METHOD FOR TREATING ARTHRITIS BY USING STEM CELL PREPARATION AND METHOD FOR PREPARING STEM CELL PREPARATION

Abstract

The present disclosure provides a method for treating arthritis by using a stem cell preparation. The stem cell preparation of the present disclosure can effectively delay cartilage degeneration caused by arthritis, and it is confirmed by whole blood analysis and blood biochemical analysis that the stem cell preparation in the form of three-dimensional stem cell spheres provides a safe treatment for arthritis. The present disclosure also provides a method for preparing the stem cell preparation.

Claims

1. A method for treating arthritis, comprising administering to a subject in need thereof a pharmaceutical composition comprising an effective amount of a stem cell preparation, wherein the stem cell preparation comprises a stem cell sphere.

2. The method according to claim 1, wherein the arthritis is degenerative arthritis.

3. The method according to claim 1, wherein the stem cell sphere has a size greater than 0 .Math.m and less than 50 .Math.m.

4. The method according to claim 1, wherein the stem cell sphere comprises a stem cell selected from the group consisting of: an adipose-derived stem cell (ADSC), an umbilical cord mesenchymal stem cell, and a combination thereof.

5. The method according to claim 4, wherein the adipose-derived stem cell is a primary human adipose-derived stem cell.

6. The method according to claim 5, wherein the stem cell sphere is prepared by seeding the primary human adipose-derived stem cell on a scaffold to form a cell scaffold copolymer, followed by sequentially performing a static cultivation and a dynamic cultivation on the cell scaffold copolymer.

7. The method according to claim 6, wherein the primary human adipose-derived stem cell is seeded on the scaffold at a concentration of at least 1 × 10.sup.6 cells/scaffold.

8. The method according to claim 1, wherein the stem cell sphere has a concentration of at least 1 × 10.sup.6 cells/50 .Math.L.

9. The method according to claim 1, wherein the stem cell sphere expresses a surface marker selected from the group consisting of: CD73, CD90, CD105, and a combination thereof.

10. The method according to claim 1, wherein the stem cell sphere expresses a cartilage-related gene selected from the group consisting of: a transforming growth factor-β1 (TCF-β1) gene, a bone morphogenetic protein-2 (BMP-2) gene, a Sox9 gene, an Aggrecan gene, a cartilage oligomeric matrix protein (COMP) gene, a collagen type II alpha 1 chain (Col2a1) gene, and a combination thereof.

11. The method according to claim 1, wherein the pharmaceutical composition is in a dosage form for parenteral administration.

12. A method for preparing a stem cell preparation, comprising: seeding a primary human adipose-derived stem cell on a scaffold to form a cell scaffold copolymer, followed by sequentially performing a static cultivation and a dynamic cultivation on the cell scaffold copolymer.

13. The method according to claim 12, wherein the primary human adipose-derived stem cell is seeded on the scaffold at a concentration of at least 1 × 10.sup.6 cells/scaffold.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0023] The following drawings form part of the present specification and are included here to further demonstrate some aspects of the present invention, which can be better understood by reference to one or more of these drawings, in combination with the detailed description of the embodiments presented herein.

[0024] FIG. 1 is a data diagram showing the size of the stem cell preparation in the form of three-dimensional stem cell spheres in the present invention.

[0025] FIG. 2 is a schematic diagram of gait analysis.

[0026] FIG. 3 is a data diagram showing the result of gait analysis of the adipose-derived stem cell preparation in the form of three-dimensional stem cell spheres in the present invention on experimental animals with degenerative arthritis.

[0027] FIG. 4 is a tissue section staining diagram showing the result of hematoxylin-eosin (H&E) staining at 4 weeks after surgery.

[0028] FIG. 5 is a tissue section staining diagram showing the result of hematoxylin-eosin (H&E) staining at 8 weeks after surgery.

[0029] FIG. 6 is a tissue section staining diagram showing the result of toluidine blue staining at 4 weeks after surgery.

[0030] FIG. 7 is a tissue section staining diagram showing the result of toluidine blue staining at 8 weeks after surgery.

[0031] FIG. 8 is a data diagram showing the result of the osteoarthritis (OA) score of the adipose-derived stem cell preparation in the form of three-dimensional stem cell spheres in the present invention on experimental animals with degenerative arthritis four weeks after surgery.

[0032] FIG. 9 is a data diagram showing the result of the osteoarthritis (OA) score of the adipose-derived stem cell preparation in the form of three-dimensional stem cell spheres in the present invention on experimental animals with degenerative arthritis 8 weeks after surgery.

[0033] FIG. 10 is a data diagram showing that after human adipose-derived stem cells (hADSCs) have been cultured in two and three dimensions (that is, the adipose-derived stem cell preparation in the form of three-dimensional stem cell spheres in the present invention) on day 14, the expression level of mRNA in the cells.

[0034] FIG. 11A is a data diagram showing that after human umbilical cord mesenchymal stem cells have been cultured in two and three dimensions (that is, the stem cell preparation in the form of three-dimensional stem cell spheres in the present invention prepared by referring to the method described in Example 1) on day 14, the expression level of mRNA in the cells.

[0035] FIG. 11B is a data diagram showing that after human umbilical cord mesenchymal stem cells have been cultured in two and three dimensions (that is, the stem cell preparation in the form of three-dimensional stem cell spheres in the present invention prepared by referring to the method described in Example 1) on day 14, the expression level of mRNA in the cells.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

[0036] In the following detailed description of the embodiments of the present invention, reference is made to the accompanying drawings, which are shown to illustrate the specific embodiments in which the present disclosure may be practiced. These embodiments are provided to enable those skilled in the art to practice the present disclosure. It is understood that other embodiments may be used and that changes can be made to the embodiments without departing from the scope of the present invention. The following description is therefore not to be considered as limiting the scope of the present invention.

Definition

[0037] As used herein, the data provided represent experimental values that can vary within a range of ±20%, preferably within ±10%, and most preferably within ±5%.

[0038] According to the present invention, the term “adipose-derived stem cells (ADSCs)” refers to mesemchymal stem cells separated from fat, which are multipotent stem cells having high plasticity. After induction, they can be differentiated into cells of many different tissues.

[0039] As used herein, the term “treating” or “treatment” refers to alleviating, reducing, ameliorating, relieving, or controlling one or more clinical signs of a disease or disorder, and lowering, stopping, or reversing the progression of severity regarding the condition or symptom being treated.

[0040] According to the present invention, the pharmaceutical composition can be manufactured to a dosage form suitable for parenteral administration, using techniques well known to those skilled in the art, including, but not limited to, injection (e.g., sterile aqueous solution or dispersion), sterile powder, tablet, troche, lozenge, pill, capsule, dispersible powder or granule, solution, suspension, emulsion, syrup, elixir, slurry, and the like.

[0041] The pharmaceutical composition according to the present invention may be administered by a parenteral route selected from the group consisting of: intraperitoneal injection, subcutaneous injection, intraepidermal injection, intradermal injection, intramuscular injection, intravenous injection, and intralesional injection.

[0042] According to the present invention, the pharmaceutical composition may further comprise a pharmaceutically acceptable carrier which is widely used in pharmaceutically manufacturing techniques. For example, the pharmaceutically acceptable carrier can comprise one or more reagents selected from the group consisting of solvent, emulsifier, suspending agent, decomposer, disintegrating agent, dispersing agent, binding agent, excipient, stabilizing agent, chelating agent, diluent, gelling agent, preservative, lubricant, absorption delaying agent, liposome, and the like. The selection and quantity of these reagents fall within the scope of professionalism and routine techniques of those who are familiar with this technology.

[0043] According to the present invention, the pharmaceutically acceptable carrier comprises a solvent selected from the group consisting of water, normal saline, phosphate buffered saline (PBS), sugar-containing solution, aqueous solution containing alcohol, and combinations thereof.

[0044] The experimental animals used in the following examples are SD (Sprague-Dawley) male rats. The day before the experiment, the rats to be tested were given a preoperative analgesic injection (ketoprofen, 5 mg/kg). Each rat was first anesthetized with isoflurane, the shaved area was disinfected with iodine, and then 75% alcohol was used for secondary disinfection. 27 G needle was used to inject 50 .Math.L of mono-iodoacetate (MIA) (6 mg/mL)(Sigma, product number A2512) in the left knee joint cavity to induce degenerative arthritis. Sham group was injected with normal saline (50 .Math.L). Ketoprofen (5 mg/kg) would be given daily for postoperative pain relief in three days after surgery.

[0045] In the following examples, adipose-derived stem cells (ADSCs) are selected as examples of stem cells. In another example, the stem cells may also be umbilical cord mesenchymal stem cells or a combination of adipose-derived stem cells and umbilical cord mesenchymal stem cells.

[0046] In the following examples, degenerative arthritis is selected as an illustration of arthritis. In an example, arthritis may also be arthritis caused by other causes.

Example 1

Preparation And Size Of Adipose-Derived Stem Cell (ADSC) Preparations In The Form Of Three-Dimensional Stem Cell Spheres

[0047] The human adipose-derived stem cells (ADSCs) used in the examples of the present invention were obtained from donors during abdominal surgery in accordance with the “Human Cell Therapy Product Donor Eligibility Determination Criteria” issued by the Food and Drug Administration of the Ministry of Health and Welfare. The donors undergo liposuction and 2-5 g of adipose tissue from the subcutaneous fat of the abdominal wall is collected. Written consent was provided by all donors. The human adipose tissue was placed in Ca.sup.2+ /Mg.sup.2+ -free phosphate buffered saline (PBS) and immediately transferred to the good tissue practice (GTP) laboratory. In the GTP laboratory, human adipose tissue is removed from the transport medium and placed in a petri dish, and cut into small pieces (about 1-2 mm.sup.3 in volume) without the presence of Ca.sup.2+ /Mg.sup.2+ in PBS. The tissue was dissociated with 0.1% collagenase and incubated at 37° C. for 60 minutes. After collagenase digestion, the resulting cells were collected and cultured in a proliferation medium. The proliferation medium is a serum-free medium, usually a keratinocyte serum-free medium including at least 10% fetal bovine serum, N-acetyl-L-cysteine, L2 ascorbic acid, and phosphate. After culturing for 2 days, the supernatant and debris were removed from the petri dish to obtain primary cultured adipose-derived stem cells.

[0048] Next, a two-dimensional cultured stem cell preparation (2D) was prepared as a comparative group. On the day before the animal experiment, two plates of 75T-flask of human primary cultured adipose-derived stem cells (number: 13A001, passage 6) were provided. On the day of the animal experiment, the trypsin solution (containing 0.25% trypsin and EDTA (Sigma, product number: 34549-1KG) solution, purchased from Gibco, product number: 25200-072) was used to collect cells. After collecting cells, a hemocytometer was used to perform cell counting, and the two-dimensional cultured stem cell preparation was prepared for in vivo injection.

[0049] Next, the stem cell preparation in the form of three-dimensional stem cell spheres of the present invention (3D) was prepared as an experimental group. Two plates of 75T-flask of human primary cultured adipose-derived stem cells (number: 13A001, passage 5 and passage 6) were provided, respectively. The trypsin solution was used to collect cells, followed by seeding the cells on the scaffold. The number of seeded cells is at least 1×10.sup.6 cells/scaffold. In one embodiment of the present invention, the number of seeded cells is 1 × 10.sup.6 cells/scaffold. The cell scaffold copolymer was cultured in a 24-well culture plate. After 24 hours of culture to stabilize the cells, the cell scaffold copolymer was transferred to a 50 mL centrifuge tube and a static cultivation was performed for 48 hours. Subsequently, the cell scaffold copolymer was transferred to a bioreactor, and a dynamic cultivation was performed at 37° C., 5% CO.sub.2 incubator (Thermo) for 14 days.

[0050] After 14 days of dynamic cultivation, the scaffold was dissolved with EDTA/PBS to collect cell spheres, and the size of cell spheres was counted with a cell counter (Cellometer® Auto T4). The size of each of the resulting cell spheres is less than 50 .Math.m. The experimental result is shown in FIG. 1.

Example 2

Determination Of Cell Number And Dosage Of Adipose-Derived Stem Cell Preparation In The Form Of Three-Dimensional Stem Cell Spheres

[0051] A commercially available cell viability detection kit (CellTiter-Glo 3D Cell Viability Assay Kit, Promega, product number: G9682) was used to detect the mitochondrial activity with a known cell number, the average mitochondrial activity of the cells was calculated as a reference standard, and the calculation benchmark value was used to convert the number of cell proliferation after 14 days of dynamic cultivation. The number is 2.35 × 10.sup.6 cells/scaffold. This result is used to calculate the number of cells in the cell sphere group in animal experiments.

[0052] In addition, the dosages of the two-dimensional cultured cell particles (i.e., the two-dimensional cultured stem cell preparation described in Example 1) and the stem cell preparation in the form of three-dimensional stem cell spheres were found respectively. The dosage of two-dimensional cultured cell particles is as follows. The dosage of cell therapy injected into an animal is 1 × 10.sup.6 cells/50 .Math.L, 8 experimental animals in one group, the minimum requirement is 8× 10.sup.6 cells, and the total demand for the experiment is 2× 10.sup.7 cells/mL. The number of cells calculated by hemocytometer (3.08× 10.sup.7 cells/mL) divided by 2× 10.sup.7 cells/mL is the cell re-dissolution volume of 1.54 mL. 1.54 mL of cell injection provided in this example, with 1.2× 10.sup.7 cells/600 .Math.L, was dispensed into 1 mL syringes (27 G) and provided for subsequent animal experiments (each experimental animal receives 50 .Math.L of cell injection, that is, the cell dose is 1 × 10.sup.6 cells/50 .Math.L).

[0053] The dosage of the stem cell preparation in the form of three-dimensional stem cell spheres is as follows. The dosage of cell therapy injected into an animal is 1× 10.sup.6 cells/50 .Math.L, 8 experimental animals in one group, the minimum requirement is 8× 10.sup.6 cells, and the total demand for the experiment is 2× 10.sup.7 cells/mL. The number of cells per scaffold is 2.35× 10.sup.6 cells multiplied by 10 scaffolds, the total number of cells is 2.35 × 10.sup.7 cells, the total number of cells (2.35 × 10.sup.7 cells) divided by the total demand for the experiment (2× 10.sup.7 cells/mL) is the cell re-dissolution volume of 1.17 mL. 1.17 mL of cell injection for animal experiments provided in this example, with 1× 10.sup.7 cells/500 .Math.L, was dispensed into 1 mL syringes (27 G) and provided for subsequent animal experiments (each experimental animal receives 50 .Math.L of cell injection, that is, the cell dose is 1× 10.sup.6 cells/50 .Math.L).

Example 3

Result of Gait Analysis of Adipose-Derived Stem Cell Preparation in the Form of Three-Dimensional Stem Cell Spheres on Experimental Animals With Degenerative Arthritis

[0054] Four groups of experimental animals in this example and the following examples were prepared, including Sham group, mono-iodoacetate (MIA) group, 2D group (i.e. the comparative group) and 3D group (i.e. the experimental group). The treatment methods of each group are shown in Table 1.

TABLE-US-00001 Group Sham MIA 2D 3D MIA - + + + 2D - - + - 3D - - - + Normal saline + + - -

[0055] Three days after the operation, the rats were anesthetized with isoflurane, and the left knee of each rat was surface-disinfected. The two-dimensional cultured stem cell preparation (2D) (1 × 10.sup.6 cells/50 .Math.L), the stem cell preparation in the form of three-dimensional stem cell spheres (3D) (1× 10.sup.6 cells/50 .Math.L) and normal saline (50 .Math.L) injected with a 27G needle were injected into the left knee joint cavity of each rat in 2D, 3D and MIA groups respectively. Sham group was injected with normal saline again. A gait analyzer (Clever Sys Inc.) analysis was performed four weeks after the operation.

[0056] The operation flow of gait analysis is as follows. At 4 weeks after the operation of the SD rats, the SD rats were placed on the track of the gait analyzer, and the test was performed at a rate of 23 cm/sec. TreadScan™ 2.0 was used to determine reaction time of the stance, brake and propulsion (see FIG. 2).

[0057] The result of gait analysis (see Table 2) confirms that compared with the reaction time of the stance, brake and propulsion of rats in Sham group (without MIA induction and without any treatment), the reaction time of the stance, brake and propulsion of rats induced by MIA without any treatment (MIA group) was increased (see FIG. 3). After 2D and 3D treatment, the reaction time of the stance, brake and propulsion of rats induced by MIA was decreased compared with that of the MIA group. In addition, the reaction time of the 3D group was lower than that of the 2D group, confirming that the stem cell preparation in the form of three-dimensional stem cell spheres of the present invention can indeed improve the walking performance of MIA-induced degenerative arthritis. The gait analysis initially confirms that the prognosis of the 3D group was better than that of the 2D group, in which the stance time was reduced by 39.84%, the brake time was reduced by 43.62%, and the propulsion time was reduced by 36.39%.

TABLE-US-00002 Time (ms) Normal saline (Sham) MIA 2D 3D Stance 244.20 ± 53.71 418.88 ± 89.24 308.96 ± 110.03 185.86 ± 56.35 Brake 93.43 ± 46.75 168.91 ± 116.78 147.46 ± 96.26 83.14 ± 40.19 propulsion 150.77 ± 73.77 249.96 ± 156.67 161.50± 103.61 102.73 ± 48.04

Example 4

Result of Hematoxylin-Eosin (H&E) Staining Regarding Tissue Section of Adipose-Derived Stem Cell Preparation in the Form of Three-Dimensional Stem Cell Spheres on Experimental Animals With Degenerative Arthritis

[0058] The grouping and treatment of experimental animals are the same as those described in Example 3. The experimental animals in each group were sacrificed at four and eight weeks after the operation, the sampled tissues were immersed and fixed in 10% formaldehyde (Macron) (product number: H121-08) solution for 3 days and then decalcified with 5% nitric acid solution for 2 weeks, followed by replacing with formaldehyde solution. After that, the sampled tissues were sent to the pathology core laboratory of the National Health Research Institutes for dehydration, paraffin embedding and tissue sectioning, and then the paraffin sections were serially stained.

[0059] Next, the paraffin sections were deparaffinized with xylene (Sigma) (product number: 534056) and serially rehydrated. The sections were immersed in hematoxylin (Sigma) (product number: GHS3) solution for staining for 5 minutes. The sections were washed in running water for 2 minutes, and then rinsed with deionized water. The sections were immersed in eosin (Sigma) (product number: E4382) solution for staining for 3 minutes. The sections were dehydrated twice with 99% absolute alcohol for 3 minutes each time. The sections were immersed in xylene, and then mounted with mounting medium.

[0060] The result of H&E staining at 4 weeks after surgery is shown in FIG. 4. It was found in the cartilage section induced by MIA that chondrocytes were destroyed and there were no obvious lacunae in the articular cartilage, proving that degenerative arthritis can be successfully induced with this animal model. Normal cartilage shape was maintained in the Sham group, and chondrocytes were encapsulated in lacunae. It was shown in the sections of rats treated with the two-dimensional cultured stem cell preparation (2D) and the stem cell preparation in the form of three-dimensional stem cell spheres (3D) that the cartilage patterns were more normal compared to those in the MIA group, and there were no significant differences in the cartilage patterns of the 3D group and the Sham group.

[0061] The result of H&E staining at 8 weeks after surgery is shown in FIG. 5. The normal cartilage shape was still maintained in the Sham group. However, the cartilages in the MIA group were destroyed. It was shown in the 8 weeks of 2D and 3D treatment that the cartilage type was significantly improved compared with that of the MIA group, and there was no significant difference between the 3D group and the Sham group. The result of H&E staining confirms that the stem cell preparation in the form of three-dimensional stem cell spheres (3D) of the present invention has a better effect of ameliorating degenerative arthritis regardless of whether it is in rats at 4 or 8 weeks.

Example 5

Result Of Toluidine Blue Staining Regarding Tissue Section Of Adipose-Derived Stem Cell Preparation In The Form Of Three-Dimensional Stem Cell Spheres On Experimental Animals With Degenerative Arthritis

[0062] The grouping and treatment of experimental animals are the same as those described in Example 3. The experimental animals in each group were sacrificed at four and eight weeks after the operation, the sampled tissues were immersed and fixed in 10% formaldehyde (Macron) (product number: H121-08) solution for 3 days and then decalcified with 5% nitric acid solution for 2 weeks, followed by replacing with formaldehyde solution. After that, the sampled tissues were sent to the pathology core laboratory of the National Health Research Institutes for dehydration, paraffin embedding and tissue sectioning, and then the paraffin sections were serially stained.

[0063] Next, the paraffin sections were deparaffinized with xylene and rehydrated. The sections were immersed in 0.04% toluidine blue (Sigma) (product number: T3260) solution for staining for 10 minutes. The sections were washed with running water for 1 minute and then allowed to stand for 10 minutes to dry. The sections were immersed in xylene, and then mounted with mounting medium.

[0064] The result of toluidine blue staining 4 weeks after surgery is shown in FIG. 6. The length from the cartilage surface to the tidemark is 131.81, 64.47, 51.32 and 153.95 .Math.m in the Sham, MIA, 2D and 3D groups, respectively. The length of the 3D group is increased by 299.98% compared to that of the 2D group. The toluidine blue staining result of the cartilage of rats induced by MIA is negative. The color of the Sham group and the 3D group was toluidine blue positive, and the color of the 2D group was toluidine blue negative.

[0065] The result of toluidine blue staining 8 weeks after surgery is shown in FIG. 7. The length from the cartilage surface to the tidemark is 192.10, 73.69, 63.16 and 184.87 .Math.m in the Sham, MIA, 2D and 3D groups, respectively. The length of the 3D group is increased by 292.7% compared to that of the 2D group. Among them, it was positive for toluidine blue only in the cartilage of the Sham group and the 3D group. The result of toluidine blue staining confirms that the stem cell preparation in the form of three-dimensional stem cell spheres (3D) of the present invention can effectively ameliorate cartilage degeneration caused by degenerative arthritis.

Example 6

Result Of Whole Blood Analysis And Blood Biochemical Analysis Of Adipose-Derived Stem Cell Preparation In The Form Of Three-Dimensional Stem Cell Spheres On Experimental Animals With Degenerative Arthritis

[0066] The grouping and treatment of experimental animals are the same as those described in Example 3. The experimental animals in each group were collected and sacrificed four weeks and eight weeks after the operation, and the blood was analyzed for whole blood and blood biochemical analysis. Blood samples were obtained from the tail vein at four and eight weeks after the operation, and the whole blood analysis and blood biochemical analysis were performed respectively. The items for whole blood analysis are red blood cell (RBC), white blood cell (WBC), platelet (PLT), monocyte and lymphocyte. The items for blood biochemical analysis items are glutamic oxaloacetic transaminase (GOT), glutamic pyruvic transaminase (GPT), alkaline phosphatase (ALP), blood urea nitrogen (BUN) and creatinine (CRE). The result shows that there was no significant difference between the four-week and eight-week groups (Table 3 to Table 6). This result also confirms that injection of the two-dimensional cultured stem cell preparation (2D) and the stem cell preparation in the form of three-dimensional stem cell spheres of the present invention (3D) into the joint cavity of rats would not cause obvious inflammation and liver and kidney toxicity.

TABLE-US-00003 Whole blood analysis (4 weeks) Item Unit Sham MIA 2D 3D RBC 10.sup.6/.Math.L 7.67 ± 0.29 7.73 ± 0.88 7.63 ± 0.51 7.63 ± 0.38 WBC 10.sup.3/.Math.L 9.08 ± 0.93 9.86 ± 3.39 9.92 ± 1.92 10.18 ± 0.96 PLT 10.sup.3/.Math.L 866.50 ± 209.56 819.50 ± 454.21 906.75 ± 391.95 911.67 ± 198.61 monocyte 10.sup.3/.Math.L 0.46 ± 0.10 0.41 ± 0.14 0.45 ± 0.10 0.49 ± 0.07 lymphocyte 10.sup.3/.Math.L 6.53 ± 0.65 6.61 ± 3.03 5.96 ± 2.04 7.15 ± 0.47

TABLE-US-00004 Blood biochemical analysis (4 weeks) Item Unit Sham MIA 2D 3D GOT U/L 88.33 ± 9.29 110.00 ± 1.41 108.50 ± 2.12 87.50 ± 7.78 GPT U/L 28.75 ± 2.22 35.75 ± 5.74 34.00 ± 3.46 30.00 ± 4.40 ALP U/L 1127.50 ± 428.25 1085.00 ± 268.52 1082.75 ± 224.38 1055.00 ± 344.34 BUN mg/dL 17.75 ± 2.00 19.80 ± 1.42 19.75 ± 1.42 18.65 ± 2.51 CRE mg/dL 0.34 ± 0.04 0.28 ± 0.02 0.25 ± 0.03 0.28 ± 0.05

TABLE-US-00005 Whole blood analysis (8 weeks) Item Unit Sham MIA 2D 3D RBC 10.sup.6/.Math.L 8.11 ± 0.54 7.98 ± 0.59 7.99 ± 0.22 8.32 ± 0.44 WBC 10.sup.3/.Math.L 6.36 ± 1.50 6.76 ± 0.88 6.65 ± 0.49 6.54 ± 0.75 PLT 10.sup.3/.Math.L 943.50 ± 67.18 939.33 ± 273.35 937.33 ± 290.18 868.50 ± 62.66 monocyte 10.sup.3/.Math.L 0.41 ± 0.09 0.32 ± 0.09 0.37 ± 0.11 0.39 ± 0.05 lymphocyte 10.sup.3/.Math.L 4.48 ± 0.86 4.03 ± 0.66 4.17 ± 0.52 4.19 ± 0.46

TABLE-US-00006 Blood biochemical analysis (8 weeks) Item Unit Sham MIA 2D 3D GOT U/L 71.25 ± 3.40 67.50 ± 21.02 81.50 ± 19.84 83.50 ± 6.36 GPT U/L 27.70 ± 2.38 27.75 ± 6.29 27.50 ± 5.92 32.50 ± 1.00 ALP U/L 890.50 ± 53.03 850.67 ± 154.57 877.00 ± 150.79 928.00 ± 64.63 BUN mg/dL 18.40 ± 1.14 19.33 ± 3.44 19.27 ± 4.10 18.20 ± 0.84 CRE mg/dL 0.23 ± 0.02 0.28 ± 0.03 0.27 ± 0.04 0.26 ± 0.03

Example 7

OA Score of Adipose-Derived Stem Cell Preparation in the Form of Three-Dimensional Stem Cell Spheres on Experimental Animals With Degenerative Arthritis

[0067] The grouping and treatment of experimental animals are the same as those described in Example 3. The experimental animals in each group were sacrificed at four and eight weeks after the operation, the sampled tissues were immersed and fixed in 10% formaldehyde (Macron) (product number: H121-08) solution for 3 days and then decalcified with 5% nitric acid solution for 2 weeks, followed by replacing with formaldehyde solution. After that, the sampled tissues were sent to the pathology core laboratory of the National Health Research Institutes for dehydration, paraffin embedding and tissue sectioning, and then the paraffin sections were serially stained.

[0068] Next, the paraffin sections were deparaffinized with xylene and rehydrated. The sections were immersed in 0.04% toluidine blue (Sigma) (product number: T3260) solution for staining for 10 minutes. The sections were washed with running water for 1 minute and then allowed to stand for 10 minutes to dry. The sections were immersed in xylene, and then mounted with mounting medium.

[0069] Toluidine blue stained sections were scored for degenerative arthritis according to the scoring method disclosed by the Osteoarthritis Research Society International, and the score is the product of Grade × Stage.

[0070] Four weeks and eight weeks of toluidine blue stained sections were evaluated for degenerative arthritis score. The scores at four weeks after surgery are 0 ± 0.14 ± 2.31, 11.5 ± 6.40 and 0.75 ± 0.96 in the Sham, MIA, 2D and 3D groups, respectively (FIG. 8). The scores at eight weeks after surgery are 0 ± 0.16 ± 1.63, 12.5 ± 5.74 and 0.5 ± 0.57 in the Sham, MIA, 2D and 3D groups, respectively (FIG. 9). The result of this example shows that the adipose-derived stem cell preparation in the form of three-dimensional stem cell spheres of the present invention can effectively ameliorate degenerative arthritis.

Example 8

Cartilage-Related Gene Expression Of Stem Cell Preparation In The Form Of Three-Dimensional Stem Cell Spheres

[0071] The human umbilical cord mesenchymal stem cells (Wharton’s jelly) used in this example were purchased from the Bioresource Collection and Research Center (BCRC) of the Food Industry Research and Development Institute.

[0072] Human adipose-derived stem cells (hADSCs) and human umbilical cord mesenchymal stem cells were prepared according to the preparation method in Example 1. After 14 days of culture, the mRNAs of the cells were isolated using an mRNA isolation kit (Quick-RNA™ MiniPrep, ZYMO RESEARCH), and reverse-transcribed into cDNA using a reverse transcription kit (PrimeScript™ RT reagent Kit, Takara). SYBR® Premix Ex Taq™ II (Takara) was used to mix 100 ng of cDNA samples and appropriate primers for the transforming growth factor β1 (TGF-β1) gene, the bone morphogenetic protein-2 (BMP-2) gene, the Sox9 gene, the Aggrecan gene, the cartilage oligomeric matrix protein (COMP) gene, and the Collagen, type II alpha 1 (Col2al) gene were selected for real-time polymerase chain reaction (real-time PCR). The primer sequence of each gene is shown in Table 7.

TABLE-US-00007 Gene name SEQ ID NO TGF-β1 Forward GGT TGA GCC GTG GAG GGG AAAT SEQ ID NO: 1 Reverse TGC CAT GAA TGG TGG CCA GGT SEQ ID NO: 2 BMP-2 Forward CGC TGT CTT CTA GCG TTG C SEQ ID NO: 3 Reverse TCG TCA GAG GGC TGG GAT G SEQ ID NO: 4 Sox9 Forward GGC AAG CTC TGG AGA CTT CTG SEQ ID NO: 5 Reverse CTG CAG CGC CTT GAA GAT G SEQ ID NO: 6 Aggrecan Forward TCG AGG ACA GCG AGG CC SEQ ID NO: 7 Reverse TCG AGG GTG TAG CGT GTA GAGA SEQ ID NO: 8 COMP Forward ACA ATG ACG GAG TCC CTG AC SEQ ID NO: 9 Reverse TCT GCA TCA AAG TCG TCC TG SEQ ID NO: 10 Col2al Forward GAG ACA GCA TGA CGC CGA G SEQ ID NO: 11 Reverse GCG GAT GCT CTC AAT CTG GT SEQ ID NO: 12 GAPDH Forward TGC CCC CGA CCG TCT AC SEQ ID NO: 13 Reverse ATG CGG TTC CAG CCT ATC TG SEQ ID NO: 14

[0073] After the mRNAs isolation of the adipose-derived stem cell preparation in the form of three-dimensional stem cell spheres prepared in Example 1, the mRNAs were reverse transcribed into cDNAs, and real-time PCR was performed. The expression level of each gene was analyzed and recorded, as shown in Table 8 and FIG. 10. Table 8 and FIG. 10 show the expression level of mRNA in the cells after human adipose-derived stem cells (hADSCs) have been cultured in two (2D) and three dimensions (3D) (that is, the adipose-derived stem cell preparation in the form of three-dimensional stem cell spheres in the present invention) on day 14.

TABLE-US-00008 Relative mRNA Expression 2D 3D TGF-β1 1 2.43 ± 0.03 BMP-2 1 20.84 ± 5.76 Sox9 1 8.14 ± 1.37 Aggrecan 1 4.52 ± 1.53 COMP 1 23.85 ± 10.21 Col2al 1 23.76 ± 7.74

[0074] As shown in Table 8 and FIG. 10, the expression levels of the TGF-β1 gene, the BMP-2 gene, the Sox9 gene, the Aggrecan gene, the COMP gene and the Col2al gene in the three-dimensional cultured cells of hADSC after 14 days of culture are significantly increased compared to those of the two-dimensional cultured cells. The range of increase is about 2 to 25 times.

[0075] Table 9, FIG. 11A and FIG. 11B show the expression level of mRNA in the cells after human umbilical cord mesenchymal stem cells have been cultured in two (2D) and three dimensions (3D) (that is, the stem cell preparation in the form of three-dimensional stem cell spheres in the present invention prepared by referring to the method described in Example 1) on day 14. The mRNA expression of the 2D group is taken as 1, and the relative mRNA expression is calculated. It should be noted that in FIG. 11B, due to the large difference in the relative mRNA expressions between the two groups and the widening of the Y-axis spacing, the mRNA expressions of the 2D group cannot be clearly displayed, but it does not mean that there is no COMP gene expression in the 2D group.

TABLE-US-00009 Relative mRNA Expression 2D 3D TGF-β1 1 3.70 ± 1.47 BMP-2 1 0.9 ± 0.7 Sox9 1 0.9 ± 0.22 Aggrecan 1 2.6 ± 0.25 COMP 1 1386 ± 186 Col2al 1 0.9 ± 0.43

[0076] As shown in Table 9, FIG. 11A and FIG. 11B, the expression levels of the TGF-β1 gene, the Aggrecan gene, and the COMP gene in the three-dimensional cultured human umbilical cord mesenchymal stem cells after 14 days of culture are significantly increased compared to those of the two-dimensional cultured cells. The range of increase is about 2.4 to 1572 times.

[0077] In summary, the effect of the stem cell preparation of the present invention is that treating rats with arthritis induced by mono-iodoacetate (MIA), using a gait analyzer and hematoxylin & eosin (H&E) staining and toluidine blue staining for treatment evaluation, and evaluating the feasibility of applying the stem cell preparation to the treatment of arthritis. It was found that the stem cell preparation of the present invention can effectively delay arthritis (especially degenerative arthritis) caused by cartilage degradation, and whole blood analysis and blood biochemical analysis confirmed that the stem cell preparation in the form of three-dimensional stem cell spheres can provide a safe treatment for arthritis (especially degenerative arthritis). In addition, the current cell therapy products are mostly two-dimensional cells. However, under the two-dimensional culture system, the cell morphology, differentiation, cell-cell interaction, cell-matrix interaction, and in vivo physiological conditions, there are obvious differences. The stem cell preparation of three-dimensional stem cell spheres is used in the present invention for the treatment of arthritis (especially degenerative arthritis). Taking into account the manufacturing specifications of drugs, the closed biological response system and the biological scaffold with three-dimensional porous material can be used to achieve the advantages of sterility, multi-batch, stable production, and the like. It is believed to increase the chance of success and provide new treatment strategies for arthritis (especially degenerative arthritis) in the future.

[0078] Although the present invention has been described with reference to the preferred embodiments, it will be apparent to those skilled in the art that a variety of modifications and changes in form and detail may be made without departing from the scope of the present invention defined by the appended claims.