INJECTION FORMULATION COMPOSITION CONTAINING MESENCHYMAL STEM CELL-HYDROGEL AND METHOD FOR PREPARING, FREEZING AND DEFROSTING SAME

Abstract

The present invention relates to a composition containing injectable mesenchymal stem cell-hydrogel and a method of preparing the same. Specifically, in a mesenchymal stem cell-hydrogel composition for injection prepared by a method of the present invention, since stem cells are attached to scaffolds in hydrogel beads, the stem cells are not easily lost or killed after the injection, and thus there is an advantage that an engraftment rate increases since the paracrine effect of the stem cells is continuously exhibited, and the stem cells are gradually released as hydrogel is degraded. In addition, the present invention has an advantage that healthy cells can be used without damages in cell membranes since injection formulation can be prepared without a treatment with proteolytic enzymes, and also a cryopreservation solution is easily removed from the mesenchymal stem cell-hydrogel beads even after freezing and thawing.

Claims

1. A cell-hydrogel composition comprising: cells; and hydrogel, wherein the cell-hydrogel composition has a bead form, and the cells and the hydrogel have a diameter of 0.1 mm to 5 mm.

2. The composition according to claim 1, wherein the cells and the hydrogel have a diameter of 1 mm to 4 mm.

3. The composition according to claim 1, wherein the cell is any one selected from the group consisting of a stem cell, a somatic cell, and a germ cell.

4. The composition according to claim 1, wherein the hydrogel is any one selected from the group consisting of fibrin glue, hyaluronic acid, gelatin, collagen, alginic acid, chitosan, cellulose, pectin, 2-hydroxyethyl methacrylate derivative or a copolymer thereof, polyethylene oxide, and polyvinyl alcohol, or a complex of two or more thereof.

5. A pharmaceutical composition for preventing or treating a musculoskeletal disease, a fistula disease, or an inflammatory disease comprising: the composition according to claim 1 as an active ingredient.

6. The pharmaceutical composition for preventing or treating a musculoskeletal disease, a fistula disease, or an inflammatory disease according to claim 5, wherein the musculoskeletal disease is any one selected from the group consisting of an injury of a joint, a bone disease, muscle weakness, an injury of a joint caused by a nerve damage of the joint, a bone disease, muscle weakness, myositis caused by a nerve damage of a joint, a myofascial pain syndrome, tendinitis, tenosynovitis, bursitis, ganglion tumor, a carpal tunnel syndrome, Guyon's canal syndrome, wrist tendonitis, a hand-arm vibration syndrome, trigger finger, ganglion tumor, white finger, Raynaud's syndrome, lateral epicondylitis, medial epicondylitis, ulnar tunnel syndrome, olecranon bursitis, median nerve entrapment, a shoulder impingement syndrome, adhesive capsulitis, degenerative arthritis, turtle neck syndrome, cervical neuropathy, lumbar sprain, disc herniation, spondylolysis, spondylolisthesis, a degenerative lumbar disease, a degenerative disease, urinary incontinence, and a ligament and tendon damage.

7. The pharmaceutical composition for preventing or treating a musculoskeletal disease, a fistula disease, or an inflammatory disease according to claim 5, wherein the inflammatory disease is any one selected from the group consisting of atopic dermatitis, systemic lupus erythematosus, lupus, chilblain lupus, tuberculous lupus, lupus nephritis, dystrophic epidermolysis bullosa, psoriasis, rheumatoid fever, rheumatoid arthritis, back pain, fibromyalgia, myofascial disease, undifferentiated spondyloarthropathy, undifferentiated arthropathy, arthritis, inflammatory osteolysis, reactive arthritis, osteoarthritis, scleroderma, osteoporosis, chronic inflammatory disease caused by viral or bacterial infection, colitis, ulcerative colitis, inflammatory bowel disease, fungal infection, burns, a wound by a surgical or dental surgery, diabetic foot ulcer, type 1 diabetes, type 2 diabetes, ulcerative skin disease, sinusitis, rhinitis, conjunctivitis, asthma, dermatitis, inflammatory collagen vascular disease, glomerulonephritis, encephalitis, inflammatory enteritis, chronic obstructive pulmonary disease, sepsis, septic shock, pericarditis, cystic fibrosis, Hashimoto's thyroiditis, Graves' disease, leprosy, syphilis, Lyme disease, borreliosis, neurogenic borreliosis, tuberculosis, sarcoidosis, macular degeneration, macular degeneration, uveitis, irritable bowel syndrome, Crohn's disease, Sjogren's syndrome, a chronic fatigue syndrome, chronic fatigue immunodeficiency syndrome, myalgic encephalomyelitis, amyotrophic lateral sclerosis, Parkinson's disease, and multiple sclerosis.

8. An injection comprising: the composition according to claim 1.

9. The injection according to claim 8, wherein the injection is administered by any one selected from the group consisting of transdermal injection, subcutaneous injection, intramuscular injection, submucosal injection, and intraperitoneal injection.

10. A method of preparing a cell-hydrogel composition, the method comprising: (a) a step of culturing cells; (b) a step of mixing the cultured cells and a hydrogel solution to form a cell hydrogel bead of 0.1 mm to 5 mm; and (c) a step of culturing the cell hydrogel bead.

11. The method of preparing a cell-hydrogel composition according to claim 10, wherein the hydrogel is any one selected from the group consisting of fibrin glue, hyaluronic acid, gelatin, collagen, alginic acid, chitosan, cellulose, pectin, 2-hydroxyethyl methacrylate derivative of a copolymer thereof, polyethylene oxide, and polyvinyl alcohol, or a complex of two or more thereof.

12. The method of preparing a cell-hydrogel composition according to claim 10, wherein the hydrogel is fibrin glue.

13. The method of preparing a cell-hydrogel composition according to claim 10, wherein the fibrin glue contains fibrinogen at a concentration of 1.8 to 90 mg/mL.

14. The method of preparing a cell-hydrogel composition according to claim 10, wherein the cell hydrogel bead is 1 mm to 4 mm.

15. The method of preparing a cell-hydrogel composition according to claim 10, further comprising: a step of culturing the cell hydrogel bead of the step (c) and then freezing and thawing the cell hydrogel bead.

16. The method of preparing a cell-hydrogel composition according to claim 10, further comprising: (d) a step of filling the cell-hydrogel composition into a syringe after the step (c).

17. A method of preventing or improving a musculoskeletal disease, a fistula disease, or an inflammatory disease, the method comprising: a step of administering a cell-hydrogel composition including cells and hydrogel in pharmaceutically effective amounts, wherein the cell-hydrogel composition has a bead form, and the cells and the hydrogel have a diameter of 0.1 mm to 5 mm.

18. A method of treating a musculoskeletal disease, a fistula disease, or an inflammatory disease, the method comprising: a step of administering a cell-hydrogel composition including cells and hydrogel in pharmaceutically effective amounts, wherein the cell-hydrogel composition has a bead form, and the cells and the hydrogel have a diameter of 0.1 mm to 5 mm.

19. (canceled)

Description

BRIEF DESCRIPTION OF DRAWINGS

[0034] FIG. 1A is a view showing a photograph obtained by observing a cultured cells-hydrogel bead.

[0035] FIG. 1B is a view showing a micrograph of cell-hydrogel beads cryopreserved at −80° C., thawed, filled into a syringe, and sprayed by using a 17-gauge needle.

[0036] FIG. 2 is a view confirming cell characteristics in the cell-hydrogel bead.

[0037] FIG. 3 is a view confirming an activating factor captured in the cell-hydrogel bead.

[0038] FIG. 4 is a diagram confirming the paracrine factor secretion effect of the cell-hydrogel bead:

[0039] y axis: relative value (fold increase).

[0040] FIG. 5 is a view showing changes in volume for 7 days after subcutaneous injection of the cell-hydrogel bead of the present invention into a mouse.

[0041] FIG. 6 is a view confirming a chondrocyte apoptosis inhibition effect due to oxidative stress of the cell-hydrogel bead of the present invention.

[0042] FIG. 7 is a view confirming an anti-inflammatory effect of the cell-hydrogel bead of the present invention.

DESCRIPTION OF EMBODIMENTS

[0043] Hereinafter, the present invention is specifically described.

[0044] The present invention provides a cell-hydrogel composition including cells; and hydrogel, in which the cell-hydrogel composition has a bead form.

[0045] The present invention provides a use of a cell-hydrogel composition

[0046] including cells and hydrogel and having

[0047] a bead form,

[0048] as a pharmaceutical composition for preventing and treating a musculoskeletal disease, a fistula disease, or an inflammatory disease.

[0049] According to an aspect of the present invention, the diameter of the cell and the hydrogel is preferably 0.1 to 5 mm, more preferably 0.5 mm to 5 mm, and most preferably 1 mm to 4 mm.

[0050] According to an aspect of the present invention, the cell is preferably any one selected from the group consisting of a stem cell, a somatic cell, and a germ cell, but the present invention is not limited thereto.

[0051] According to an aspect of the present invention, the hydrogel is any one of two or more selected from the group consisting of fibrin glue, hyaluronic acid, gelatin, collagen, alginic acid, chitosan, cellulose, pectin, 2-hydroxyethyl methacrylate derivative or a copolymer thereof, polyethylene oxide, and polyvinyl alcohol, or two or more thereof.

[0052] According to an aspect of the present invention, the musculoskeletal disease is preferably any one selected from the group consisting of an injury of a joint, a bone disease, muscle weakness, myositis caused by a nerve damage of a joint, a myofascial pain syndrome, tendinitis, tenosynovitis, bursitis, ganglion tumor, carpal tunnel syndrome, Guyon's canal syndrome, wrist tendonitis, a hand-arm vibration syndrome, trigger finger, ganglion tumor, white finger, Raynaud's syndrome, lateral epicondylitis, medial epicondylitis, a radial tunnel syndrome, ulnar tunnel syndrome, olecranon bursitis, median nerve entrapment, a shoulder impingement syndrome, adhesive capsulitis, degenerative arthritis, turtle neck syndrome, cervical neuropathy, lumbar sprain, disc herniation, spondylolysis, spondylolisthesis, a degenerative lumbar disease, a degenerative disease, urinary incontinence, and a ligament and tendon damage, but the present invention is not limited thereto.

[0053] According to an aspect of the present invention, the fistula disease may be fistula Crohn's disease, but the present invention is not limited thereto.

[0054] According to an aspect of the present invention, the inflammatory disease is any one selected from the group consisting of atopic dermatitis, systemic lupus erythematosus, lupus, chilblain lupus, tuberculous lupus, lupus nephritis, dystrophic epidermolysis bullosa, psoriasis, rheumatoid fever, rheumatoid arthritis, back pain, fibromyalgia, myofascial disease, undifferentiated spondyloarthropathy, undifferentiated arthropathy, arthritis, inflammatory osteolysis, reactive arthritis, osteoarthritis, scleroderma, osteoporosis, chronic inflammatory disease caused by viral or bacterial infection, colitis, ulcerative colitis, inflammatory bowel disease, fungal infection, burns, a wound by a surgical or dental surgery, diabetic foot ulcer, type 1 diabetes, type 2 diabetes, ulcerative skin disease, sinusitis, rhinitis, conjunctivitis, asthma, dermatitis, inflammatory collagen vascular disease, glomerulonephritis, encephalitis, inflammatory enteritis, chronic obstructive pulmonary disease, sepsis, septic shock, pulmonary fibrosis, atherosclerosis, myocarditis, endocarditis, pericarditis, cystic fibrosis, Hashimoto's thyroiditis, Graves' disease, leprosy, syphilis, Lyme disease, borreliosis, neurogenic borreliosis, tuberculosis, sarcoidosis, macular degeneration, macular degeneration, uveitis, irritable bowel syndrome, Crohn's disease, Sjogren's syndrome, a chronic fatigue syndrome, chronic fatigue immunodeficiency syndrome, myalgic encephalomyelitis, amyotrophic lateral sclerosis, Parkinson's disease, and multiple sclerosis, but the present invention is not limited thereto.

[0055] The composition of the present invention exhibits a constant bead form having a diameter of 5 mm or less, and thus is easily filled into a syringe, and a cryopreservation solution can be removed by a physically simple method, so that DMSO that is the cryopreservation solution component that can cause various side effects in the body can be easily removed prior to administration. Specifically, in an example of a physically simple method, a cryopreservation solution and bead mixture are introduced into a syringe, a filter having a pore size of about 100 ul is mounted at the front end of the syringe, the solution is pushed, beads are caught in the filter, and only the cryopreservation solution can be removed.

[0056] In addition, after being filled into a syringe, the composition can be administered by transdermal injection, subcutaneous injection, intramuscular injection, submucosal injection, intraperitoneal injection, and the like.

[0057] In addition, in the present invention, the concentration of the hydrogel can be used without any particular limitation, can be adjusted according to various therapeutic purposes, and preferably contains fibrinogen at a concentration of 1.8 to 90 mg/mL.

[0058] The present invention provides a method of preparing a cell-hydrogel composition, including:

[0059] (a) a step of culturing cells;

[0060] (b) a step of mixing the cultured cells and a hydrogel solution to form a cell hydrogel bead; and

[0061] (c) a step of culturing the cell hydrogel bead.

[0062] According to an aspect of the present invention, as the hydrogel, any one selected from the hydrogel group consisting of fibrin glue, hyaluronic acid, gelatin, collagen, alginic acid, chitosan, cellulose, pectin, 2-hydroxyethyl methacrylate derivative or a copolymer thereof, polyethylene oxide, and polyvinyl alcohol, or a complex of two or more thereof may be used, and more specifically, more specifically, fibrin glue may be used.

[0063] According to an aspect of the present invention, the fibrin glue may contain fibrinogen in a concentration of 1.8 to 90 mg/mL.

[0064] According to an aspect of the present invention, the cell hydrogel bead is preferably 0.1 to 5 mm, more preferably 0.5 mm to 5 mm, and most preferably 1 mm to 4 mm.

[0065] According to an aspect of the present invention, after the step (c), a step (d) of filling the cell-hydrogel composition into a syringe can be further included. The cell-hydrogel composition prepared by the preparation method according to the present invention can be directly administered locally using a needle of 10 to 25 gauges.

[0066] Meanwhile, in the method according to the present invention, after the cell hydrogel of the step (c) is cultured, a freezing and thawing step can be additionally added, and a cryopreservation solution can be removed by a physically simple method after the freezing and thawing.

[0067] When the cell-hydrogel composition of the present invention is used for injection, the physical properties or physical strength of the hydrogel can be variously adjusted and used according to purposes, and the hydrogel concentration is not specifically limited.

[0068] In a specific example of the present invention, the present inventors cultured human adipose-derived mesenchymal stem cells, added the mesenchymal stem cells into a thrombin solution, and dripped fibrinogen and the cell-thrombin solution each in an amount of 1 to 10 uL into a culture vessel by using a dispenser, respectively, to prepare fibrin glue hydrogel beads containing cells (hereinafter, referred to as cell-hydrogel beads) (see FIGS. 1A and 1B).

[0069] As a result of checking the number of cells in a cell-hydrogel bead, viability, and characteristics of the cells, the present inventors confirmed that the cells cultured in a hydrogel bead according to the present invention confirmed immunological characteristics showing positive for CD73, CD90, and CD105 which were representative mesenchymal stem cell markers and showing negative for CD34 and CD45 which were hematopoietic cell markers were maintained (see Table 1, and FIG. 2).

[0070] In addition, as a result of checking activating factors whether the activating factors secreted from the cell were captured in the bead in a cell-hydrogel bead preparation step, the present inventors confirmed that HGF and Type II Collagen secreted in the mesenchymal stem cells were significantly captured (see FIG. 3).

[0071] As a result of checking a paracrine factor secretion effect of the cell-hydrogel beads, the present inventors confirmed that the cell-hydrogel bead of the present invention gradually secreted paracrine factors in an inflammatory environment induced with Interleukin 1β and exhibited significant therapeutic effects (see FIG. 4).

[0072] In the mesenchymal stem cell-hydrogel composition for injection prepared by the method of the present invention, the stem cells are attached to the scaffolds of the hydrogel beads, and thus the stem cells are not easily lost or killed after injection. Therefore, there is an advantage that an engraftment rate increases since the paracrine effect of the stem cells is continuously exhibited, and the stem cells are gradually released as hydrogel is degraded (see FIG. 5). In addition, there is an advantage that healthy cells can be used without damages in cell membranes since injection formulation can be prepared without a treatment with proteolytic enzymes, and also a cryopreservation solution is easily removed from the mesenchymal stem cell-hydrogel beads even after freezing and thawing.

[0073] In addition, the present invention provides a cell therapeutic agent for preventing and treating a musculoskeletal disease, a fistula disease, or an inflammatory disease which contains the composition according to the present invention as an active ingredient.

[0074] In the present invention, the term “cell therapeutic agent” refers to cells and tissues separated from humans and prepared by culture and special manipulation which are pharmaceuticals used for the purpose of treatment, diagnosis, and prevention. In particular, the term “cell therapeutic agent” refers to pharmaceuticals used for treatment, diagnosis and prevention by a series of actions such as proliferating and selecting live autologous, allogeneic, or xenogeneic cells in vitro in order to restore the function of cells or tissues or changing biological characteristics of cells by other methods. The cell therapeutic agent is largely classified into a somatic cell therapeutic agent and a stem cell therapeutic agent according to the degree of cell differentiation, and the present invention more specifically relates to an adipose-derived stem cell therapeutic agent.

[0075] In the present invention, the term “individual” refers to all animals including humans which have already developed or can develop a disease that can be prevented or treated by administration of the cell therapeutic agent according to the present invention.

[0076] The composition can be applied to various diseases such as an injury of a joint, a bone disease, muscle weakness, degenerative diseases caused by nerve damage in a joint, urinary incontinence, degenerative arthritis, ligament and tendon damage, diabetic foot ulcer, lower-extremity ischemic ulcers, and fistula disease.

[0077] In addition, the present invention provides a method of preventing or improving a musculoskeletal disease, a fistula disease, or an inflammatory disease, the method including a step of administering, to an individual, a cell-hydrogel composition containing

[0078] cells and hydrogel in pharmaceutically effective amounts

[0079] and having a bead form,

[0080] and the cell and hydrogel have a diameter of 0.1 mm to 5 mm.

[0081] In addition, the present invention provides a method of treating a musculoskeletal disease, a fistula disease, or an inflammatory disease including a step of administering, to an individual, a cell-hydrogel composition containing

[0082] cells and hydrogel, and

[0083] having a bead form, and

[0084] a diameter of the cell and the hydrogel is 0.1 mm to 5 mm, in pharmaceutically effective amounts.

[0085] The descriptions of the cell-hydrogel, the musculoskeletal disease, the fistula disease, and the inflammatory disease are the same as described for the cell-hydrogel composition, and thus specific descriptions thereof are incorporated herein by reference.

[0086] Meanwhile, in the mesenchymal stem cell-hydrogel composition for injection prepared by the method of the present invention, the stem cells are attached to the scaffolds of the hydrogel beads, and thus the stem cells are not easily lost or killed after injection. Therefore, there is an advantage that an engraftment rate increases since the paracrine effect of the stem cells is continuously exhibited, and the stem cells are gradually released as hydrogel is degraded. In addition, advantages that healthy cells can be used without damages in cell membranes since injection formulation can be prepared without a treatment with proteolytic enzymes, and also that a cryopreservation solution is easily removed from the mesenchymal stem cell-hydrogel beads even after freezing and thawing are confirmed. Therefore, the cell-hydrogel composition according to the present invention can be usefully used for preventing, treating, or improving a musculoskeletal disease, a fistula disease, or an inflammatory disease.

[0087] Hereinafter, the present invention is described in more detail through examples.

[0088] However, the following examples are provided for easier understanding of the present invention, and are not intended to limit the scope of the present invention thereto.

Example 1. Method of Culturing Human Adipose-Derived Mesenchymal Stem Cell

[0089] Adipose tissues can usually be obtained by liposuction, but the method is not limited thereto.

[0090] Adipose-derived mesenchymal stem cells were separated from adipose tissues obtained by liposuction as follows: adipose tissues were washed 3 to 4 times with an equal volume of PBS to remove the blood. A collagenase solution of the same volume as the adipose tissues was added and reacted in a water bath at 37° C. This was transferred to a centrifuge tube and centrifuged at 20° C. and 1,500 rpm for 10 minutes. The fat layer which was the supernatant was removed, and the collagenase solution which was the lower layer was carefully separated so as not to be shaken. The cell culture medium was added for suspension and then centrifuged at 20° C. and 1,200 rpm for 5 minutes. At this time, what was sunk to the bottom was a stroma-vascular fraction, and the supernatant was removed. The stroma-vascular fraction was suspended in a cell culture medium, inoculated into a culture vessel, and cultured at 37° C. in a 5%-CO.sub.2 incubator for 24 hours. After removing the culture solution, the resultant was washed with a phosphate buffer solution, and was proliferated by using a cell culture medium or a culture medium containing cell growth factors such as a basic fibroblast growth factor (bFGF) or an epidermal growth factor (EGF) in a cell culture medium. When the adipose-derived mesenchymal stem cells grew to about 80% to 90% of the culture vessel, the mesenchymal stem cells were treated with trypsin to separate and obtain single cells.

Example 2. Preparing and Freezing Cell-Fibrin Glue Hydrogel Bead

[0091] 1) Preparation of Cell-Fibrin Glue Hydrogel Bead

[0092] 1 to 3×10.sup.5 cells/mL of the adipose-derived mesenchymal stem cells obtained in Example 1 were added with a thrombin solution. 1.8 to 90 mg/mL of Fibrinogen was prepared. Fibrinogen and a cell-thrombin solution each in an amount of 1 to 10 uL were dripped into a culture vessel by using a dispenser to prepare fibrin glue hydrogel beads containing cells (hereinafter, referred to as cell-hydrogel beads).

[0093] 2) Culture Vessel Attachment Culture Method

[0094] When the cell-hydrogel beads were completely hardened on the culture vessel, the cell culture medium was added without change, and then the resultant was cultured for 4 to 8 days in a 5%-CO.sub.2 incubator at 37° C., and it was confirmed that a hemispherical shape is exhibited.

[0095] 3) Suspension Culture Method

[0096] In addition, instead of the culture vessel attachment culture method of 2), a suspension culture method can be used. After the cell-hydrogel beads were completely hardened, the beads were removed from the bottom of the culture vessel and transferred to the culture vessel. The beads were suspended by adding a cell culture medium and cultured in a 5%-CO.sub.2 incubator at 37° C. for 4 to 8 days. Meanwhile, in the case of the suspension culture, there is an advantage that culturing in a large volume is easy.

[0097] 4) Washing and Freezing Cell-Hydrogel Beads

[0098] The culture medium was removed, and cell-hydrogel beads were collected from the culture vessel. The collected beads were mixed with human serum albumin containing 10% to 20% DMSO in a ratio of 1:1 and cryopreserved at −80° C.

[0099] Meanwhile, the cryopreservation solution and bead mixture were put into a syringe, a filter having a pore size of about 100 ul was mounted at the front end of the syringe, the solution was pushed, and then the beads were caught in the filter, so that only the cryopreservation solution was able to be removed in a physically simple method.

[0100] FIG. 1A is a photograph obtained by observing a cultured cells-hydrogel bead. It was confirmed that the diameter of the beads was 3 to 5 mm and that cells were proliferated well in the beads and were well compatible with the fibrin glue hydrogel.

[0101] FIG. 1B is a view showing a micrograph of cell-hydrogel beads cryopreserved at −80° C., thawed, filled into a syringe, and sprayed by using a 17-gauge needle. It was confirmed that the shape and size of the beads were all kept constant even after freezing, thawing, and injecting using a needle.

Example 3. The Number of Cells in Cell-Hydrogel Bead, Viability, and Characteristics of Cells

[0102] As a result of observing the cell-hydrogel bead cultured, washed, frozen, and thawed according to Example 2 under a microscope, it was confirmed that cells in the beads were homogeneously distributed (FIGS. 1A and 1B). Then, an enzyme was added to the cell-hydrogel bead cultured, washed, frozen, and thawed according to Example 2 to dissolve fibrin glue, and cells were obtained. As a result of analyzing the number of cells and the cell viability by staining the cells with trypan blue, about 15,000 cells were contained per bead, and the cell viability was 95% or more.

[0103] The obtained cells were stained with CD73, CD90, CD105, CD34, and CD45 and analyzed by flow cytometry. As a result, as shown in FIGS. 1A and 1B, it was confirmed that immunological characteristics in which the cells cultured in the hydrogel bead according to the present invention were positive for CD73, CD90, and CD105 that were representative mesenchymal stem cell markers and were negative for CD34 and CD45 that were hematopoietic cell markers were maintained (Table 1 and FIG. 2).

TABLE-US-00001 TABLE 1 CD90 97.4 CD73 98.8 CD105 95.4 CD45 1.4 CD34 0.2

Example 4. Containing Activating Factor in Cell-Hydrogel Bead

[0104] It is known that mesenchymal stem cells secrete various activating factors during culture. Therefore, in order to check activating factors whether activators secreted in the cells in the cell-hydrogel bead preparation step were captured in the beads, an enzyme was added to the cell-hydrogel bead cultured, washed, frozen, and thawed according to Example 2 to obtain an eluate in which fibrin glue was dissolved, and the activating factors in the cell-hydrogel bead were analyzed.

[0105] As a result, as shown in FIG. 3, it was confirmed that about 10 pg of hepatocyte growth factors (HGF) that were representative activating factors secreted in the mesenchymal stem cell were captured per bead, and about 2 pg of Type II Collagen that was a major cartilage component was captured per bead (FIG. 3).

Example 5. Secretion of Paracrine Factor of Cell-Hydrogel Bead

[0106] It is known that mesenchymal stem cells have anti-inflammatory, anti-apoptotic, and cell proliferation promoting effects by a secretion action (paracrine action).

[0107] Accordingly, DMEM was added to the cell-hydrogel bead cultured, washed, frozen, and thawed according to Example 2, the resultant was cultured for 72 hours, an enzyme was added to the cell-hydrogel beads remaining after obtaining the culture supernatant, an eluate in which fibrin glue was dissolved was obtained, and the factors secreted from the cell-hydrogel beads were analyzed.

[0108] In addition, in order to create a microenvironment at a lesion site (inflammatory environment), a treatment with Interleukin 1β (Interleukin 1 beta; IL-1β) was performed, and the culture supernatant and the eluate were obtained in the same manner and analyzed.

[0109] As a result, as shown in FIG. 4, it was confirmed that, prostaglandin E2 (PGE2), known as a representative inflammatory regulator secreted from mesenchymal stem cells, was not secreted in a normal environment, but secretion was promoted in an inflammatory environment (FIG. 4).

[0110] In addition, it was confirmed that hepatocyte growth factors (HGF) and vascular endothelial growth factors (VEGF) that exhibit anti-inflammatory, anti-apoptotic, cell proliferation promotion, and angiogenesis promotion effects among representative growth factors secreted from mesenchymal stem cells were similarly secreted in both the normal environment and the inflammatory environment, and Type II Collagen that was major structural protein of articular cartilage, which was insufficiently secreted in the normal environment, rapidly increased in the inflammatory environment (FIG. 4).

[0111] Therefore, it was confirmed that the cell-hydrogel beads of the present invention can exhibit a significant therapeutic effect by gradually secreting paracrine factors when being administered to a lesion environment.

Example 6. Animal Test

[0112] 30 cell-hydrogel beads cultured, washed, frozen, and thawed according to Example 2 were subcutaneously injected in two sites of on the left and right sides of a mouse, and volume changes were measured for seven days.

[0113] As a result, as shown in FIG. 5, it was confirmed that stem cells were not easily lost or killed even after subcutaneous injection, thus the paracrine effect of the stem cells was continuously exhibited, and the stem cells were gradually released as the hydrogel was degraded.

Example 7. Chondrocyte Apoptosis Inhibition Ability of Cell-Hydrogel Bead

[0114] It is known that osteoarthritis that is a representative musculoskeletal disease becomes worse as chondrocytes are killed. Accordingly, the chondrocyte apoptosis inhibition ability of the cell-hydrogel beads prepared according to Example 2 was confirmed.

[0115] Specifically, human chondrocytes were inoculated and attached to a 48-well plate, the cell-hydrogel beads prepared according to Example 2 and hydrogel beads without cells were added, and then the beads were co-cultured for 24 hours. Then, t-butyl hydroperoxide (tBOOH) was treated at concentrations of 100 μM, 200 μM, and 400 μM for 16 hours, respectively, to induce oxidative apoptosis of human chondrocytes. Thereafter, water soluble tetrazolium salt (WST-1) was added and cultured for about 3 hours, and then absorbance was measured to measure the number of living cells.

[0116] As a result, as shown in FIG. 6 and Table 2, it was confirmed that the cell-hydrogel beads inhibited apoptosis of chondrocytes due to oxidative stress (Table 2 and FIG. 6).

TABLE-US-00002 TABLE 2 tBOOH treatment Concen- Concen- Concen- Concen- tration tration tration tration of 0 μM of 100 μM of 200 μM of 400 μM Untreated control 1.00 0.42 0.07 0.00 group Cell-hydrogel bead 1.00 1.32 0.72 0.56 Hydrogel bead 1.00 0.41 0.05 0.01

Example 8. Anti-Inflammatory Macrophage Differentiation Promoting Ability of Cell-Hydrogel Beads

[0117] Human monocytes were treated with phorbol 12-myristate 13-acetate (PMA) for 24 hours to induce differentiation into macrophages, then the cell-hydrogel beads prepared according to Example 2 were added, and the resultant was additionally cultured for 48 hours. Thereafter, the supernatant was collected, an amount of TNF-α secreted from activated M1 macrophages having pro-inflammatory characteristics and an amount of IL-10 secreted from activated M2 macrophages having anti-inflammatory characteristics were measured by enzyme-linked immunosorbent assay (ELISA).

[0118] As a result, as shown in FIG. 7, it was confirmed that when the amount of TNF-α secreted in case of adding PMA to human monocytes is 1.00, if the cell-hydrogel beads were further added thereto, the amount of TNF-α decreased to 0.73, while when the amount of IL-10 secreted in case of adding PMA is 1.00, if the cell-hydrogel beads were further added thereto, the amount of IL-10 increased to 1.61. Therefore, it was confirmed that the cell-hydrogel beads have an effect of inhibiting the inflammatory reaction (FIG. 7).

INDUSTRIAL APPLICABILITY

[0119] The present invention relates to a composition containing injectable mesenchymal stem cell-hydrogel and a preparation method thereof. Specifically, in the mesenchymal stem cell-hydrogel composition for injection prepared by the method of the present invention, the stem cells are attached to the scaffolds of the hydrogel beads, and thus the stem cells are not easily lost or killed after injection. Therefore, there is an advantage that an engraftment rate increases since the paracrine effect of the stem cells is continuously exhibited, and the stem cells are gradually released as hydrogel is degraded. In addition, there is an advantage that healthy cells can be used without damages in cell membranes since injection formulation can be prepared without a treatment with proteolytic enzymes, and also, a cryopreservation solution is easily removed from the mesenchymal stem cell-hydrogel beads even after freezing and thawing, thus the mesenchymal stem cell-hydrogel composition can be usefully used.