stem cells that secrete TGF-β1 at a concentration exceeding a specific value in the culture.

[0023] In the method for manufacturing a cell therapy product according to the foregoing embodiments, the method may include measuring the concentration of TGF-β1 in a culture of stem cells passaged 2 or fewer times.

[0024] In the method for manufacturing a cell therapy product according to any one of the foregoing embodiments, the method may further include culturing stem cells that secrete TGF-β1 at a concentration exceeding a specific value in a culture.

[0025] In the method for manufacturing a cell therapy product according to any one of the foregoing embodiments, the method is characterized by predicting the chondrogenic differentiation ability of stem cells at passage 6 or higher by measuring the TGF-β1 secretion ability at passage 2 or lower (corresponding to early passage).

[0026] In the method for manufacturing a cell therapy product according to any one of the foregoing embodiments, the method may include: culturing stem cells that secrete TGF-β1 at a concentration exceeding a specific value in a culture; and proliferating the stem cells.

[0027] In the method for manufacturing a cell therapy product according to any one of the foregoing embodiments, the method may include additionally passaging stem cells that secrete TGF-β1 at a concentration exceeding a specific value in a culture of stem cells passaged 2 or fewer times.

[0028] In the method for manufacturing a cell therapy product according to any one of the foregoing embodiments, the method may include: culturing stem cells that secrete TGF-β1 at a concentration exceeding a specific value in a culture; and proliferating and differentiating the stem cells.

[0029] In the cell therapy product according to any one of the foregoing embodiments, the cell therapy product may include: stem cells that secrete TGF-β1 at a concentration exceeding a specific value in a culture of stem cells passaged 2 or fewer times; or cells differentiated or proliferated from the stem cells.

[0030] In the method for manufacturing a cell therapy product according to any one of the foregoing embodiments, the method may include isolating stem cells that secrete TGF-β1 at a concentration exceeding 1046.7 pg / 1x10 5 cells in a culture of stem cells passaged 2 or fewer times.

[0031] In the method for manufacturing a cell therapy product according to any one of the foregoing embodiments, the method may include isolating stem cells that secrete TGF-β1 at a concentration not less than 1132.12 pg / 1x10 5 cells in a culture of stem cells passaged 2 or fewer times.

[0032] In the method for manufacturing a cell therapy product according to any one of the foregoing embodiments, the method may include isolating stem cells that secrete TGF-β1 at a concentration of not less than 1486.7 pg / 1x10 5 cells in a culture of stem cells passaged 2 or fewer generations.

[0033] In the method for manufacturing a cell therapy product according to any one of the foregoing embodiments, the stem cells may be mesenchymal stem cells derived from umbilical cord blood.

[0034] In the method for manufacturing a cell therapy product according to any one of the foregoing embodiments, the method may include: isolating stem cells that secrete TGF-β1 at a concentration exceeding 1046.7 pg / 1x10 5 cells in a culture of stem cells passaged 2 or fewer generations; and proliferating and differentiating the stem cells.

[0035] In the method for manufacturing a cell therapy product according to any one of the foregoing embodiments, the method may include isolating stem cells that secrete TGF-β1 at a concentration exceeding 1046.7 pg / 1x10 5 cells in a culture of stem cells passaged 2 or fewer generations to select the stem cells as an active ingredient of the cell therapy product.

[0036] In the method for manufacturing a cell therapy product according to any one of the foregoing embodiments, in a culture of stem cells passaged 2 or fewer generations, the stem cells may secrete TGF-β1 at a concentration exceeding 1046.7 pg / 1x10 5 cells.

[0037] In the method for manufacturing a cell therapy product according to any one of the foregoing embodiments, in a culture of stem cells passaged 2 or fewer generations, the stem cells may secrete TGF-β1 at a concentration exceeding 1046.7 pg / 1x10 5 cells.

[0038] In the method for manufacturing a cell therapy product according to any one of the foregoing embodiments, the cell therapy product contains stem cells or differentiated cells that are proliferated or differentiated from stem cells that secrete TGF-β1 at a concentration of not less than 1132.12 pg / 1x10 5 cells in a culture of stem cells passaged 2 or fewer generations.

[0039] In the method for manufacturing a cell therapy product according to any one of the foregoing embodiments, the cell therapy product contains stem cells or differentiated cells that are proliferated or differentiated from stem cells that secrete TGF-β1 at a concentration of not less than 1486.7 pg / 1x105 of TGF-β1 in a single cell for stem cell proliferation or differentiation.

[0040] In the method for manufacturing a cell therapy product according to any one of the foregoing embodiments, stem cells that secrete TGF-β1 at a concentration exceeding a specific value in the culture may exhibit excellent chondrogenic differentiation ability compared to stem cells that secrete TGF-β1 at a concentration not exceeding the specific value in the culture. In the method for manufacturing a cell therapy product according to any one of the foregoing embodiments, the cell therapy product is a cell therapy product for preventing or treating arthritis.

[0041] In the method for manufacturing a cell therapy product according to any one of the foregoing embodiments, the method may include differentiating isolated stem cells into chondrocytes.

[0042] Another aspect of the present invention relates to a pharmaceutical composition for preventing or treating arthritis, which contains stem cells or cells differentiated therefrom as an active ingredient.

[0043] In one embodiment, the cells contained in the pharmaceutical composition may be those proliferated or differentiated from stem cells that secrete TGF-β1 at a concentration exceeding a specific value in the culture.

[0044] In the pharmaceutical composition according to the foregoing embodiment, the cells contained in the pharmaceutical composition may be those proliferated or differentiated from stem cells that secrete TGF-β1 at a concentration exceeding a specific value in the stem cell culture at passage 2 or less.

[0045] In the pharmaceutical composition according to any one of the foregoing embodiments, the cells contained in the pharmaceutical composition may be those proliferated or differentiated from stem cells that secrete TGF-β1 at a concentration exceeding 1046.7 pg / 1x10 5 of TGF-β1 in a single cell.

[0046] Another aspect of the present invention relates to a method for selecting stem cells suitable for preventing or treating arthritis.

[0047] Another aspect of the present invention relates to a method for selecting stem cells for preventing or treating arthritis.

[0048] In one embodiment, the method may include: measuring the concentration of TGF-β1 in the culture of stem cells; measuring the concentration of TGF-β1 in the culture of stem cells; and isolating stem cells that secrete TGF-β1 at a concentration exceeding a specific value in the culture as stem cells for preventing or treating arthritis.

[0049] In the method of selecting stem cells for preventing or treating arthritis according to the foregoing embodiments, the method may include selecting stem cells that secrete TGF-β1 at a concentration exceeding 1046.7 pg / 1x10 5 cells in culture.

[0050] The present invention will be described in detail below. Each description and exemplary embodiment disclosed herein is also applicable to other descriptions and exemplary embodiments. That is, all combinations of the various elements disclosed herein fall within the scope of the present invention. In addition, the scope of the present invention is not limited by the following detailed description.

[0051] In addition, those skilled in the art will also recognize or be able to determine, using only routine experimentation, numerous equivalent forms of the specific embodiments of the invention described herein. In addition, these equivalent forms are intended to be included in the present invention.

[0052] In addition, throughout the specification, reference has been made to a number of papers and patent documents and their citations are provided. The disclosures of the cited papers and patent documents are hereby incorporated by reference in their entirety, and the level of the art to which the present invention pertains and the details of the present invention are more clearly illustrated.

[0053] As used herein, the term "cell therapy product" refers to cells and tissues that have been isolated and cultured from a subject and prepared by special manipulation, and are used as a pharmaceutical product for therapeutic, diagnostic, and prophylactic purposes. The term is intended to mean a pharmaceutical product for treating, diagnosing, and preventing diseases through a series of actions that include in vitro proliferation and selection of viable autologous cells, allogeneic cells, or xenogeneic cells, or otherwise altering the biological characteristics of the cells for the purpose of restoring the function of the cells or tissues.

[0054] The cell therapy product of the present invention can be used without lyophilization or after lyophilization for subsequent use. When lyophilization is required, a standard cryoprotectant (e.g., DMSO, glycerol, or Epilife cell lyophilization medium (Cascade Biologics)) can be added to the cell population before lyophilization. Additionally, the cell therapy product can be administered after being formulated into a unit dosage form suitable for administration to a patient's body according to conventional methods in the pharmaceutical field, and the formulation can include a dose effective after one or several administrations. Examples of formulations suitable for this purpose and used as parenteral administration formulations can preferably be injections (such as injection ampoules), infusions (such as infusion bags), and sprays (such as aerosol formulations). Injection ampoules can be prepared by mixing with an injection solution immediately before use. For injection solutions, normal saline, glucose, mannitol, Ringer's solution, or the like can be used. In addition, for infusion bags, polyvinyl chloride or polyethylene materials can be used, and infusion bags produced by Baxter, Becton Dickinson, Medcep, National Hospital Products, or Terumo can be used as examples.

[0055] The cells used in the cell therapy product can be stem cells or cells differentiated from stem cells.

[0056] In one embodiment, the cells contained in the cell therapy product of the present invention can be: stem cells with an endocrine concentration of TGF-β1 exceeding 1046.7 pg / 1 x 10 5 cells in the culture of passage stem cells at passage 2 or less; or stem cells or differentiated cells proliferated or differentiated therefrom.

[0057] In one embodiment, the cells contained in the cell therapy product of the present invention can be: stem cells with an endocrine concentration of TGF-β1 exceeding 1046.7 pg / 1 x 10 5 cells in the stem cell culture at passage 2 (P2); or stem cells or differentiated cells proliferated or differentiated therefrom.

[0058] In one embodiment, the cells contained in the cell therapy product of the present invention can be: stem cells obtained by further passaging the culture at P2 (passage 2) with an endocrine concentration of TGF-β1 exceeding 1046.7 pg / 1 x 10 5 cells to expand the stem cells to P6 (passage 6); or stem cells or cells differentiated therefrom.

[0059] In one embodiment, the concentration of TGF-β1 can exceed 1046.7 pg / 1 x 10 5 cells or exceed 1109.49 pg / 1 x 10 5cells, or may be not less than 1132.12 pg / 1x10 5 cells, not less than 1146.8 pg / 1x10 5 cells, not less than 1450.57 pg / 1x10 5 cells, or not less than 1486.7 pg / 1x10 5 cells.

[0060] The cell therapy product of the present invention can be administered together with other stem cells used in the art for transplantation and other purposes by conventional administration methods, or administered in a mixture with such stem cells, and can preferably be directly implanted (engrafted) or transplanted into the diseased area, or directly transplanted or infused into the abdominal cavity of a patient in need of treatment, but not limited thereto. In addition, the administration can be carried out by a non-surgical administration method using a catheter or by a surgical administration method (such as injection or transplantation after incision in the diseased area). In addition, in addition to direct administration to the lesion, the cell therapy product can also be administered parenterally, for example, by intravenous injection, which is one of the common methods for hematopoietic stem cell transplantation.

[0061] The cell therapy product can be administered in a single dose or in split doses. However, it should be understood that the actual dose of the active ingredient is determined by considering various relevant factors, such as the disease to be treated, the severity of the disease, the administration route, and the weight, age and gender of the patient, so the dose should not be construed as limiting the scope of the present invention in any way.

[0062] As used herein, the term "stem cell" refers to a cell with the potential to differentiate into various tissues, i.e., an undifferentiated cell. Stem cells can be derived from humans or animals, or can be derived from the umbilical cord, umbilical cord blood, bone marrow, fat, muscle, nerve, skin, amnion or placenta. As an example, the stem cell can be a mesenchymal stem cell.

[0063] As used herein, the term "mesenchymal stem cell" refers to a heterogeneous population of stem cells with the potential for self-renewal and differentiation into mesodermal lineages and different embryonic lineages such as endodermal and ectodermal lineages. Mesenchymal stem cells can be used interchangeably with pluripotent undifferentiated cells, and can be adult stem cells with the potential to differentiate into various mesodermal cells (such as adipocytes, osteoblasts, chondrocytes, cardiomyocytes or myocytes) or ectodermal cells (such as neuronal cells).

[0064] Specifically, the stem cells of the present invention can be umbilical cord blood-derived mesenchymal stem cells.

[0065] As used herein, the term "subculture" is one of the methods of cell proliferation and refers to a culturing method of periodically (e.g., every 1 to 7 days) transferring cells from a previous culture to a new culture medium. Subculture is a culturing method for preserving cell lines and maintaining cell generations, and this method is called subculture or passage. This subculture allows for the removal of accumulated toxic metabolites and the replenishment of depleted nutrients, thereby preventing cell death and promoting growth and proliferation. Subculture can be performed when the cells cover approximately 70% to 90% of the culture surface area. As used herein, the term "passage 2" refers to the state in which the cells have undergone two rounds of subculture for proliferation.

[0066] As used herein, the term "differentiation" refers to the process of specialization in structure and function of cells during cell division, proliferation, and growth, i.e., the cells, tissues, etc. of an organism are transformed in form or function to perform their given roles. For example, in ontogeny, the state in which qualitative differences arise between initially homologous parts of any biological system or the system is divided into qualitatively distinguishable partial systems is called differentiation.

[0067] The term "differentiated cell" refers to any cell that is in the stage of differentiating into somatic cell lineages or has been finally differentiated. In other words, differentiated cells are cells that make up an adult and have limited differentiation potential and self-renewal.

[0068] For the stem cell cultures of the present invention, media known for stem cell culture can be used, and specifically, KSB-3 medium can be used.

[0069] In one embodiment of the present invention, the measurement result of the concentration of TGF-β1 in the culture obtained by culturing umbilical cord blood-derived stem cells in KSB-3 complete medium to P2 is that the concentration of TGF-β1 in the culture exceeds 1046.7 pg / 1x10 5 The group of cells showed a significantly high ability to differentiate into chondrocytes, while the group with a concentration at or below the above value showed a low ability to differentiate into chondrocytes. Therefore, the present invention is completed by providing this value as a standard for selecting cells that can be used as cell therapy products due to their excellent differentiation ability.

[0070] As used herein, the term "arthritis" can refer to chronic inflammation of joints in the tissues around the joints (such as tendons, ligaments, and muscles) and other organs. Arthritis can be at least one selected from osteoarthritis, osteochondritis dissecans, joint ligament injury, meniscus injury, infectious arthritis, psoriatic arthritis, ankylosing spondylitis, rheumatoid arthritis, and juvenile rheumatoid arthritis.

[0071] Osteoarthritis, also known as degenerative arthritis, is a type of arthritis caused by degenerative changes in the cartilage of synovial joints and the surrounding bones. Osteoarthritis can be caused by cartilage damage due to aging or excessive physical stress (e.g., obesity, trauma), and is characterized by progressive loss of articular cartilage, subchondral bone hypertrophy, marginal bone formation at the joints, and nonspecific synovial inflammation.

[0072] Rheumatoid arthritis is a chronic autoimmune disease characterized by synovial cell inflammation and proliferation, leading to osteoporosis and bone erosion of the bones around the joints. When rheumatoid arthritis progresses to a certain extent, the articular cartilage is gradually damaged, resulting in narrowing of the joint space and loss of tension in the joint capsule and ligaments.

[0073] As used herein, the term "prevention" includes any action that inhibits or delays the onset of a disease.

[0074] As used herein, the term "treatment" includes any action that alleviates or favorably modifies the symptoms of a disease.

[0075] The pharmaceutical composition of the present invention may further comprise a pharmaceutically acceptable salt.

[0076] The term "pharmaceutically acceptable carrier" refers to a carrier or diluent that does not inhibit the biological activity and properties of the compound being injected, and at the same time does not cause irritation to the organism. The carriers that can be used in the present invention are not particularly limited to their types, and any carrier can be used as long as it is commonly used in the art and is pharmaceutically acceptable. Non-limiting examples of carriers may include saline, sterile water, Ringer's solution, buffered saline, albumin infusion solution, dextran solution, maltodextrin solution, glycerol, ethanol, etc. These can be used alone, or in a mixture of two or more of them.

[0077] In an embodiment of the present invention, it was determined that: when the concentration of TGF-β1 in the culture obtained by subculturing stem cells to P2 exceeds 1046.7 pg / 1x10 5 cells, the stem cells show excellent chondrogenic differentiation ability when subcultured to P6. Therefore, the cell therapy products and pharmaceutical compositions manufactured by the method of the present invention can be advantageously used for the prevention and treatment of arthritis.

[0078] Examples

[0079] Hereinafter, the present invention will be described in detail with reference to examples and experimental examples. However, these examples and experimental examples are given to specifically illustrate the present disclosure, and the scope of the present disclosure is not limited thereto.

[0080] Example 1: Comparative observation of the TGF-β1 secretion ability of early passage umbilical cord blood-derived mesenchymal stem cells of different batches

[0081] Umbilical cord blood-derived mesenchymal stem cells were subjected to primary culture, with 2,000 - 10,000 cells / cm inoculated per passage 2 , and then cultured for 2 - 6 days, followed by subculture.

[0082] To compare the TGF-β1 secretion capacity of early-passage umbilical cord blood-derived mesenchymal stem cells, the concentration of TGF-β1 secreted per 1x10 5 cells of eight batches of umbilical cord blood-derived mesenchymal stem cells at P2 was analyzed.

[0083] P2 umbilical cord blood-derived mesenchymal stem cells were cultured in KSB-3 complete medium, inoculated at 40,000 cells / 400 μL in a 48-well plate, and cultured at 37 °C in a 5% CO2 incubator for 96 hours. At the end of the culture, the culture was obtained, centrifuged at 500 g for 5 minutes, and the supernatant was collected. The cells were treated with TrypLE TM to obtain single cells, and then cell counting was performed. The TGF-β1 concentration of the collected supernatant was measured using a TGF-1 Quantikine ELISA kit. The result values were corrected by the measured cell count and expressed as pg / 1x10 5 cells. All data were analyzed using Student T-test and one-way ANOVA. This analysis was performed using GraphPad Prism software, where p < 0.05 or p < 0.01 was considered statistically significant. The results are shown in Table 1 and Figure 1 .

[0084] Table 1

[0085] Batch 1 Batch 2 Batch 3 Batch 4 Batch 5 Batch 6 Batch 7 Batch 8 Average 1486.7 1383.9 1213.8 1146.8 1046.7 810.9 805.7 753.9 s.d.(±) 36.13 88.75 53.46 14.72 62.75 26.27 38.25 19.92

[0086] (Unit: pg / 1x10 5 cells)

[0087] The analysis results confirmed that the TGF-β1 secretion capacities of umbilical cord blood-derived mesenchymal stem cells from different batches were different, within the range of 1486.7 pg / 1x10 5 cells to 753.9 pg / 1x10 5 cells. When these batches were arranged in descending order of TGF-β1 secretion and classified into high and low groups, the mean values of these groups were different, 1290.2 pg / 1x10 5 cells and 854.3 pg / 1x10 5 cells respectively, and this difference was statistically significant.

[0088] These results indicate that the TGF-β1 secretion capacity values of umbilical cord blood-derived mesenchymal stem cells from multiple batches are significantly different. (Figure 1 )。

[0089] Example 2: Comparative observation of the chondrogenic differentiation ability of late-passage umbilical cord blood-derived mesenchymal stem cells based on the TGF-β1 secretion ability of early-passage umbilical cord blood-derived mesenchymal stem cells

[0090] To compare the chondrogenic differentiation ability of late-passage umbilical cord blood-derived mesenchymal stem cells based on the TGF-β1 secretion ability of early-passage umbilical cord blood-derived mesenchymal stem cells, the chondrogenic differentiation ability of umbilical cord blood-derived mesenchymal stem cells from eight batches at passage P6 was analyzed.

[0091] To compare the chondrogenic differentiation ability, the eight batches of umbilical cord blood-derived mesenchymal stem cells in Example 1 were at passage P6 and dispensed into 15 mL tubes at a concentration of 3×10 5 cells / mL, centrifuged, and then cultured in a 37 °C, 5% CO2 incubator for 1 day. The resulting pellets were transferred to 96-well round bottom plates, and the medium was replaced with chondrogenic differentiation medium (Promocell Mesenchymal Stem Cell Chondrogenic Medium Kit). Subsequently, the pellets were induced to differentiate for three weeks, with the medium being changed three times a week. After differentiation was completed, the pellets were washed with PBS, fixed with 4% PFA, and then allowed to be stained with safranin-O by an analytical service company.

[0092] The safranin-O staining results confirmed that the chondrogenic differentiation ability values of umbilical cord blood-derived mesenchymal stem cells from different batches were different. In particular, the chondrogenic differentiation ability of the four batches of umbilical cord blood-derived mesenchymal stem cells classified as the high group according to the higher ranking of TGF-β1 secretion was generally higher than that of the low group ( Figure 2 )。

[0093] Example 3: Quantitative evaluation of the chondrogenic differentiation ability of late-passage umbilical cord blood-derived mesenchymal stem cells from different batches

[0094] To more detailedly compare and analyze the chondrogenic differentiation ability, a quantitative evaluation was performed based on the safranin-O staining results indicating the chondrogenic differentiation ability. For the quantitative evaluation, the values were obtained by calculating the safranin-O positive area (%) within the entire pellet area using the color threshold tool of ImageJ software. All data were analyzed using Student T-test and one-way ANOVA. This analysis was performed using GraphPad Prism software, where p < 0.05 or p < 0.01 was considered statistically significant.

[0095] As Figure 3As shown, the quantitative evaluation results confirmed that the safranin O positive area values (%) of the eight batches were different, ranging from approximately 90.0% to 0.0%. Among them, the four batches in the high group showed an average safranin O positive area value (%) of approximately 79.2% for TGF-β1 secretion, and the four batches in the low group showed an average safranin O positive area value (%) of approximately 14.1%, with a significant difference.

[0096] From this, it can be determined that the TGF-β1 secretion ability of early passage umbilical cord blood-derived mesenchymal stem cells is related to the chondrogenic differentiation ability of early passage umbilical cord blood-derived mesenchymal stem cells. In addition, based on the results of the 4th and 5th batches, the cut-off value of the TGF-β1 concentration in the culture of early passage umbilical cord blood-derived mesenchymal stem cells can be determined, which significantly affects the chondrogenic differentiation ability.

[0097] Example 4: Evaluation of the correlation coefficient between the TGF-β1 secretion ability of early passage umbilical cord blood-derived mesenchymal stem cells and the chondrogenic differentiation ability of late passage umbilical cord blood-derived mesenchymal stem cells

[0098] To analyze the correlation between the TGF-β1 secretion ability of early passage umbilical cord blood-derived mesenchymal stem cells and the results of the chondrogenic differentiation ability of late passage umbilical cord blood-derived mesenchymal stem cells, the correlation coefficient was examined. The results are shown in Figure 4 below.

[0099] The analysis result of the correlation coefficient between the TGF-β1 secretion ability result value of the P2 generation and the safranin O positive area (%) result value of the P6 generation from a total of eight batches of umbilical cord blood-derived mesenchymal stem cells was that the correlation coefficient was 0.67934.

[0100] From this, it can be determined that the TGF-β1 secretion ability of early passage umbilical cord blood-derived mesenchymal stem cells has a "positive correlation" with the chondrogenic differentiation ability of late passage umbilical cord blood-derived mesenchymal stem cells, and the TGF-β1 secretion ability at early passage can predict the in vitro chondrogenic differentiation ability at late passage.

[0101] Example 5: Non-clinical evaluation of the chondrogenic regeneration efficacy based on the TGF-β1 secretion ability of early passage umbilical cord blood-derived mesenchymal stem cells in an arthritis model

[0102] To compare the chondrogenic regeneration ability after in vivo administration between the high cell group (batch 1) and the low cell group (batch 7) (classified from the umbilical cord blood-derived mesenchymal stem cells in Example 1) in terms of the TGF-β1 secretion ability at early passage, the chondrogenic regeneration effect was evaluated.

[0103] After inducing arthritis in rabbits by transection of the anterior cruciate ligament, umbilical cord blood-derived mesenchymal stem cells with high TGF-β1 secretion capacity and umbilical cord blood-derived mesenchymal stem cells with low TGF-β1 secretion capacity were administered intra-articularly at week 6. Eight weeks after administration, knee joint cartilage tissues were collected, fixed with 4% PFA, and then allowed to be stained with safranin-O by an analytical service company.

[0104] Evaluation of the regeneration efficacy by safranin-O staining confirmed that the group administered with umbilical cord blood-derived mesenchymal stem cells with relatively high TGF-β1 secretion capacity (MSC(high)) showed a higher degree of cartilage regeneration compared to the group administered with umbilical cord blood-derived mesenchymal stem cells with relatively low TGF-β1 secretion capacity (MSC(low)). In addition, the quantitative evaluation results based on histological findings obtained by Mankin scoring confirmed a similar trend, where the group administered with umbilical cord blood-derived mesenchymal stem cells with relatively high TGF-β1 secretion capacity (MSC(high)) showed a higher degree of regeneration compared to the group administered with umbilical cord blood-derived mesenchymal stem cells with relatively low TGF-β1 secretion capacity (MSC(low)).

[0105] Thus, it was able to be determined that the TGF-β1 secretion capacity of early passage (P2) umbilical cord blood-derived mesenchymal stem cells could predict the in vitro cartilage differentiation ability and in vitro cartilage tissue regeneration ability of late passage (P6) umbilical cord blood-derived mesenchymal stem cells, and could be used as a criterion to select cells for the purpose of treating arthritis (<x Figure 5 ).

[0106] Although the present invention has been described with reference to specific exemplary embodiments, those skilled in the art to which the present invention pertains can understand that the present invention can be implemented in other specific forms without departing from the technical spirit or important features of the present invention. Therefore, the above embodiments should be construed as exemplary and not as limiting the present invention. The scope of the present invention should be construed as the meaning and scope of the appended claims, rather than the detailed description, and all variations or modifications obtained by equivalent concepts fall within the scope of the present invention.

Claims

1. A method for manufacturing a cell therapy product, the method comprising: Measuring the concentration of TGF-β1 in a culture of stem cells passaged 2 or fewer passages; and Isolate stem cells that secrete TGF-β1 at a concentration exceeding 1046.7 pg / 1x10 5 cells in the culture.

2. The method according to claim 1, further comprising culturing or proliferating the stem cells that secrete TGF-β1 at a concentration exceeding 1046.7 pg / 1x10 5 cells in a stem cell culture that has been passaged 2 or fewer times, wherein the cell therapy product includes the stem cells that secrete TGF-β1 at a concentration exceeding 1046.7 pg / 1x10 5 cells in a stem cell culture subcultured for 2 passages or fewer; or stem cells or differentiated cells proliferated or differentiated therefrom.

3. The method according to claim 1, wherein the method comprises separating stem cells that secrete TGF-β1 at an endocrine concentration of not less than 1132.12 pg / 1x10 5 cells in a stem cell culture passaged 2 or fewer passages.

4. The method according to claim 1, wherein the stem cells are mesenchymal stem cells derived from umbilical cord blood.

5. The method according to claim 1, wherein the cell therapy product is a cell therapy product for preventing or treating arthritis.

6. The method according to claim 1, wherein the culture medium is KSB-3 medium.

7. A pharmaceutical composition for preventing or treating arthritis, the pharmaceutical composition comprising stem cells or cells differentiated therefrom as an active ingredient, wherein the cell is a stem cell that secretes TGF-β1 at a concentration exceeding 1046.7 pg / 1 x 10 5 cells in a stem cell culture subcultured for 2 passages or fewer; or a cell proliferated or differentiated therefrom.

8. A method for selecting stem cells for preventing or treating arthritis, the method comprising: Measuring the concentration of TGF-β1 in a culture of stem cells passaged 2 or fewer passages; and Select stem cells that secrete TGF-β1 at a concentration exceeding 1046.7 pg / 1x10 5 cells as stem cells for preventing or treating arthritis.

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