Injectable composition containing mesenchymal stem cells-hydrogel, and methods for producing, freezing and thawing the same

Abstract

The present invention relates to a composition containing an injectable mesenchymal stem cell-hydrogel and a method for producing the same. Specifically, the injectable mesenchymal stem cell-hydrogel composition produced by the method of the present invention has the advantage that the stem cells are not easily lost or killed even after injection because the stem cells are attached within the scaffold of the hydrogel beads, thereby continuing the paracrine effect of the stem cells, and the stem cells are gradually released as the hydrogel is degraded, thereby increasing the engraftment rate. Furthermore, since the composition can be prepared into an injectable dosage form without treatment with protease, the cell membrane is not damaged and healthy cells can be used. Furthermore, the composition has the advantage that the cryopreservation solution can be easily removed from the mesenchymal stem cell-hydrogel beads after freezing and thawing.

Description

[Technical field]

[0001] The present invention relates to an injectable mesenchymal stem cell-hydrogel composition, and methods for producing, freezing, and thawing the same. Specifically, the present invention relates to a method for producing a stem cell-hydrogel in a fixed shape with a diameter of 5 mm or less, which can be developed into a dosage form that is easy to fill into a syringe, and which can be frozen and stored, and can be immediately thawed and used as needed.

[0002] More specifically, when stem cells are injected into the body alone, there is a problem that the stem cells are easily lost at the injection site and the engraftment rate is low. The mesenchymal stem cell-hydrogel composition prepared by the method of the present invention has the advantage that the stem cells are not easily lost or killed even after injection because the stem cells are attached within the scaffold of the hydrogel beads, so the paracrine effect of the stem cells continues to be exerted, and the stem cells are gradually released as the hydrogel decomposes, thereby increasing the engraftment rate.

[0003] In addition, the method of the present invention allows the adherent mesenchymal stem cells to be prepared in an injectable form without treatment with protease, which is advantageous in that the cell membrane is not damaged and healthy cells can be used.Furthermore, the method of the present invention has the advantage that the freezing solution can be easily removed from the mesenchymal stem cell-hydrogel beads after thawing. [Background technology]

[0004] Conventional first-generation stem cell therapeutics are isolated cells obtained by treating with protease such as trypsin or dispase. During the treatment with protease, all proteins exposed on the cell membrane are non-selectively degraded, so that the intercellular bonds and basement membrane proteins are hardly maintained. In addition, animal-derived substances such as FBS are added to inactivate the protease, and washing and centrifugation processes are performed several times to remove them, but usually about 5 to 10% of the cells are lost in one washing and centrifugation process. Therefore, the cell recovery process using protease is a very inefficient method.

[0005] In addition, when isolated single cells are transplanted to the diseased area, they will flow out of the target area due to diffusion and absorption, resulting in a low cell adhesion rate. In addition, since mesenchymal stem cells have strong adhesive properties, they die within 6 to 24 hours when separated into single cells, resulting in a very low engraftment rate.

[0006] Patent Document 1 discloses a method for de novo synthesis of soft tissue of a predetermined shape and size in vivo from adult mesenchymal stem cells (MSCs) in a biocompatible scaffold, and a composition produced by the method. The document discloses a method using a hydrogel polymer, more specifically, polyethylene glycol diacrylate, as a biocompatible scaffold. However, the document only indicates that the composition is for reconstruction of soft tissue connection, and that when polyethylene glycol diacrylate is used as a scaffold, it is required that there is little change in diameter, and does not disclose or suggest a manufacturing process that allows the composition to be filled into a syringe after culture and immediately administered.

[0007] Patent Document 2 discloses a cellular therapeutic agent for sphincter regeneration that contains amniotic fluid-derived stem cells, and discloses that the effect of the cellular therapeutic agent is enhanced when the agent is injected into a hydrogel complex, specifically, alginate / PF-127 / hyaluronic acid. However, the document only discloses a form in which stem cells and hydrogel are mixed and injected at the time of use, and is manufactured by injecting the mixture into a hydrogel complex, and does not disclose culturing stem cells in hydrogel beads.

[0008] Patent Document 3 discloses a method for differentiating mesenchymal stem cells into chondrocytes, more specifically, a method for immobilizing and culturing mesenchymal stem cells on a mixed support containing the biodegradable polymer fibrin / HA. However, the document merely discloses that the use of fibrin / HA as a support promotes differentiation of mesenchymal stem cells into chondrocytes without the addition of TGF-beta, which has been conventionally added for cell differentiation.

[0009] Patent Document 4 discloses a method for the stable mass production of chondrocytes and cells with chondrocyte differentiation ability. More specifically, the method discloses a method for uniformly mass-producing a chondrocyte therapeutic agent without a support through a process of dispensing cells into a 96-well plate with a V-shaped bottom, a process of ultra-high density culture, and a process of recovering pellets, and then transplanting the agent into a cartilage injury site. However, this method produces spheres using only chondrocytes without using a support, and therefore has a structural difference from the present invention in which stem cells are cultured on a hydrogel bead support.

[0010] Furthermore, in conventional methods, the stem cell therapeutic agent is produced by treating it with proteolytic enzymes, which can damage the bonds between cells and basement membrane proteins, and can result in cell loss during the steps of cell collection, washing, filling the vial, and filling the vial into a syringe. In addition, since only single cells are collected and injected after enzyme treatment, there is a problem that active substances such as collagen synthesized during cell culture are almost entirely removed.

[0011] Meanwhile, in order to solve these problems, Patent Document 5 discloses a method of preparing a hydrogel mass containing stem cells and filling the hydrogel mass into a syringe. However, the hydrogel mass has a drawback in that the shape of the hydrogel becomes irregular during the process of filling the hydrogel mass into a syringe and injecting it, making it impossible to maintain a constant dosage form. In addition, although cells can grow within the hydrogel scaffold, there is a limitation in the concentration of the hydrogel, which requires a hardness that allows it to be filled into a syringe.

[0012] Patent Document 6 discloses a method for culturing stem cells or primary cultured cells using a porous membrane and a hydrogel. More specifically, a method for culturing cells in three dimensions by placing a porous membrane in a cell culture vessel without contact and coating the membrane with a hydrogel. However, the document only clearly states that the system promotes cell proliferation and growth, and does not disclose the preparation of a hydrogel containing cells in the form of beads and culturing it, or the administration of the hydrogel by filling it into a syringe.

[0013] To solve the above problems, recent studies have focused on methods for placing cells or stem cells on a support and creating them in the form of beads (Elizabeth et al., 2012; Christian et al., 2013; Havva et al., 2016; Medi et al., 2017). Alginate beads are the most commonly used type of beads.

[0014] Patent Document 7 discloses a method for producing a hydrogel matrix containing chondrogenic cells and a method for forming the matrix into beads. The document discloses a method for forming an alginate solution and a chitosan solution, mixing the solution with chondrogenic cells to form a hydrogel matrix, forming the matrix into beads, and culturing the matrix. However, this is a study using general chondrocytes, not stem cells, and the average size of the beads is adjusted by adjusting the diameter of the needle, so that the size of the beads cannot be made constant, and the number of cells in the chondrocyte-hydrogel complex cannot be made constant.

[0015] Patent Document 8 discloses a method of mixing mesenchymal stem cells with a hydrogel solution and culturing the mixture and a method of filling the mixture into a syringe. However, this method has the limitation that it is difficult to fill a desired number of cells into a syringe. In contrast, the present invention has the advantage that a desired number of cells can be administered to the cartilage damage site by placing a certain number of cells in one bead and culturing the cells.

[0016] It is important to prepare stem cells or cells in the form of beads so that they can be injected into the body, but it is also important to freeze them and store them for a long time, so that they can be thawed and used easily whenever and wherever they are needed. Therefore, research is being conducted on freezing and thawing methods that can reduce cell damage and increase cell viability (Dominique et al., 2017).

[0017] Patent Documents 9 and 10 disclose compositions for cryopreservation of stem cells. However, these disclose methods for increasing cell viability during freezing / thawing of 2D-cultured stem cells, which differs from the present invention, which discloses a method for freezing and thawing a hydrogel-beads-cell mixture.

[0018] Conventional stem cell therapeutic agents used after cryopreservation have difficulty in removing the cryopreservation solution after thawing, and are therefore used by injection together with the cryopreservation solution. In this case, there is a problem that DMSO, a component of the cryopreservation solution, causes various side effects in the body. The hydrogel-bead cell mixture according to the present invention has a size of about 0.1 to 5 mm, and therefore has the advantage that the cryopreservation solution can be removed by a physically simple method.

[0019] Therefore, in the present invention, by manufacturing the stem cell-hydrogel into a fixed shape with a diameter of 5 mm or less, a dosage form that can be easily loaded into a syringe was developed. The injectable dosage form, which is used as a therapeutic agent after freezing and thawing, can be used for various therapeutic purposes because the physical properties or physical strength of the hydrogel can be adjusted in various ways according to the purpose, and the present invention was completed. [Prior art documents] [Patent documents]

[0020] [Patent Document 1] International Publication No. 2006 / 004951 [Patent Document 2] Korean Patent No. 10-1289834 [Patent Document 3] Korean Patent No. 10-0684940 [Patent Document 4] Korean Patent No. 10-1814440 [Patent Document 5] Korean Patent No. 10-1613478 [Patent Document 6] Korean Patent No. 10-1687291 [Patent Document 7] Korean Patent No. 10-1740298 [Patent Document 8] Korean Patent No. 10-1585032 [Patent Document 9] Korean Patent No. 10-1321144 [Patent Document 10] Korean Patent No. 10-1407355 Summary of the Invention [Problem to be solved by the invention]

[0021] The present invention provides a composition containing an injectable stem cell-hydrogel, which is a ready-made preparation that can be cryopreserved for long periods of time, and is produced in a fixed shape with a diameter of 5 mm or less, making it easy to fill into a syringe and allowing the freezing solution to be removed by a simple physical method, and a method for producing the same.

[0022] The present invention also provides a method for preventing, treating or ameliorating a musculoskeletal disease, fistulous disease or inflammatory disease, comprising administering to an individual a pharma- tically effective amount of a composition containing a stem cell-hydrogel.

[0023] Furthermore, the present invention provides a use of the composition containing stem cells-hydrogel as a pharmaceutical composition for preventing or treating a musculoskeletal disease, a fistulous disease, or an inflammatory disease. [Means for solving the problem]

[0024] The present invention provides a cell-hydrogel composition comprising cells and a hydrogel, the cell-hydrogel composition being in the form of a bead.

[0025] The present invention also provides a method for preparing a cell-hydrogel composition, comprising the steps of: (a) culturing cells; (b) mixing the cultured cells with a hydrogel solution to form cell-hydrogel beads; and (c) culturing the cell-hydrogel beads.

[0026] Additionally, the present invention provides a method for producing a cell-hydrogel composition, further comprising the steps of freezing and thawing the cell-hydrogel beads of the present invention.

[0027] Furthermore, the present invention provides a method for preventing or ameliorating a musculoskeletal disease, a fistulous disease or an inflammatory disease, comprising the step of administering to an individual a pharma- ceutically effective amount of cell-hydrogel beads.

[0028] Additionally, the present invention provides a method for treating a musculoskeletal disease, a fistulous disease or an inflammatory disease, comprising administering to an individual a pharma- ceutically effective amount of cell-hydrogel beads.

[0029] Furthermore, the present invention provides the use of the cell-hydrogel beads as a pharmaceutical composition for the prevention or treatment of musculoskeletal diseases, fistulous diseases or inflammatory diseases. Effect of the Invention

[0030] The injectable mesenchymal stem cell-hydrogel composition prepared by the method of the present invention has the advantage that the stem cells are not easily lost or killed even after injection because the stem cells are attached within the scaffold of the hydrogel beads, so the paracrine effect of the stem cells continues to be exerted, and the stem cells are gradually released as the hydrogel is decomposed, thereby increasing the engraftment rate. In addition, since it can be prepared into an injectable form without treatment with proteolytic enzymes, the cell membrane is not damaged and healthy cells can be used. Furthermore, it has the advantage that the freezing storage solution can be easily removed from the mesenchymal stem cell-hydrogel beads after freezing and thawing. [Brief description of the drawings]

[0031] [Figure 1A] Photographs showing the cultured cells-hydrogel beads. [Figure 1B] This is a micrograph of cell-hydrogel beads that were frozen and stored at -80°C and then thawed, loaded into a syringe, and ejected using a 17 gauge needle. [Diagram 2] FIG. 1 shows confirmation of cell characteristics within cell-hydrogel beads. [Diagram 3] FIG. 14 confirms that active factors are captured within cell-hydrogel beads. [Figure 4]This is a diagram confirming the paracrine factor secretion effect of cells-hydrogel beads. Y axis: relative value (fold increase) [Diagram 5] FIG. 1 shows the volume change measured over a 7-day period following subcutaneous injection of cell-hydrogel beads of the present invention into mice. [Figure 6] FIG. 1 is a graph confirming the effect of the cell-hydrogel beads of the present invention in inhibiting chondrocyte death caused by oxidative stress. [Figure 7] FIG. 1 is a graph confirming the inflammation suppressing effect of the cell-hydrogel beads of the present invention. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0032] The present invention will be described in detail below.

[0033] The present invention provides a cell-hydrogel composition comprising cells and a hydrogel, the cell-hydrogel composition being in the form of a bead.

[0034] The present invention also provides a use of a cell-hydrogel composition, comprising cells and a hydrogel, in the form of beads, as a pharmaceutical composition for the prevention or treatment of a musculoskeletal disease, a fistulous disease or an inflammatory disease.

[0035] In one embodiment of the present invention, the cells and hydrogel preferably have a diameter of 0.1 to 5 mm, more preferably 0.5 mm or more and 5 mm or less, and most preferably 1 mm or more and 4 mm or less.

[0036] In one aspect of the present invention, the cells are preferably any one selected from the group consisting of stem cells, somatic cells, and germ cells, but are not limited thereto.

[0037] In one aspect of the present invention, the hydrogel is preferably one or more selected from the group consisting of fibrin glue, hyaluronic acid, gelatin, collagen, alginic acid, chitosan, cellulose, pectin, 2-hydroxyethyl methacrylate derivatives and copolymers thereof, polyethylene oxide, and polyvinyl alcohol.

[0038] In one aspect of the present invention, the musculoskeletal disease is preferably any one selected from the group consisting of joint trauma, bone disease, muscle weakening, myositis due to nerve damage in a joint, myofascial pain syndrome, tendonitis, tendonitis, bursitis, ganglion, carpal tunnel syndrome, Guyon canal syndrome, wrist tendonitis, hand-arm vibration syndrome, trigger finger, ganglion, white wax disease, Raynaud's syndrome, lateral epicondylitis, medial epicondylitis, radial tunnel syndrome, ulnar tunnel syndrome, olecranon bursitis, median nerve entrapment disorder, shoulder impingement syndrome, adhesive capsulitis of the shoulder, degenerative arthritis, forward head posture, cervical radiculopathy, lumbosacral contusion, intervertebral disc herniation, spondylolysis, spondylolisthesis, lumbar spondylosis degenerative, degenerative disease, urinary incontinence, and ligament and tendon injury, but is not limited thereto.

[0039] In one embodiment of the present invention, the fistulizing disease is, but is not limited to, fistulizing Crohn's disease.

[0040] In one embodiment of the present invention, the inflammatory disease is atopic dermatitis, systemic lupus erythematosus, lupus, lupus pernio, lupus tuberculosis, lupus nephritis, dystrophic epidermolysis bullosa, psoriasis, rheumatic fever, rheumatoid arthritis, low back pain, fibromyalgia, fascial disease, undifferentiated spondyloarthropathy, undifferentiated arthropathy, arthritis, inflammatory osteolysis, reactive arthritis, osteoarthritis, scleroderma, osteoporosis, chronic inflammatory diseases due to viral or bacterial infection, colitis, ulcerative colitis. Inflammation, inflammatory bowel disease, mycoses, burns, wounds from surgery or dentistry, diabetic foot ulcers, type 1 diabetes, type 2 diabetes, ulcerative skin disease, sinusitis, rhinitis, conjunctivitis, asthma, dermatitis, inflammatory collagen vascular disease, glomerulonephritis, encephalitis, inflammatory bowel disease, chronic obstructive pulmonary disease, sepsis, septic shock, pulmonary fibrosis, atherosclerosis, myocarditis, endocarditis, pericarditis, cystic fibrosis, Hashimoto's disease, Graves' disease, leprosy, syphilis, Lyme disease It is preferable that the disease is any one selected from the group consisting of chronic inflammation, pulmonary tuberculosis, borreliosis, neuroborreliosis, tuberculosis, sarcoidosis, macular degeneration, uveitis, irritable bowel syndrome, Crohn's disease, Sjogren's syndrome, chronic fatigue syndrome, chronic fatigue immune deficiency syndrome, myalgic encephalomyelitis, amyotrophic lateral sclerosis, Parkinson's disease, and multiple sclerosis, but is not limited thereto.

[0041] The composition of the present invention has a certain bead shape with a diameter of 5 mm or less, so it is easy to fill a syringe, and the cryopreservation solution can be removed by a physically simple method, so that DMSO, a component of the cryopreservation solution that causes various side effects in the body, can be easily removed before administration. Specifically, as an example of the physically simple method, a mixture of the cryopreservation solution and beads is placed in a syringe, and a filter with a pore size of about 100 ul is attached to the front of the syringe, and the solution is pushed, so that the beads do not pass through the filter and only the cryopreservation solution can be removed.

[0042] In addition, the composition can be filled into a syringe and administered by transdermal injection, subcutaneous injection, intramuscular injection, submucosal injection, abdominal injection, or the like.

[0043] Furthermore, the concentration of the hydrogel in the present invention is not particularly limited and can be adjusted according to various therapeutic purposes, and it is preferable that the hydrogel contains fibrinogen at a concentration of 1.8 to 90 mg / mL.

[0044] The present invention further provides a method for preparing a cell-hydrogel composition, comprising the steps of: (a) culturing cells; (b) mixing the cultured cells with a hydrogel solution to form cell-hydrogel beads; and (c) culturing the cell-hydrogel beads.

[0045] In one aspect of the present invention, the hydrogel may be one or more complexes selected from the group consisting of fibrin glue, hyaluronic acid, gelatin, collagen, alginic acid, chitosan, cellulose, pectin, 2-hydroxyethyl methacrylate derivatives and copolymers thereof, polyethylene oxide, and polyvinyl alcohol, and more specifically, fibrin glue may be used.

[0046] In one embodiment of the present invention, the fibrin glue may contain fibrinogen at a concentration of 1.8 to 90 mg / mL.

[0047] In one embodiment of the present invention, the cell hydrogel beads are preferably 0.1 to 5 mm, more preferably 0.5 to 5 mm, and most preferably 1 to 4 mm.

[0048] In one embodiment of the present invention, after step (c), a step (d) of loading the cell-hydrogel composition into a syringe may be further included. The cell-hydrogel composition produced by the production method of the present invention can be immediately administered locally using a 10-25 gauge needle.

[0049] In addition, the method of the present invention may further include a step of freezing and thawing the cell hydrogel after culturing in step (c), and the cryopreservation solution can be removed by a simple physical method after freezing and thawing.

[0050] Furthermore, when the cell-hydrogel composition of the present invention is used for injection, the physical properties or physical strength of the hydrogel can be adjusted in various ways depending on the purpose, and therefore the concentration of the hydrogel is not limited to a specific one.

[0051] In a specific embodiment of the present invention, the inventors cultured human adipose-derived mesenchymal stem cells, then added them to a thrombin solution, and then used a dispenser to inject 1 to 10 uL of fibrinogen and cell-thrombin solution into a culture vessel to produce fibrin glue hydrogel beads containing cells (hereinafter referred to as cell-hydrogel beads) (see FIG. 1).

[0052] In addition, the present inventors observed the number of cells, viability, and characteristics of the cells in the cell-hydrogel beads, and confirmed that the cells cultured in the hydrogel beads according to the present invention maintained immunological characteristics that showed positive reactions to representative mesenchymal stem cell markers CD73, CD90, and CD105, and negative reactions to hematopoietic cell markers CD34 and CD45 (see Table 1 and FIG. 2).

[0053] Furthermore, the present inventors observed whether active factors secreted from cells during the cell-hydrogel bead manufacturing process were captured within the beads, and confirmed that HGF and collagen type 2 secreted from mesenchymal stem cells were significantly captured (see Figure 3).

[0054] Furthermore, the inventors observed the paracrine factor secretion effect of the cell-hydrogel beads and confirmed that the cell-hydrogel beads of the present invention gradually secrete paracrine factors in an inflammatory environment induced by interleukin-1β, thereby exerting a significant therapeutic effect (see FIG. 4).

[0055] Therefore, the injectable mesenchymal stem cell-hydrogel composition prepared by the method of the present invention has the advantage that the stem cells are not easily lost or killed even after injection because the stem cells are attached within the scaffold of the hydrogel beads, so the paracrine effect of the stem cells continues to be exerted, and the stem cells are gradually released as the hydrogel is decomposed, thereby increasing the engraftment rate (see FIG. 5). In addition, since the composition can be prepared into an injectable form without treatment with proteolytic enzymes, the cell membrane is not damaged and healthy cells can be used. Furthermore, the composition has the advantage that the freezing storage solution can be easily removed from the mesenchymal stem cell-hydrogel beads after freezing and thawing.

[0056] Furthermore, the present invention provides a cellular therapeutic agent for the prevention and treatment of musculoskeletal diseases, fistulous diseases, or inflammatory diseases, which contains the composition of the present invention as an active ingredient.

[0057] In the present invention, the term "cell therapeutic agent" refers to a pharmaceutical product that is a cell or tissue isolated from a human, cultured, and specially manipulated, and is used for the purposes of treatment, diagnosis, and prevention. In particular, the term refers to a pharmaceutical product that is used for the purposes of treatment, diagnosis, and prevention by a series of actions of proliferating and selecting living autologous, allogeneic, or xenogeneic cells outside the body, or by changing the biological properties of cells in other ways, in order to restore the function of the cells or tissue. Cell therapeutic agents are broadly classified into somatic cell therapeutic agents and stem cell therapeutic agents according to the degree of cell differentiation, and the present invention more specifically relates to an adipose-derived stem cell therapeutic agent.

[0058] In the present invention, the term "individual" refers to any animal, including a human, that has already developed or is at risk of developing a disease that can be prevented or treated by administration of a cell therapeutic agent according to the present invention.

[0059] The composition can be applied to various diseases such as joint trauma, bone diseases, muscle weakening, degenerative diseases due to nerve damage in joints, urinary incontinence, degenerative arthritis, ligament and tendon injuries, diabetic foot ulcers, lower limb ischemic ulcers, fistula diseases, etc.

[0060] The present invention further provides a method for preventing or ameliorating a musculoskeletal disease, a fistula disease, or an inflammatory disease, comprising administering to an individual a cell-hydrogel composition comprising a pharma- tically effective amount of cells and a hydrogel, the cells and the hydrogel being in the form of beads, the cells and the hydrogel having a diameter of 0.1 mm or more and 5 mm or less.

[0061] The present invention further provides a method for treating a musculoskeletal disease, a fistulous disease or an inflammatory disease, comprising administering to an individual a cell-hydrogel composition comprising a pharma- tically effective amount of cells and a hydrogel, the cells and the hydrogel being in the form of beads, the cells and the hydrogel having a diameter of 0.1 mm or more and 5 mm or less.

[0062] The cell-hydrogel, musculoskeletal disease, fistula disease, and inflammatory disease are similar to those described for the cell-hydrogel composition, and therefore detailed descriptions thereof will be omitted.

[0063] In addition, the injectable mesenchymal stem cell-hydrogel composition prepared by the method of the present invention has the advantage that the stem cells are not easily lost or killed even after injection because the stem cells are attached within the scaffold of the hydrogel beads, so the paracrine effect of the stem cells continues to be exerted, and the stem cells are gradually released as the hydrogel is decomposed, so that the engraftment rate is increased. In addition, since it can be prepared in an injectable form without treatment with proteolytic enzymes, the cell membrane is not damaged, so healthy cells can be used, and further, it has been confirmed that the freezing storage solution can be easily removed from the mesenchymal stem cell-hydrogel beads after freezing and thawing. Therefore, the cell-hydrogel composition of the present invention is useful for preventing, treating, or improving musculoskeletal diseases, fistula diseases, or inflammatory diseases.

[0064] The present invention will now be described in more detail with reference to examples.

[0065] However, these examples are merely for the purpose of facilitating understanding of the present invention, and the present invention is not limited thereto. EXAMPLES

[0066] Method for culturing human adipose-derived mesenchymal stem cells The adipose tissue can be obtained by, but is not limited to, conventional liposuction.

[0067] Adipose-derived mesenchymal stem cells were isolated from adipose tissue obtained by liposuction as follows. To remove blood, the adipose tissue was washed 3 to 4 times with the same volume of PBS. The same volume of collagenase solution as the adipose tissue was added and reacted in a water bath at 37 ° C. This was transferred to a centrifuge tube and centrifuged at 20 ° C and 1500 rpm for 10 minutes. The supernatant fat layer was removed and the lower layer collagenase solution was carefully separated so as not to shake. Cell culture medium was added and suspended, and then centrifuged at 20 ° C and 1200 rpm for 5 minutes. Here, the stromal vascular fraction was removed because it sank to the bottom. The stromal vascular fraction was suspended in cell culture medium, inoculated into a culture vessel, and cultured for 24 hours in a 5% CO2 incubator at 37 ° C. After removing the culture medium, it was washed with phosphate buffer and grown using cell culture medium or cell culture medium containing cell growth factors such as basic fibroblast growth factor (bFGF) and epidermal growth factor (EGF). When the adipose-derived mesenchymal stem cells proliferated to approximately 80-90% of the culture vessel, they were separated into single cells by trypsinization. EXAMPLES

[0068] Preparation and freezing of cell-fibrin glue hydrogel beads 1) Preparation of cell-fibrin glue hydrogel beads 1 to 3 × 10 adipose-derived mesenchymal stem cells obtained in Example 1 5 Thrombin solution was added to cells / mL. Fibrinogen was prepared at 1.8 to 90 mg / mL. Fibrinogen and cell-thrombin solutions were dripped into the culture vessel at volumes of 1 to 10 uL each using a dispenser to produce fibrin glue hydrogel beads containing cells (hereinafter referred to as cell-hydrogel beads).

[0069] 2) Culture vessel attachment culture method Once the cell-hydrogel beads had completely solidified on the culture vessel, cell culture medium was added to them, and they were then cultured in a 5% CO2 incubator at 37°C for 4 to 8 days. It was confirmed that the beads exhibited a hemispherical morphology.

[0070] 3) Suspension culture method Also, instead of the culture vessel attachment culture method of 2), a suspension culture method may be used. When the cell-hydrogel beads were completely solidified, the beads were removed from the bottom of the culture vessel and transferred to the culture vessel. A cell culture medium was added to suspend the beads, and the beads were then cultured for 4 to 8 days in a 5% CO2 incubator at 37°C. Note that suspension culture has the advantage that it is easy to culture large volumes.

[0071] 4) Washing and Freezing of Cell-Hydrogel Beads The culture medium was removed, and the cell-hydrogel beads were collected from the culture vessel. The collected beads were mixed with human serum albumin containing 10%-20% DMSO in a 1:1 ratio, and then frozen and stored at -80°C.

[0072] Furthermore, if the mixture of freezing solution and beads is placed in a syringe and a filter with a pore size of approximately 100 ul is attached to the front of the syringe, and the solution is pushed, the beads will not pass through the filter, and only the freezing solution can be removed physically in a simple manner.

[0073] Figure 1A is a photograph of the cultured cells-hydrogel beads. The beads had a diameter of 3 to 5 mm, and it was confirmed that the cells proliferated well within the beads and were well integrated with the fibrin glue hydrogel.

[0074] Figure 1B shows a micrograph of cell-hydrogel beads that were frozen at -80°C and then thawed, loaded into a syringe, and injected through a 17-gauge needle. It was confirmed that the morphology and size of the beads all remained consistent after freezing, thawing, and injection through a needle. EXAMPLES

[0075] Cell-Hydrogel Bead Cell Count, Viability, and Cell Characteristics The cell-hydrogel beads cultured, washed, frozen, and thawed in Example 2 were observed under a microscope, and it was confirmed that the cells were distributed homogeneously within the beads (Figure 1). Next, an enzyme was added to the cell-hydrogel beads cultured, washed, frozen, and thawed in Example 2 to dissolve the fibrin glue, and the cells were then obtained. The cell count and cell viability were analyzed by staining with trypan blue, and it was found that each bead contained approximately 15,000 cells, and the cell viability was 95% or more.

[0076] The cells obtained as described above were stained with CD73, CD90, CD105, CD34, and CD45 and analyzed by a flow cytometer. As a result, as shown in Figure 1, it was confirmed that the cells cultured in the hydrogel beads according to the present invention maintained immunological characteristics that showed positive reactions to representative mesenchymal stem cell markers CD73, CD90, and CD105, and negative reactions to hematopoietic cell markers CD34 and CD45 (Table 1 and Figure 2).

[0077] [Table 1] EXAMPLES

[0078] Inclusion of active factors within cell-hydrogel beads It is known that mesenchymal stem cells secrete various active factors during culture. Therefore, in order to confirm whether the active factors secreted from the cells during the cell-hydrogel bead production process are captured within the beads, an enzyme was added to the cell-hydrogel beads that were cultured, washed, frozen, and thawed in Example 2 to dissolve the fibrin glue, and the eluate was collected and the active factors within the cell-hydrogel beads were analyzed.

[0079] As a result, as shown in Figure 3, it was confirmed that approximately 10 pg of hepatocyte growth factor (HGF), a representative active factor secreted by mesenchymal stem cells, and approximately 2 pg of collagen type 2, a major cartilage component, were captured per bead (Figure 3). EXAMPLES

[0080] Paracrine factor secretion from cells-hydrogel beads Mesenchymal stem cells are known to have anti-inflammatory effects, anti-cell death effects, and cell proliferation promoting effects due to their paracrine action.

[0081] Therefore, DMEM was added to the cell-hydrogel beads that had been cultured, washed, frozen, and thawed in Example 2, and the beads were cultured for 72 hours, after which the culture supernatant was collected. Then, an enzyme was added to the remaining cell-hydrogel beads to dissolve the fibrin glue, and the eluate was collected and the factors secreted from the cell-hydrogel beads were analyzed.

[0082] In addition, to create a microenvironment (inflammatory environment) at the lesion site, the cells were treated with interleukin 1 beta (IL-1β), and the culture supernatant and eluate were then similarly collected and analyzed.

[0083] As a result, as shown in Figure 4, it was confirmed that prostaglandin E2 (PGE2), which is known as a representative inflammatory regulator secreted by mesenchymal stem cells, was not secreted under normal conditions, but its secretion was promoted under an inflammatory environment (Figure 4).

[0084] In addition, among the representative growth factors secreted by mesenchymal stem cells, hepatocyte growth factor (HGF) and vascular endothelial growth factor (VEGF), which have anti-inflammatory, anti-cell death, cell proliferation-promoting, and angiogenesis-promoting effects, were found to be secreted in similar amounts in both normal and inflammatory environments, and it was confirmed that collagen type 2, a major component protein of articular cartilage that is secreted in small amounts in normal environments, is rapidly increased in secretion in inflammatory environments (Figure 4).

[0085] Thus, it was confirmed that the cell-hydrogel beads of the present invention exert significant therapeutic effects by gradually secreting paracrine factors when administered in a lesion environment. EXAMPLES

[0086] Animal testing Thirty cell-hydrogel beads that had been cultured, washed, frozen, and thawed as described in Example 2 were subcutaneously injected into two sites on the left and right sides of the mice, and the volume change thereafter was measured for seven days.

[0087] As a result, as shown in Figure 5, it was confirmed that the stem cells are not easily lost or killed even after subcutaneous injection, so the paracrine effect of the stem cells continues and the stem cells are gradually released as the hydrogel decomposes. EXAMPLES

[0088] Inhibitory effect of cell-hydrogel beads on chondrocyte death Osteoarthritis, a representative musculoskeletal disease, is known to be aggravated by chondrocyte death, so the ability of the cell-hydrogel beads prepared in Example 2 to inhibit chondrocyte death was examined.

[0089] Specifically, human chondrocytes were seeded and attached to a 48-well plate, and then the cell-hydrogel beads or cell-free hydrogel beads prepared in Example 2 were added and co-cultured for 24 hours. Then, the cells were treated with t-butyl hydroperoxide (tBOOH) at concentrations of 100 μM, 200 μM, and 400 μM for 16 hours, respectively, to induce oxidative death of the human chondrocytes. Then, water soluble tetrazolium salt (WST-1) was added and cultured for about 3 hours, and the absorbance was measured to measure the number of living cells.

[0090] As a result, as shown in FIG. 6 and Table 2, it was confirmed that the cell-hydrogel beads suppressed chondrocyte death caused by oxidative stress (Table 2 and FIG. 6).

[0091] [Table 2] EXAMPLES

[0092] Ability of cell-hydrogel beads to promote anti-inflammatory macrophage differentiation Human mononuclear cells were treated with phorbol 12-myristate 13-acetate (PMA) for 24 hours to induce differentiation into macrophages, and then the cell-hydrogel beads prepared in Example 2 were added and further cultured for 48 hours. Then, the supernatant was collected and the amount of TNF-α secreted from activated M1 macrophages having pro-inflammatory properties and IL-10 secreted from activated M2 macrophages having anti-inflammatory properties were measured by enzyme-linked immunosorbent assay (ELISA).

[0093] As a result, as shown in Figure 7, when the amount of TNF-α secreted by human mononuclear cells when PMA was added was taken as 1.00, the amount decreased to 0.73 when cell-hydrogel beads were further added, whereas when the amount of IL-10 secreted by human mononuclear cells when PMA was added was taken as 1.00, the amount increased to 1.61 when cell-hydrogel beads were further added, confirming that the cell-hydrogel beads have the effect of suppressing the salt hypoxia response (Figure 7). [Industrial Applicability]

[0094] The present invention relates to a composition containing an injectable mesenchymal stem cell-hydrogel and a method for producing the same. Specifically, the injectable mesenchymal stem cell-hydrogel composition produced by the method of the present invention has the advantages that stem cells are not easily lost or killed even after injection because stem cells are attached within the scaffold of hydrogel beads, so the paracrine effect of stem cells continues to be exerted, and stem cells are gradually released as the hydrogel is decomposed, thereby increasing the engraftment rate. In addition, since it can be produced in an injectable form without treatment with proteolytic enzymes, the cell membrane is not damaged and healthy cells can be used. Furthermore, it is useful because it is easy to remove the cryopreservation solution from the mesenchymal stem cell-hydrogel beads after freezing and thawing.

Claims

1. comprising cells and a hydrogel; A cell-hydrogel bead composition in the form of beads, comprising: the cells are mesenchymal stem cells; The diameter of the cell-hydrogel beads is 0.1 mm or more and 5 mm or less; the hydrogel is a fibrin glue, the fibrin glue comprising fibrinogen at a concentration of 1.8 to 90 mg / mL; The cell-hydrogel bead composition maintains bead morphology, cell shape and properties after freezing and thawing compared to before freezing.

2. The composition of claim 1, wherein the cell-hydrogel bead has a diameter of 1 mm to 4 mm.

3. A pharmaceutical composition for the prevention or treatment of musculoskeletal diseases, fistula diseases or inflammatory diseases, comprising the composition according to claim 1 or claim 2 as an active ingredient.

4. The musculoskeletal disorders include joint trauma, bone disease, muscle weakness, and nerve damage to the joints, myositis due to joint trauma, bone disease, muscle weakness, and nerve damage to the joints, myofascial pain syndrome, tendonitis, tendonitis, bursitis, ganglion, carpal tunnel syndrome, Guyon's tunnel syndrome, wrist tendonitis, hand-arm vibration syndrome, trigger finger, ganglion, white wax disease, Raynaud's syndrome, lateral epicondylitis, medial epicondylitis, radial tunnel syndrome, ulnar tunnel syndrome, and olecranon bursitis.

4. The pharmaceutical composition for preventing or treating musculoskeletal diseases, fistula diseases or inflammatory diseases as described in claim 3, characterized in that the disease is any one selected from the group consisting of musculoskeletal diseases, median nerve entrapment disorder, shoulder impingement syndrome, adhesive shoulder capsulitis, degenerative arthritis, forward head posture, cervical radiculopathy, lumbosacral contusion, intervertebral disc herniation, spondylolysis, spondylolisthesis, lumbar spondylosis, degenerative diseases, urinary incontinence, and ligament and tendon injuries.

5. The inflammatory diseases include atopic dermatitis, systemic lupus erythematosus, lupus, lupus pernio, lupus tuberculosis, lupus nephritis, dystrophic epidermolysis bullosa, psoriasis, rheumatic fever, rheumatoid arthritis, lower back pain, fibromyalgia, fascial diseases, undifferentiated spondyloarthropathy, undifferentiated arthropathy, arthritis, inflammatory osteolysis, reactive arthritis, osteoarthritis, scleroderma, osteoporosis, chronic inflammatory diseases caused by viral or bacterial infections, colitis, ulcerative colitis, inflammatory bowel disease, and inflammatory bowel disease. Diseases, fungal infections, burns, wounds from surgical or dental procedures, diabetic foot ulcers, type 1 diabetes, type 2 diabetes, ulcerative skin diseases, sinusitis, rhinitis, conjunctivitis, asthma, dermatitis, inflammatory collagen vascular disease, glomerulonephritis, encephalitis, inflammatory bowel disease, chronic obstructive pulmonary disease, sepsis, septic shock, pulmonary fibrosis, atherosclerosis, myocarditis, endocarditis, pericarditis, cystic fibrosis, Hashimoto's disease, Graves' disease, leprosy, syphilis, Lyme disease 4. The pharmaceutical composition for preventing or treating a musculoskeletal disease, fistula disease or inflammatory disease according to claim 3, wherein the disease is any one selected from the group consisting of chronic fatigue syndrome, chronic fatigue immunodeficiency syndrome, myalgic encephalomyelitis, amyotrophic lateral sclerosis, Parkinson's disease and multiple sclerosis.

6. An injectable preparation comprising the composition described in claim 1 or claim 2.

7. The injection according to claim 6, which is administered by any one selected from the group consisting of percutaneous injection, subcutaneous injection, intramuscular injection, submucosal injection and peritoneal injection.

8. (a) culturing cells; (b) mixing the cultured cells with a hydrogel solution to form cell-hydrogel beads; (c) culturing the cell-hydrogel beads, the cells are mesenchymal stem cells; The diameter of the cell-hydrogel beads is 0.1 mm or more and 5 mm or less; the hydrogel is a fibrin glue, the fibrin glue comprising fibrinogen at a concentration of 1.8 to 90 mg / mL; The method for producing the cell-hydrogel composition, wherein the bead morphology, cell shape and properties are maintained after freezing and thawing compared to before freezing.

9. The method for producing a cell-hydrogel composition comprising cells and a hydrogel according to claim 8, wherein the diameter of the cell-hydrogel beads is 1 mm or more and 4 mm or less.

10. The method for producing a cell-hydrogel composition comprising cells and a hydrogel according to claim 8, further comprising the steps of freezing and thawing the cell-hydrogel beads after culturing them in step (c).

11. 9. The method for producing a cell-hydrogel composition comprising cells and a hydrogel according to claim 8, further comprising the step (d) of loading the cell-hydrogel beads into a syringe after the step (c).

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