METHOD FOR PREPARING MATRILIN-3 PRETREATED STEM CELL SPEROIDS, AND COMPOSITION, DERIVED THEREFROM, FOR PREVENTING OR TREATING CARTILAGE DISEASES

Abstract

Provided are a method of preparing a spheroid of stem cells and a composition including the spheroid prepared by the method, the method including: culturing stem cells in a medium supplemented with matrilin-3 protein; and performing 3D cell culture on the cultured stem cells in the medium. The composition disclosed herein has effects of preventing or treating cartilage disease. In detail, the composition may be able to further promote cartilage differentiation of adult stem cells and reduce dedifferentiation and hypertrophy that may occur during cartilage regeneration, thereby providing a more effective cartilage tissue regeneration method.

Claims

1. A method of preparing a spheroid of stem cells, the method comprising: culturing stem cells in a medium supplemented with matrilin-3 (MATN-3) protein; and performing 3D cell culture on the cultured stem cells.

2. The method of claim 1, wherein the medium supplemented with the MATN-3 protein has a MATN-3 protein concentration in a range of about 5 ng/ml to about 50 ng/ml.

3. The method of claim 1, wherein a period of the culturing of the stem cells in the medium supplemented with the MATN-3 protein is in a range of about 80 hours to about 130 hours.

4. The method of claim 1, wherein the stem cells are derived from a human.

5. The method of claim 1, wherein the stem cells are adipose-derived mesenchymal stem cells.

6. The method of claim 1, wherein the 3D cell culture is selected from the group consisting of pellet culture, static suspension culture, spinner/rotational chamber culture, nano/micro pattern culture, magnetic levitation culture, solid-scaffold-in-well culture, hydrogels-in-well culture, hydrogels-on-micropillar culture, hydrogels-in-microchannel culture, hang-in-drop culture, U-shape-well culture, and V-shape well culture.

7. The method of claim 1, wherein the 3D cell culture is performed in a range of about 50 cells per microwell to about 500 cells per microwell.

8. A stem cell spheroid prepared by the method according to claim 1.

9. A pharmaceutical composition for preventing or treating cartilage disease, comprising the stem cell spheroid prepared by the method according to claim 1.

10. The pharmaceutical composition of claim 9, wherein the pharmaceutical composition promotes differentiation into chondrocytes and inhibits hypertrophy and dedifferentiation of chondrocytes.

11. The pharmaceutical composition of claim 9, wherein the cartilage disease is selected from the group consisting of degenerative intervertebral disc, degenerative disc, intervertebral disc herniation, osteoarthritis, degenerative arthritis, osteomalacia, cartilage injury, and cartilage defect.

12. A method of preventing, improving, or treating obesity or a metabolic disease, the method comprising administering an effective dose of the pharmaceutical composition of claim 9 to an individual in need thereof.

13. Use of the pharmaceutical composition of claim 9 for preparation of a composition for preventing or treating cartilage disease.

Description

BRIEF DESCRIPTION OF DRAWINGS

[0046] FIG. 1 is an image showing a spheroid formation method according to an embodiment and the sequence of spheroid efficacy confirmation.

[0047] FIG. 2 is a graph showing increased matrilin-3 mRNA and protein expression levels when adipose-derived stem cells are cultured in a culture medium that induces differentiation into cartilage.

[0048] FIG. 3 is a graph showing increase expression levels of collagen 2 and aggrecan, which are markers related to cartilaginification, in stem cells that are cultured after the stem cells are primed with MATN-3.

[0049] FIGS. 4A to 4F are graphs showing increased expression levels of collagen 2 and aggrecan, which are markers related to cartilaginification, in a 3D pellet tissue after the stem cells are respectively primed with matrilin-3 at different concentrations.

[0050] FIGS. 5A to 5C show experiment results obtained under conditions that a dose of matrilin-3 for priming adipose-derived stem cells is 10 ng/ml, 20 ng/ml, and 50 ng/ml and a period of priming is 1 day (24 hours), 3 days (72 hours), and 5 days (120 hours), wherein G1 refers to a group of stem cells only, G2 refers to a group of stem cells primed with 10 ng/ml of matrilin-3, G3 a group of stem cells primed with 20 ng/ml of matrilin-3, and G4 refers to a group of stem cells primed with 50 ng/ml of matrilin-3. Here, A shows the results obtained by live and dead assay, B shows the results obtained by proliferation assay, and C shows the results obtained by cytokine analysis based on mRNA expression levels of cartilage-related markers, such as SOX9, collagen 2, and aggrecan.

[0051] FIG. 6 is an image showing a total of six conditions designed to establish optimal experimental conditions, wherein the six conditions include: a condition of a monolayer of adipose-derived stem cells; a condition of a monolayer of matrilin-3-primed adipose-derived stem cells; a condition of a matrilin-3-primed adipose-derived stem cell spheroid formed of 125 cells per microwell; a condition of a matrilin-3-primed adipose-derived stem cell spheroid formed of 125 cells per microwell; a condition of a matrilin-3-primed adipose-derived stem cell spheroid formed of 250 cells per microwell; and a condition of a matrilin-3-primed adipose-derived stem cell spheroid formed of 500 cells per microwell.

[0052] FIG. 7 is a graph showing experiment results obtained to establish optimal experimental conditions when forming a spheroid after priming of adipose-derived stem cells with matrilin-3 for 5 days, wherein G1 refers to a MSC monolayer culture group; G2 refers to a matrilin-3-primed MSC monolayer culture group; G3 refers to a MSC spheroid group; G4 refers to a matrilin-3-primed MSC spheroid group (formed of 125 cells per microwell); and G5 refers to a matrilin-3-primed MSC spheroid group (formed of 250 cells per microwell. FIG. 7A shows results of live and dead assay; FIG. 7B shows mRNA expression levels of p53 and BAX that are apoptosis markers; FIG. 7C shows results of human cytokine array analysis used to determine growth factors and cytokines in conditioned media of Ad-MSCs and primed Ad-MSCs; FIG. 7D is an image showing signal densities normalized as a positive control; and FIG. 7E is an image showing signal densities normalized as a positive control.

[0053] FIG. 8 is a graph showing results that cartilage differentiation markers increase and cartilage hypertrophy markers decrease when co-culturing matrilin-3-primed adipose-derived stem cell spheroids and degenerative nucleus pulposus (dNP) cells. FIG. 8A shows mRNA expression levels of SOX9, collagen 2, and aggrecan, which are cartilage differentiation markers, in dNPs; and FIG. 8B shows mRNA expression levels of cartilage hypertrophy markers, such as collagen 10, collagen 1, and MMP13 in dNP (***p<0.001, **p<0.01, * p<0.05).

[0054] FIG. 9 is a graph showing results obtained by co-culturing matrilin-3-primed adipose-derived stem cell spheroids and dNP cells. FIG. 9A shows immunofluorescence images of cadherin 2 and fluorescent intensity of the dNP cells, FIG. 9B shows immunofluorescence images of chondroitin sulphate and fluorescent intensity of the dNP cells, and FIG. 9C shows immunofluorescence images of collagen I and fluorescent intensity of the dNP cells (***p<0.001, **p<0.01, * p<0.05).

[0055] FIG. 10 shows images depicting a process of preparing a rabbit model with degenerative lumbar intervertebral disc by a retroperitoneal approach, wherein an image on the right shows that an 18-gauge needle is selected for insertion as a result of using various sizes of spinal needles.

[0056] FIG. 11 is an image showing T MRI results to verify regeneration of degenerative lumbar intervertebral disc, wherein a dotted line arrow indicates a defected site, and a solid line arrow indicates a normal site in contrast to the defected site. From left to right, spinal MRI analysis images of a group of non-primed cells, a group to which only stem cells are administered, a group to which matrilin-3-primed stem cells are administered, a group to which stem cell spheroids are administered, and a group to which matrilin-3-primed stem cell spheroids are administered are shown.

[0057] FIG. 12 is a graph showing the results of Masson's trichrome staining on tissues to clarify the MRI results in more detail. From top to bottom, histological analysis results of a group of non-primed cells, a group to which only stem cells are administered, a group to which matrilin-3-primed stem cells are administered, and a group to which stem cell spheroids are administered are shown. G5 at the bottom shows a histological analysis result of a group to which adipose-derived stem cell spheroids are administered.

[0058] FIG. 13 shows a schematic diagram depicting experiment results.

MODE OF DISCLOSURE

[0059] Hereinafter, the present disclosure will be described in more detail through Examples. However, these Examples are for illustrative purposes of the present disclosure only, and the scope of the present disclosure is not limited thereto.

Example 1. Confirmation of Effect of Matrilin-3 (MATN-3) Protein on Cartilage Differentiation of Human Adipose-Derived Stem Cells

[0060] To confirm effect of MATN-3 protein on cartilage differentiation of human adipose-derived stem cells, the inventors of the present disclosure used human adipose-derived stem cells for MATN-3 priming.

[0061] 1.1. Isolation of Human Adipose-Derived Stem Cells

[0062] To isolate human adipose-derived stem cells, adipose tissues to be removed and discarded by liposuction were collected and washed with phosphate buffered saline (PBS). The washed adipose tissues were treated with 1.5 mg/ml of collagenase, and then filtered through a 70 μm-scale nylon mesh. Red blood cells were removed from the filtrate by using a hemolysis buffer solution (0.15 M NH.sub.4Cl, 10 mM KHCO.sub.3, 0.1 mM EDTA), and a washing process was performed thereon twice by using PBS, so as to obtain adipose-derived stem cells. The adipose-derived mesenchymal stem cells thus obtained were seeded at a concentration of 1×10.sup.4/cm.sup.2 on a culture plate containing a DMEM medium supplemented with 10% fetal bovine serum (FBS) and 1% antibacterial agent, and cultured with 5% CO.sub.2 at 37° C. When the cells covered about 80% of the bottom area of the culture plate, the cells attached to the bottom surface were isolated from the culture plate by using trypsin/EDTA. Then, the isolated cells were centrifuged at 1,200 rpm for 5 minutes, suspended again in the same medium, and subcultured in the same manner three times. Accordingly, passage 3 cells were used for next experiments.

[0063] 1.2. Confirmation of Increased Expression Level of MATN-3 by Chondrogenesis Induction of Stem Cells

[0064] A total of 2×10.sup.5 adipose-derived stem cells were collected in a 15 mL falcon tube, and centrifuged at 120 rpm for 3 minutes. A culture containing pellets of adipose-derived stem cells obtained by the centrifugation was cultured with 5% CO.sub.2 at 37° C. The pellets were divided into two groups, and one group was treated with a serum free (SF) medium, and the other group was treated with chondrogenic (CM) medium. The CF medium was supplemented with DMEM-high glucose, 10% fetal bovine serum, 100× insulin-transferrin-selenium (ITS), 50 ng/ml of ascorbic acid, 100-nM dexamethasone, 1× penicillin and streptomycin, and 10-ng/ml of TGF-β. Each medium was changed every 3 days with a fresh medium, and after 21 days of the culture, the increase in the MATN-3 expression of the adipose-derived stem cells cultured in each of the SF medium and the CF medium was compared.

[0065] Consequently, it was confirmed that the MATN-3 mRNA and protein expression levels increased by the induction of chondrogenesis of the stem cells (see FIG. 2). Furthermore, it was also confirmed that, when the adipose-derived stem cells were cultured in a culture medium for inducing cartilage differentiation, the MATN-3 secretion was increased (see FIG. 3). Accordingly, the inventors of the present disclosure could confirm that MATN-3 was a protein related to the cartilage differentiation.

[0066] 1.3. Analysis of Expression of Cartilage-Related Genes

[0067] To confirmed effects of the MATN-3 protein priming on the expression of cartilage genes in the adipose-derived stem cells, passage 3 cells were collected, divided into groups for every 2×10.sup.5 adipose-derived stem cells, and centrifuged at 1,200 rpm for 3 minutes. The resultant cells were cultured for 24 hours in the form of pellets in a FBS-free DMEM medium as being divided into a group with MATN-3 protein and a group without MATN-3 protein. After 24 hours of the culture, for quantitative analysis of the obtained cells after removing the medium, quantitative real-time polymerase chain reaction (qRT-PCR) was performed to measure RNA expression of the cartilage-related genes. That is, the obtained cell pellets were washed with PBS three times, and collected by using trypsin/EDTA. Then, RNA of the cells was extracted according to a TRIzol method (Life Technologies, Inc. Grand Island, N.Y.). 1 μg of the extracted RNA was used to synthesize cDNA by using a cDNA synthesis kit (AB biosystems), and qRT-PCR was performed thereon by using Master SYBR green (AB biosystems). Cell normalization was performed by using glyceraldehyde 3-phosphate dehydrogenase (GAPDH), and primer sets and respective cartilage-related gene markers used in the qRT-PCR are as shown in Table 1. Accordingly, the inventors of the present disclosure were able to confirm that the MATN-3 protein priming increased the expression of cartilage-related genes (see FIG. 4).

TABLE-US-00001 TABLE 1 Accession Amplicon Gene Primer sequence number (bp) 18S 5′-GTA ACC CGT TGA NR_003286.2 151 ACC CCA TT-3′5′-CCA TCC AAT CGG TAG TAG CG-3′ SOX9 5′- GTA CCC GCA CTT NM_000346.3 74 GCA CAA C-3′5′- TCT CGC TCT CGT TCA GAA GTC-3′ Collagen 5′- GGGAGTAATGCAAGG NM_001844.4 175 2a ACCA - 3′5′-ATCATCA CCAGGCTTTCCAG -3′ Aggrecan 5′- GCC TGC GCT CCA NM_013227.3 104 ATG ACT - 3′5′-ATG  GAA CAC GAT GCC TTT CAC -3′

Example 2. Preparation Process of MATN-3-Primed Adipose-Derived Stem Cell Spheroid and Confirmation of Regeneration Effects of the MATN-3-Primed Adipose-Derived Stem Cell Spheroid

[0068] 2.1. Determination of MATN-3 Dose and Period with Respect to Adipose-Derived Stem.

[0069] As stem cells for priming with MATN-3, human adipose-derived stem cells were used. First, cells were not fed with nutrients for 12 hours, and subjected to a priming process. Here, determining appropriate MATN-3 concentration and priming period was a significant concern. In the present experiment, to establish appropriate MATN-3 concentration for priming of the adipose-derived stem cells, MATN-3 concentration conditions were set to 10 ng, 20 ng, and 50 ng, and priming period conditions were was set to 1 day, 3 days, and 5 days. Afterwards, the cells were collected.

[0070] To determine whether the MATN-3 dose and the priming period were optimal conditions, the inventors of the present disclosure transported the cells to a 6-well plate (EZSPHERE). After 48 hours, cytokine array was performed thereon. For the cytokine array, a film customized by using C-series of RayBiotech Ltd based on the sandwich immunoassay principles was used. The customized film was designed as shown in Table 2. Regarding the results visualized in digital images after the measurement, the fold change was calculated for each protein, and the expression levels of SOX9, collagen 2, and aggrecan were compared with one another. As a result, it was confirmed that, when the human adipose-derived stem cells were isolated, the MATN-3 concentration was 10 ng/ml, and the MATN-3 priming period was 5 days, the greatest gene content and the highest synthesis degree were resulted (see FIG. 5B), and that the cartilage differentiation markers (e.g., collagen 2 and aggrecan) were expressed to the maximum. Consequently, it was confirmed that the concentration and period conditions above were optimal conditions to exhibit maximum effects on the cartilage differentiation by MATN-3 priming to the stem cells.

TABLE-US-00002 TABLE 2 A B C D E F G H I J K L 1 POS POS NEG NEG TGF-β1 TGF-β2 TGF-β3 SDF-1 bFGF EGF G-CSF RANTES (CCL5) 2 POS POS NEG NEG TGF-β1 TGF-β2 TGF-β3 SDF-1 bFGF EGF G-CSF RANTES (CCL5) 3 IL-11 IL-1β IL-6 MMP-1 MMP-9 TNF-α TIMP-1 TIMP-2 HGF VEGF IGF-1 GDF-15 4 IL-11 IL-1β IL-6 MMP-1 MMP-9 TNF-α TIMP-1 TIMP-2 HGF VEGF IGF-1 GDF-15 5 BMP-2 BMP-7 BMP-9 Adipsin MMP-13 Activin-A IL-4 IL-10 Matrilin-3 IL-1ra BLANK POS 6 BMP-2 BMP-7 BMP-9 Adipsin MMP-13 Activin-A IL-4 IL-10 Matrilin-3 IL-1ra BLANK POS

[0071] 2.2. Standardization of Spheroid Formation Conditions after MATN-3 Priming

[0072] Next, the inventors of the present disclosure carried out an experiment to determine the optimal culture environment conditions for the spheroid formation after MATN-3 priming. A total of six culture conditions were set up as follows: a condition of adipose-derived stem cell monolayer; a condition of MATN-3-primed adipose-derived stem cell monolayer; a condition of adipose-derived stem cell spheroids formed of 125 cells per microwell; a condition of MATN-3-primed adipose-derived stem cell spheroids formed of 125 cells per microwell; a condition of MATN-3-primed adipose-derived stem cell spheroids formed of 250 cells per microwell; and a condition of MATN-3-primed adipose-derived stem cell spheroids formed of 500 cells per microwell (see FIG. 6).

[0073] The human adipose-derived stem cells were placed in a cell culture plate, and cultured with 5% CO.sub.2 at 37° C. After 12 hours of the culture, the culture medium was changed to a serum starvation medium (supplemented with DMEM-LG and 1× penicillin and streptomycin), and cultured for 12 hours in a CO.sub.2 incubator. After 12 hours of serum starvation, the culture medium was supplemented with 10 ng/mL of MATN-3. The culture medium was changed every 24 hours with fresh MATN-3 supplement for 5 days. Next, the stem cells were transported to a 6-well plate (EZSPHERE), and then seeded at a density of 125 cells per microwell. 3 mL of a mixed solution containing 10% fetal bovine serum (FBS) and gentamicin (50 μg/ml) in Dulbecco's modified Eagle's medium (DMEM)-low glucose (LG) was added thereto and cultured for 24 hours in a CO.sub.2 incubator with 5% CO.sub.2 at 37° C. As such, spheroids were formed through this process. Consequently, it was confirmed that, when 125 cells were seeded, apoptosis markers were expressed the least (see FIG. 7B) while cytokines related to the cartilage formation were secreted the most (see FIGS. 7C to 7E).

[0074] As a result of comprehensive analysis on the concentration conditions, the culture environments, and the culture period for the MATN-3 priming to the adipose-derived stem cells, it was confirmed that the optimized culture environment system was established and the most excellent effects of cartilage formation were exhibited when the spheroids were formed under conditions that the MATN-3 concentration was 10 ng/ml, the culture period was 5 days, and 125 cells per microwell were seeded.

Example 3. Analysis of Regenerative Effect of MATN-3-Primed Spheroid on Degenerated Nucleus Pulposus Cells

[0075] 3.1. Co-Culture of MATN-3-Primed Spheroids and Nucleus Pulposus Cells

[0076] To confirm regenerative effect of the spheroids formed by the method disclosed herein on cartilage nucleus pulposus cells, the inventors of the present disclosure carried out the following experiments. First, spheroids were formed as described in Example 2.2. Then, nucleus pulposus cells were collected for 10 days from a patient undergoing surgery for degenerative lumbar intervertebral disc. After obtaining the approval from Institutional Review Board (IRB) in a hospital, informed consent was obtained in advance from a patient undergoing discectomy for cervical or lumbar herniation of nucleus pulposus. The nucleus pulposus and annulus fibrosus were isolated from the intervertebral disc obtained during surgery, and the nucleus pulposus cells were isolated from the nucleus pulposus. To isolate the nucleus pulposus cells from the nucleus pulposus, disc tissues were washed three times, each for 15 minutes, by using Dulbecco's phosphate-buffered saline (DPBS; Hyclone Laboratories) containing 1% penicillin and streptomycin (Gibco, BRL, USA). The tissue samples were digested with 0.05% (w/v) type 2 collagenase (Sigma Aldrich, St Luis, N.J., USA) for 6 hours. The digested mixture was transported to a cell strainer (40 μm pore size, Becton Dickinson, Franklin Lakes, N.J., USA), centrifuged at 1,000 rpm for 5 minutes, and washed with HBSS twice to remove the remaining collagenase. The cells were suspended in DMEM-LG supplemented with 10% FBS, 0.1 mg/ml of streptomycin, and 100 μg/ml of penicillin, and cultured until the cells were 85% confluent. The nucleus pulposus cells and the MATN-3-primed spheroids were used for co-culture experiments.

[0077] 3.2. Confirmation of Regenerative Effect of MATN-3-Primed Spheroid on Nucleus Pulposus Cell

[0078] The inventors of the present disclosure carried out the co-culture method of Example 3.1, and confirmed the expression of the cartilage-related markers for quantitative analysis of cells. The quantitative analysis was carried out by measuring RNA levels of the cartilage-related markers. RNA was extracted by using a TRIzol kit (ThermoFisher Scientific, Inc., Waltham, Mass., USA). Then, complementary DNA was subsequently prepared by using 0.5 μg of RNA with the Primescript RT reagent kit (Takara Bio Inc, Japan). RT-PCR amplification was performed on the complementary DNA by using the StepOnePlus Real Time PCR System. After the amplification, relative mRNA expression levels were calculated for each target gene. Such calculation used a 2.sub.−ΔCt method with the expression level of 18-S as an internal control. Target primers used for real-time (RT)-PCR analysis are shown in Table 3.

TABLE-US-00003 TABLE 3 Accession Amplicon Gene Primer sequence number (bp) 18S 5′- GTA ACC CGT NR_003286.2 151 TGA ACC CCA TT- 3′5′-CCA TCC AAT CGG TAG TAG CG- 3′ SOX9 5′- GTA CCC GCA NM_000346.3 74 CTT GCA CAA C- 3′5′- TCT CGC TCT CGT TCA GAA GTC -3′ Collagen 5′- GGGAGTAATGCA NM_001844.4 175 2a AGGACCA - 3′5′ - ATCATCACCAGGCTTT CCAG - 3′ Aggrecan 5′- GCC TGC GCT NM_013227.3 104 CCA ATG ACT - 3′ 5′- ATG GAA CAC GAT GCC TTT CAC - 3′ Collagen 5′- CCC CTG GAA NM_000088.3 148 A1 AGA ATG GAG ATG- 3′5 -TCC AAA CCA CTG AAA CCT CTG - 3′ MMP13 5′- TCA CCA ATT NM_002427.3 95 CCT GGG AAG TCT - 3′5′- TCA GGA AAC CAG GTC TGG AG -3′ Collagen 5′- ACG CTG AAC NM_000493.3 101 10 GAT ACC AAA TG - 3′5′- TGC TAT ACC TTT ACT CTT TAT GGT GTA- 3′ p53 5′- GGCCCACTTCAC NM_000546 156 CGTACTAA - 3′5′- GTGGTTTCAAGGCCAG ATGT - 3′ BAX 5′- TTTGCTTCAGGG NM_001291428.1 246 TTTCATCC - 3′5′- CAGTTGAAGTTGCCGT CAGA - 3′

[0079] As a result, it was confirmed that, when the MATN-3-primed adipose-derived stem cell spheroids were co-cultured with the degenerated nucleus pulposus cells, the expression levels of the cartilage differentiation markers, i.e., SOX9, Collagen 2, and Aggrecan, were increased, whereas the expression levels of cartilage hypertrophy marker, i.e., Collagen 10 and Collagen 1, were decreased (see FIG. 8). Furthermore, immunohistochemical staining was performed to confirm the effect of the MATN-3-primed adipose-derived stem cell spheroids on the degenerated nucleus pulposus cells. For the immunohistochemical staining, the degenerative nucleus pulposus cells were co-cultured for 10 days with MATN-3-primed adipose-derived stem cell spheroids and MATN-3-non-primed adipose-derived stem cell spheroids. These cells were fixed for 10 minutes with 4% paraformaldehyde at room temperature, washed three times with 1×PBS, and permeabilized with 0.5% Triton-X for 10 minutes. The resultant cells were washed with PBS and blocked for 45 minutes in blocking buffer (5% BSA and 0.5% Tween-20 in 1×PBS) containing 10% normal goat serum (5% BSA and 0.5% Tween-20 in 1×PBS) at room temperature. For immunostaining, the cells were cultured overnight at 4° C. with cadherin-2 (1:200, Abcam), chondroitin sulfate (1:100, Abcam), and collagen 1 (1:200, Abcam). Then, the cells were incubated at a constant temperature with secondary antibodies, i.e., goat anti-rabbit Alexa Fluor® 568 and goat anti-mouse Alexa Fluor® 488 (Abcam), for 1 hour at room temperature. The cells were counter-stained with DAPI (Vector Laboratories, Burlingame, Calif., USA), and images were obtained by using Cytation 3 Cell Imaging Multi-mode Reader (Biotek Instruments, Inc., Winooski, Vt., USA). The detected autofluorescence intensities were used to analyze the expression.

[0080] When the MATN-3-primed adipose-derived stem cell spheroids were co-cultured with the degenerated nucleus pulposus cells, the regeneration of the degenerated nucleus pulposus cells and the recovery of the extracellular matrix components were observed. In particular, it was confirmed that, among the extracellular matrix components, the expression levels of cadherin 2 and chondroitin sulfate increased (see FIGS. 9A and 9B), whereas the expression level of collagen 1 decreased (FIG. 9C).

Example 4. Confirmation of Cartilage Regeneration Effect of MATNn-3-Primed Adipose-Derived Stem Cell Spheroids in Rabbit Model with Degenerative Disc Disease

[0081] 4.1 Preparation of Rabbit Model and Transplantation of Therapeutic Material

[0082] Effects of the MATN-3-primed adipose-derived stem cell spheroids on disc regeneration in an animal model with degenerative disc disease were evaluated.

[0083] Here, a total of five classified groups were used as follows: G1 refers to a group to which only adipose-derived stem cells were injected; G2 refers to a group to which MATN-3-primed adipose-derived stem cells were injected; G3 refers to a group to which adipose-derived stem cell spheroids were injected; refers to a group to which MATN-3-primed adipose-derived stem cell spheroids were injected; and G5 refers to a group to which MATN-3-primed adipose-derived stem cell spheroids were injected. All groups were set to include three female rabbits.

[0084] For the preparation of the rabbit model, New Zealand white rabbits (2.5 kg or more in weight) were used as animal models with degenerative lumbar intervertebral disc in accordance with permission from the Institutional Animal Care and Use Committee of CHA University (see FIG. 10). The experiments carried out herein were non-clinical experiments on the intervertebral disc regeneration therapy by using MATN-3-primed stem cell spheroids. For surgery, the rabbits were anesthetized by intramuscular injection of a mixture of 15 mg/kg of Zoletil and 5 mg/kg of Rompun. The incision was made after anesthesia, and the lumbar disc was directly exposed through a retroperitoneal approach after the incision. The procedure was performed on L3/4 discs, L4/5 discs, and L5/6 discs. In detail, the intervertebral discs between the L3/4 discs, the L4/5 discs, and the L5/6 discs were exposed. Then, an 18-gauge spin needle was inserted to inflict damage to the intervertebral discs. At the time of induction of degenerative lumbar intervertebral discs, a therapeutic substance was inserted into each experimental group. Regarding the route of administration, to induce degenerative lumbar intervertebral disc, the intervertebral disc of the lumbar number was exposed through a skin incision on the opposite side of the surgery site, a therapeutic material was directed injected. The administration period was 2 weeks after model the preparation of the disc model, and the number of administration was a single administration. The administration method was to inject 26-gauge spinal needle to death of 5 mm after anesthesia. No immunosuppressants were administered at the time of administration. At 12 weeks after the first insertion of the therapeutic material, MRI and histological examination were performed for efficacy evaluation.

[0085] 4.2. Confirmation of MRI Results

[0086] In the present experiment, MRI was performed to evaluate the degree of degenerative changes in the intervertebral discs. Among the prepared rabbit models, rabbits graded as Pfirrmann grade 3, which represents the standard of degeneration, were used. In the present experiments, as imaging index, T2-weighted images through MRI (wherein time to repetition of 2,000 ms and time to echo of 120 ms) was used. MRI scans of rabbit models in five groups were performed. As a result, it was confirmed that the regeneration of the degenerative lumbar intervertebral disc was confirmed in all groups including a Sham group, an adipose-derived cell injection group, a matrilin-3-primed adipose-derived cell injection group, and a matrilin-3-primed adipose-derived cell spheroid. However, regarding the degree of the effect, it was confirmed that the matrilin-3-primed adipose-derived spheroid injection group of the present disclosure showed the highest signal intensity level and the greatest regenerative effect as compared with the other groups. As a result, the effect of the matrilin-3-primed adipose-derived spheroids on the degenerative lumbar intervertebral disc was confirmed. It addition, it was also confirmed that the effect above may be obtained superior to that of the matrilin-3-primed adipose-derived stem cells.

[0087] 4.3. Confirmation of Histological Analysis Results

[0088] To clarify the MRI results in more detail, tissue staining was performed by Masson's trichrome method. The staining was carried out as follows: cell nucleus was stained in a Weigert iron hematoxylin solution for 10 minutes, cytoplasm and muscles were stained in a Biebrich scarlet-acid fuchsin solution for 15 minutes, and collagen fibers were stained in 2% Aniline blue solution for 3 minutes. After performing the staining, the results were observed under a microscope. As a result, it was confirmed that the group to which the matrilin-3-primed adipose-derived stem cell spheroids were injected showed better regeneration of intervertebral discs than other groups. In addition, it was confirmed that the matrilin-3-primed adipose-derived stem cell spheroids exhibited better effects than the matrilin-3-primed stem cells (see FIG. 12).