Injectable composition containing mesenchymal stem cells-hydrogel and preparation, freezing and thawing methods thereof

Abstract

The present invention relates to a composition comprising an injectable mesenchymal stem cell-hydrogel and a preparation method thereof. Specifically, the injectable mesenchymal stem cell-hydrogel composition prepared by the method of the present invention has the following advantages: stem cells are attached to the scaffold of the hydrogel beads, so the stem cells are not easy to disappear or apoptosis after injection, and the paracrine effect of the stem cells is continuously exerted. The stem cells are slowly released while the hydrogel is decomposed, thereby increasing the survival rate. In addition, it can be prepared into an injectable dosage form without using proteolytic enzymes, so that healthy cells can be used without damaging the cell membrane. At the same time, 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 a preparation, freezing and thawing method thereof, and specifically to the following method: by preparing the stem cell-hydrogel into a specified shape with a diameter of less than 5 mm, a dosage form that can be easily filled into a syringe is developed and can be frozen and stored, and can be immediately thawed for use when needed.

[0002] Specifically, when stem cells are injected into the body alone, there are problems that stem cells easily disappear at the injection site and the rate of adhesion is low. The mesenchymal stem cell-hydrogel composition prepared by the method of the present invention has the following advantages: stem cells are attached to the scaffold of the hydrogel beads, so the stem cells are not easy to disappear or apoptosis after injection, and the paracrine effect of stem cells is continuously exerted. The stem cells are slowly released as the hydrogel decomposes, thereby increasing the rate of adhesion.

[0003] Furthermore, the present invention has the following advantages: Adherent mesenchymal stem cells can be prepared into an injectable dosage form without using a proteolytic enzyme by the method of the present invention, so healthy cells with undamaged cell membranes can be used. Furthermore, the cryopreservation solution can be easily removed from the mesenchymal stem cell-hydrogel beads after thawing. Background Art

[0004] The existing first-generation stem cell therapy uses isolated cells treated with proteolytic enzymes such as trypsin or dispase. During the proteolytic enzyme treatment, all proteins exposed to the cell membrane are non-selectively degraded, so that intercellular bonds and basement membrane proteins are almost not retained. In addition, animal-derived substances such as fetal bovine serum (FBS) are added to inactivate proteolytic enzymes, and the washing-centrifugation process is performed several times to remove them. Usually, about 5% to 10% of cells are lost during the first washing-centrifugation process. Therefore, the cell collection process using proteolytic enzymes is a very inefficient method.

[0005] Furthermore, if the isolated single cells are transplanted to the diseased site, they will flow out of the target site by diffusion and absorption, so the proportion of successfully transplanted cells is low. In addition, mesenchymal stem cells are cells with strong adhesion, and when they are separated into single cells, they die within 6 to 24 hours, so they have a disadvantage of very low adhesion rate.

[0006] WO 2006 / 004951 discloses a method for synthesizing soft tissue of a specified morphology and specification from adult mesenchymal stem cells (MSCs) in a biocompatible scaffold in vivo and a composition prepared by the method. The above document discloses a method of using a hydrogel polymer as a biocompatible scaffold, and more specifically, discloses a method of using polyethylene glycol diacrylate as a biocompatible scaffold. However, the above composition is used to restore soft tissue bonding, and in the case of using polyethylene glycol diacrylate as a scaffold, it is only predetermined that there is almost no change in diameter, and there is no disclosure or suggestion about the preparation process that can be filled into a syringe for direct administration after culture.

[0007] Patent No. 1289834 granted in Korea discloses a sphincter regeneration cell therapy agent containing stem cells derived from amniotic fluid, and discloses that the cell therapy agent can be injected into a hydrogel composite, specifically, into alginate / PF-127 / hyaluronic acid to increase its effect. However, the above document discloses that the stem cells and hydrogel are mixed and injected when used, and only discloses the form of preparation by injection into a hydrogel composite, and does not disclose the content of culturing stem cells in hydrogel beads.

[0008] Patent No. 684940 granted in Korea discloses a method for differentiating mesenchymal stem cells into chondrocytes, and more specifically, discloses a method for fixing and culturing mesenchymal stem cells on a mixed scaffold containing fibrin / HA as a biodegradable polymer. However, the above document only discloses the following: when fibrin / HA is used as a scaffold, even without adding transforming growth factor β (TGF-beta) which is conventionally added for cell differentiation, mesenchymal stem cells can be promoted to differentiate into chondrocytes.

[0009] Korean Patent No. 10-1814440 discloses a method for stably and massively preparing chondrocytes and cells with chondrogenic differentiation ability. More specifically, the following method is disclosed: a chondrocyte therapeutic agent without a scaffold is uniformly and massively prepared by inoculating cells in a 96-well culture plate with a "v"-shaped bottom, culturing at ultra-high density, and recovering microparticles, and then transplanted to the site of cartilage damage. However, this method is a method of preparing a bead form using only chondrocytes without a scaffold, which is structurally different from the present invention of culturing stem cells in a hydrogel bead scaffold.

[0010] Furthermore, in the case of the existing method, during the preparation of stem cell therapeutic agents, the treatment with proteolytic enzymes will cause damage to cell-to-cell bonds and basement membrane proteins, and cell losses will occur in the steps of cell collection, washing, vial filling, and refilling from the vial to the syringe. In addition, only single cells are collected and injected after enzyme treatment, which has the problem of removing most of the active substances such as collagen synthesized during cell culture.

[0011] On the other hand, Korean Patent No. 10-1613478 for solving the above-mentioned problems discloses a method for preparing a hydrogel mass containing stem cells and filling the hydrogel mass into a syringe. However, during the process of filling the hydrogel mass into the syringe and injecting it, the hydrogel has the disadvantages of irregular morphology and inability to maintain a consistent dosage form. In addition, the concentration of the hydrogel at which cells can grow within the hydrogel scaffold and have a stiffness that can be filled into the syringe is limited.

[0012] Patent No. 10-1687291 granted in Korea discloses a method for culturing stem cells or primary cells using a porous membrane and a hydrogel. More specifically, the following method is disclosed: a porous membrane is placed inside a cell culture container in a non-contact manner, and a hydrogel is applied thereon to culture cells in a three-dimensional manner. However, the above document only explicitly states that the proliferation and growth of cells in the above system are increased, and does not disclose that the hydrogel containing cells can be prepared in the form of beads for culturing, or that it can be filled into a syringe and then administered.

[0013] In order to solve the above problems, research on methods of placing cells or stem cells in a scaffold to prepare them in the form of beads is currently underway (Elizabeth et al., 2012; Christian et al., 2013; Havva et al., 2016; Medi et al., 2017). Alginate beads are a very widely used type of beads.

[0014] Korean Patent No. 10-1740298 discloses a method for preparing a hydrogel matrix containing chondrogenic cells and a method for preparing beads. The above document discloses the following method: after preparing an alginate solution and a chitosan solution, mixing with chondrogenic cells to prepare a hydrogel matrix, and then preparing beads and culturing. However, the above content is a study using general chondrocytes other than stem cells, and the average size of the beads is adjusted by adjusting the diameter of the needle, and beads cannot be prepared in a specified size. Therefore, there is a limitation that the cells in the chondrocyte-hydrogel complex cannot be prepared in a specified number.

[0015] Korean Patent No. 10-1585032 discloses a method of mixing and culturing mesenchymal stem cells with a hydrogel solution and a method of filling the solution into a syringe. However, the above invention has the limitation that it is difficult to fill the required number of cells into a syringe. In contrast, the present invention uses a method of culturing a specified number of cells in a bead, which has the advantage of being able to administer the required number of cells to the cartilage injury site.

[0016] Although it is important to prepare stem cells or cells in the form of beads for injection into the body, freezing them so that they can be stored for a long time and easily thawed for use whenever and wherever needed is also an important factor in the development of therapeutic agents. Therefore, research on freezing and thawing methods that reduce cell damage and improve cell survival is underway (Dominique et al., 2017).

[0017] Korean Patent Nos. 10-1321144 and 10-1407355 disclose compositions for cryopreservation of stem cells. However, the above documents disclose methods for improving cell survival rates of two-dimensional (2D) cultured stem cells during freezing / thawing, which is different from the present invention which discloses a method for freezing and thawing a hydrogel-bead cell mixture.

[0018] Existing cryopreserved and used stem cell therapeutic agents use a method of injecting the cryopreservation solution together with the cryopreservation solution because the method of removing the cryopreservation solution after thawing is inappropriate. In this case, dimethyl sulfoxide (DMSO), which is a component of the cryopreservation solution, can cause various side effects in the body. The size of the hydrogel-bead cell mixture of the present invention is about 0.1mm to 5mm, so it has the advantage of being able to remove the cryopreservation solution by a simple physical method.

[0019] Therefore, in the present invention, a dosage form is developed in which stem cell-hydrogel is prepared into a specified shape with a diameter of less than 5 mm so as to be easily filled into a syringe. The above-mentioned injectable dosage form used as a therapeutic agent by freezing and thawing can adjust the physical properties or physical strength of the hydrogel differently according to the purpose, and it is found that it can be used in a manner that meets various therapeutic purposes, thereby completing the present invention. Summary of the invention

[0020] Technical issues

[0021] The object of the present invention is to provide a composition comprising an injectable stem cell-hydrogel and a preparation method thereof, wherein the stem cell-hydrogel is prepared into a specified shape with a diameter of less than 5 mm, is easy to fill into a syringe, can remove the cryopreservation solution by a simple physical method, and is a ready-made preparation that can be stored for a long time.

[0022] Furthermore, the present invention aims to provide a method for preventing, treating or ameliorating musculoskeletal diseases, fistula diseases or inflammatory diseases, comprising the step of administering a pharmaceutically effective amount of a composition comprising stem cells-hydrogel to an individual.

[0023] At the same time, the present invention aims to provide the use of a composition comprising stem cells-hydrogel as a pharmaceutical composition for preventing or treating musculoskeletal diseases, fistula diseases or inflammatory diseases.

[0024] Technical means

[0025] The invention provides a cell-hydrogel composition, comprising cells and hydrogel in the form of beads.

[0026] In addition, the present invention provides a method for preparing a cell-hydrogel composition, comprising: step (a), culturing cells; step (b), mixing the cultured cells with a hydrogel solution to form cell-hydrogel beads; and step (c), culturing the cell-hydrogel beads.

[0027] Furthermore, the present invention provides a method for preparing a cell-hydrogel composition, further comprising the steps of freezing and thawing the cell-hydrogel beads of the present invention.

[0028] Furthermore, the present invention provides a method for preventing or improving musculoskeletal diseases, fistula diseases or inflammatory diseases, comprising the step of administering a pharmaceutically effective amount of cell-hydrogel beads to an individual.

[0029] Furthermore, the present invention provides a method for treating a musculoskeletal disease, a fistula disease or an inflammatory disease, comprising the step of administering a pharmaceutically effective amount of cell-hydrogel beads to a subject.

[0030] Meanwhile, the present invention provides a use of the cell-hydrogel beads as a pharmaceutical composition for preventing or treating musculoskeletal diseases, fistula diseases or inflammatory diseases.

[0031] Effects of the Invention

[0032] The injectable mesenchymal stem cell-hydrogel composition prepared by the method of the present invention has the following advantages: the stem cells are attached to the scaffold of the hydrogel beads, so the stem cells are not easy to disappear or apoptosis after injection, and the paracrine effect of the stem cells is continuously exerted. The stem cells are slowly released as the hydrogel decomposes, thereby increasing the survival rate. In addition, it can be prepared into an injectable dosage form without using protease treatment, and healthy cells can be used without damaging the cell membrane. At the same time, the cryopreservation solution can be easily removed from the mesenchymal stem cell-hydrogel beads after freezing and thawing. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1aThe figure shows a photograph of observation of cultured cell-hydrogel beads.

[0034] Figure 1b The figure shows a microscope photograph of cell-hydrogel beads cryopreserved at -80°C, which were thawed, filled into a syringe and ejected using a 17-gauge needle.

[0035] Figure 2 A diagram confirming the identity of cells within cell-hydrogel beads.

[0036] Figure 3 Figure 2 is a graph confirming the capture of active factors within cell-hydrogel beads.

[0037] Figure 4 This is a graph confirming the secretion effect of paracrine factors of cell-hydrogel beads. The y-axis is relative value (fold increase).

[0038] Figure 5 This graph shows the volume changes within 7 days after the cell-hydrogel beads of the present invention were subcutaneously injected into mice.

[0039] Figure 6 This is a diagram confirming the effect of suppressing chondrocyte apoptosis caused by oxidative stress of the cell-hydrogel beads of the present invention.

[0040] Figure 7 This is a diagram confirming the inflammation suppressing effect of the cell-hydrogel beads of the present invention. DETAILED DESCRIPTION

[0041] Hereinafter, the present invention will be described in detail.

[0042] The invention provides a cell-hydrogel composition, comprising cells and hydrogel in the form of beads.

[0043] Furthermore, the present invention provides a use of a cell-hydrogel composition as a pharmaceutical composition for preventing or treating musculoskeletal diseases, fistula diseases or inflammatory diseases, wherein the cell-hydrogel composition comprises cells and hydrogel in the form of beads.

[0044] In one embodiment of the present invention, preferably, the diameter of the cells and the hydrogel is 0.1 mm to 5 mm, more preferably 0.5 mm to 5 mm, and most preferably 1 mm to 4 mm.

[0045] In one embodiment of the present invention, preferably, the cell is one selected from the group consisting of stem cells, somatic cells and germ cells, but is not limited thereto.

[0046] In one embodiment of the present invention, preferably, the hydrogel is one or more selected from the group consisting of fibrin glue, hyaluronic acid, gelatin, collagen, alginate, chitosan, cellulose, pectin, methacrylate-2-hydroxyethyl ester derivatives and copolymers thereof, polyethylene oxide and polyvinyl alcohol.

[0047] In one embodiment of the present invention, preferably, the above-mentioned musculoskeletal disease is selected from the group consisting of myositis, myofascial pain syndrome, tendinitis, tenosynovitis, bursitis, ganglion cyst, carpal tunnel syndrome, Guyon's canal syndrome, wrist tendinitis, wrist vibration syndrome, trigger finger, ganglion cyst, white finger disease, Raynaud's syndrome, lateral epicondylitis, medial epicondylitis, radial tunnel syndrome, ulnar tunnel syndrome, olecranon bursitis, median nerve recurrent branch entrapment, shoulder impingement syndrome, adhesive capsulitis, degenerative arthritis, turtle neck syndrome, radiculopathy, lumbar strain, lumbar disc herniation, spondylolisthesis, spondylolisthesis, degenerative lumbar disease, degenerative disease, urinary incontinence, ligament and tendon injury, but is not limited to this.

[0048] In one embodiment of the present invention, the fistula disease may be fistula Crohn's disease, but is not limited thereto.

[0049] In one embodiment of the present invention, preferably, the inflammatory disease is selected from atopic dermatitis, systemic lupus erythematosus, lupus erythematosus, pernio lupus erythematosus, tuberculous lupus erythematosus, lupus erythematosus nephritis, dystrophic bullous epidermolysis, 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 caused by viral or bacterial infection, colitis, Ulcerative colitis, inflammatory bowel disease, fungal infections, burns, wounds caused by surgery or dental procedures, diabetic foot ulcers, type I diabetes, type II 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 thyroiditis, Graves' disease, leprosy, syphilis, Lyme disease disease), Borreliosis, neuroborreliosis, tuberculosis, sarcoidosis, macular degeneration, uveitis, irritable bowel syndrome, Crohn's disease, Sjögren's syndrome, chronic fatigue syndrome, chronic fatigue immune dysfunction syndrome, myalgic encephalomyelitis, amyotrophic lateral sclerosis, Parkinson's disease, and multiple sclerosis, but is not limited to this.

[0050] The composition of the present invention is in the form of a predetermined bead with a diameter of less than 5 mm, is easy to be filled into a syringe, and can remove the cryopreservation solution by a simple physical method, and dimethyl sulfoxide as a component of the cryopreservation solution that can cause various side effects in the body can be easily removed before administration. Specifically, as an example of the above-mentioned simple physical method, after the mixture of the cryopreservation solution and the beads is loaded into a syringe, a filter with a pore size of about 100 μL is installed at the front end of the syringe, and the solution is pushed, so that the beads are blocked by the filter, so that only the cryopreservation solution can be removed.

[0051] Furthermore, after being filled into a syringe, the drug can be administered by transdermal injection, subcutaneous injection, intramuscular injection, submucosal injection, intraperitoneal injection, or the like.

[0052] Furthermore, the concentration of the hydrogel of the present invention can be used without particular limitation and can be adjusted according to various therapeutic purposes. Preferably, the hydrogel can contain fibrinogen at a concentration of 1.8 mg / mL to 90 mg / mL.

[0053] Furthermore, the present invention provides a method for preparing a cell-hydrogel composition, comprising: step (a), culturing cells; step (b), mixing the cultured cells with a hydrogel solution to form cell-hydrogel beads; and step (c), culturing the cell-hydrogel beads.

[0054] In one embodiment of the present invention, the hydrogel can use one or a complex of two or more selected from the group consisting of fibrin glue, hyaluronic acid, gelatin, collagen, alginate, chitosan, cellulose, pectin, methacrylate-2-hydroxyethyl ester derivatives and copolymers thereof, polyethylene oxide and polyvinyl alcohol. More specifically, fibrin glue can be used.

[0055] In one embodiment of the present invention, the fibrin glue may contain fibrinogen at a concentration of 1.8 mg / mL to 90 mg / mL.

[0056] In one embodiment of the present invention, the cells and the hydrogel are preferably 0.1 mm to 5 mm, more preferably 0.5 mm to 5 mm, and most preferably 1 mm to 4 mm.

[0057] In one embodiment of the present invention, after the above step (c), a step (d) may be further included, in which the cell-hydrogel composition is filled into a syringe. The cell-hydrogel composition prepared by the preparation method of the present invention can be directly administered topically using a needle of 10 gauge to 25 gauge.

[0058] On the other hand, the method of the present invention may further include freezing and thawing steps after culturing the cell hydrogel in step (c), and the cryopreservative solution may be removed by simple physical methods after freezing and thawing.

[0059] Meanwhile, 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 differently depending on the purpose, and therefore it is not limited to a specific hydrogel concentration.

[0060] In a specific embodiment of the present invention, the inventors cultured mesenchymal stem cells derived from human fat, added them to a thrombin solution, and then used a dispenser to drip fibrinogen and cell-thrombin solution into a culture container at a volume of 1 μL to 10 μL, respectively, to prepare cell-containing fibrin glue hydrogel beads (hereinafter referred to as cell-hydrogel beads) (refer to Figure 1).

[0061] Furthermore, the present inventors confirmed the number of cells, survival rate and cell characteristics in the cell-hydrogel beads, and confirmed that the cells cultured in the hydrogel beads according to the present invention showed positive reactions with CD73, CD90 and CD105, which are representative mesenchymal stem cell markers, and maintained negative immunological characteristics with CD34 and CD45, which are hematopoietic cell markers (see Table 1, Figure 2 ).

[0062] Furthermore, the present inventors confirmed whether active factors secreted from cells were captured in the beads during the preparation process of cell-hydrogel beads, and found that hepatocyte growth factor (HGF) and collagen type II (type 2) secreted from mesenchymal stem cells were significantly captured. (See Figure 3 ).

[0063] At the same time, the inventors confirmed the results of the secretion effect of paracrine factors of cell-hydrogel beads, and confirmed that the cell-hydrogel beads of the present invention slowly release paracrine factors in the inflammatory environment induced by interleukin-1β, thereby showing a significant therapeutic effect (refer to Figure 4 ).

[0064] Therefore, the mesenchymal stem cell-hydrogel composition for injection prepared by the method of the present invention has the following advantages: the stem cells are attached to the scaffold of the hydrogel beads, the stem cells are not easy to disappear or apoptosis after injection, the paracrine effect of the stem cells is continuously exerted, and the stem cells are slowly released as the hydrogel decomposes, thereby increasing the survival rate (refer to Figure 5 ), and can be prepared as an injectable formulation without using a proteolytic enzyme treatment, can use healthy cells without damaging the cell membrane, and can easily remove the cryopreservation solution from the mesenchymal stem cell-hydrogel beads after freezing and thawing.

[0065] Furthermore, the present invention provides a cell therapy agent for preventing and treating musculoskeletal diseases, fistula diseases or inflammatory diseases, comprising the composition of the present invention as an active ingredient.

[0066] In the present invention, the term "cell therapy agent" refers to cells and tissues separated, cultured and prepared by special operations from the human body, and used for the purpose of treatment, diagnosis and prevention. In particular, it refers to a series of actions such as proliferating and screening living self, allogeneic or heterologous cells in vitro or changing the biological characteristics of cells by other methods in order to restore the function of cells or tissues, and used for the purpose of treatment, diagnosis and prevention. According to the degree of cell differentiation, cell therapy agents are generally divided into somatic cell therapy agents and stem cell therapy agents. More specifically, the present invention relates to adipose-derived stem cell therapy agents.

[0067] In the present invention, the term "subject" refers to all animals including humans who have suffered from a disease that can be prevented or treated by administering the cell therapeutic agent of the present invention or who may suffer from the above disease.

[0068] The above composition can be used for various diseases such as joint trauma, bone diseases, muscle weakness, degenerative diseases caused by joint nerve damage, urinary incontinence, degenerative arthritis, ligament and tendon injuries, diabetic foot ulcers, lower limb ischemic ulcers, fistula diseases, etc.

[0069] Furthermore, the present invention provides a method for preventing or improving musculoskeletal diseases, fistula diseases or inflammatory diseases, comprising the step of administering a pharmaceutically effective amount of a cell-hydrogel composition to an individual, wherein the cell-hydrogel composition comprises cells and hydrogel in the form of beads, and the diameter of the cells and hydrogel is greater than 0.1 mm and less than 5 mm.

[0070] At the same time, the present invention provides a method for treating musculoskeletal diseases, fistula diseases or inflammatory diseases, comprising the step of administering a pharmaceutically effective amount of a cell-hydrogel composition to an individual, wherein the cell-hydrogel composition comprises cells and hydrogel in the form of beads, and the diameter of the cells and hydrogel is greater than 0.1 mm and less than 5 mm.

[0071] The description of the cell-hydrogel, musculoskeletal diseases, fistula diseases and inflammatory diseases is the same as the description of the cell-hydrogel composition, so the specific description will be cited above.

[0072] On the other hand, it is confirmed that the injectable mesenchymal stem cell-hydrogel composition prepared by the method of the present invention has the following advantages, namely, the stem cells are attached to the scaffold of the hydrogel beads, so the stem cells are not easy to disappear or apoptosis after injection, and the paracrine effect of the stem cells is continuously exerted. The stem cells are slowly released as the hydrogel decomposes, thereby increasing the survival rate. In addition, it can be prepared as an injectable dosage form without using protease treatment, and healthy cells can be used without damaging the cell membrane. At the same time, the cryopreservative 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 can be usefully used to prevent, treat or improve musculoskeletal diseases, fistula diseases or inflammatory diseases.

[0073] Hereinafter, the present invention will be described in more detail by way of examples.

[0074] However, the following embodiments are only used to help the understanding of the present invention and are not intended to limit the scope of protection of the present invention to these embodiments.

[0075] Example 1. Method for culturing mesenchymal stem cells derived from human adipose tissue

[0076] Adipose tissue may be obtained by conventional liposuction, but is not limited thereto.

[0077] Adipose-derived mesenchymal stem cells were isolated from adipose tissue obtained by liposuction in the following manner: In order to remove blood, the adipose tissue was washed 3 to 4 times with a phosphate buffered saline (PBS) of the same volume. A collagenase solution of the same volume as the adipose tissue was added and reacted in a water bath at a temperature of 37°C. The adipose tissue was transferred to a centrifugal test tube and centrifuged at 1500 rpm for 10 minutes at a temperature of 20°C. After removing the fat layer as the upper layer, the collagenase solution as the lower layer was carefully separated without shaking. After being suspended in a cell culture medium, the adipose tissue was centrifuged at 1200 rpm for 5 minutes at a temperature of 20°C. In this case, the precipitate at the bottom was separated from the upper layer by stromal-vascular fractionation. The stromal-vascular fraction was suspended in a cell culture medium and inoculated into a culture container, and cultured in an incubator at 37°C and 5% CO2 for 24 hours. After the culture medium is removed, the cells are washed with a phosphate buffer solution and proliferated using a cell culture medium or a culture medium containing a cell growth factor such as basic fibroblast growth factor (bFGF) or epidermal growth factor (EGF). When the adipose-derived mesenchymal stem cells grow to 80% to 90% of the culture container, they are separated and single cells are obtained by treatment with trypsin.

[0078] Example 2. Preparation and freezing of cell-fibrin glue hydrogel beads

[0079] 1) Preparation of cell-fibrin hydrogel beads

[0080] To 1×10 5 / mL~3×10 5 The adipose-derived mesenchymal stem cells obtained in Example 1 above are added with a thrombin solution of 1.8 mg / mL to 90 mg / mL of fibrinogen. Fibrinogen and cell-thrombin solution are dripped into a culture container with a volume of 1 μL to 10 μL using a dispenser to prepare cell-containing fibrin glue hydrogel beads (hereinafter referred to as cell-hydrogel beads).

[0081] 2) Culture container attachment culture method

[0082] When the cell-hydrogel beads were completely solidified in the culture container, the cell culture medium was directly added and cultured in an incubator at 37°C and 5% CO2 for 4 to 8 days to confirm that they had a hemispherical morphology.

[0083] 3) Suspension culture method

[0084] In addition, a suspension culture method can be used instead of the culture vessel attachment culture method described in 2). When the cell-hydrogel beads are completely solidified, the beads are removed from the bottom of the culture vessel and placed in the culture vessel. After adding a cell culture medium to suspend the beads, the beads are cultured in an incubator at 37°C and 5% CO2 for 4 to 8 days. On the other hand, in the case of suspension culture, it has the advantage of being easy to culture in large volumes.

[0085] 4) Washing and freezing of cell-hydrogel beads

[0086] After the culture medium was removed, the cell-hydrogel beads were collected from the culture container, mixed with human serum albumin containing 10% to 20% dimethyl sulfoxide at a ratio of 1:1, and then frozen and stored at -80°C.

[0087] On the other hand, after a mixture of cryopreservative solution and beads is loaded into a syringe and a filter having a pore size of about 100 μL is attached to the front end of the syringe, the solution is pushed so that the beads are blocked by the filter, thereby removing only the cryopreservative solution by a simple physical method.

[0088] Figure 1a This figure shows a photograph of the cultured cell-hydrogel beads. It was confirmed that the diameter of the beads was 3 mm to 5 mm, and the cells proliferated well in the beads and fused well with the fibrin glue hydrogel.

[0089] Figure 1b This is a microscopic photograph of cell-hydrogel beads frozen and stored at -80°C, thawed, filled into a syringe, and injected using a 17-gauge needle. It was confirmed that all beads maintained a predetermined shape and size after freezing, thawing, and injection using a needle.

[0090] Example 3. Number of cells, survival rate and characteristics of cells in cell-hydrogel beads

[0091] The results of observing the cell-hydrogel beads cultured-washed-frozen-thawed according to Example 2 using a microscope confirmed that the cells in the beads were evenly distributed (Figure 1). Then, enzymes were added to the cell-hydrogel beads cultured-washed-frozen-thawed according to Example 2 to dissolve the fibrin glue and obtain cells. The results of analyzing the cell number and cell viability using trypan blue staining confirmed that each bead contained approximately 15,000 cells and the cell viability was more than 95%.

[0092] The cells obtained by staining with CD73, CD90, CD105, CD34, and CD45 were analyzed using a flow cytometer. The results are shown in FIG1 , and it was confirmed that the cells cultured in the hydrogel beads according to the present invention showed positive reactions with CD73, CD90, and CD105, which are representative mesenchymal stem cell markers, and maintained negative immunological characteristics with CD34 and CD45, which are hematopoietic cell markers (Table 1 and Figure 2 ).

[0093] Table 1

[0094] CD90 97.4 CD73 98.8 CD105 95.4 CD45 1.4 CD34 0.2

[0095] Example 4. Active factors contained in cell-hydrogel beads

[0096] It is known that mesenchymal stem cells secrete a variety of active factors during culture. Therefore, in order to confirm whether the active factors secreted from the cells during the preparation process of the cell-hydrogel beads are captured in the beads, the eluate after adding enzyme-dissolving fibrin glue to the cell-hydrogel beads cultured-washed-frozen-thawed according to Example 2 was obtained, and the active factors in the cell-hydrogel beads were analyzed.

[0097] The results are as follows Figure 3 As shown in the figure, it was confirmed that about 10 picograms (pg) of hepatocyte growth factor (HGF), a representative active factor secreted from mesenchymal stem cells, was captured per bead, and about 2 pg of collagen type II, a major cartilage component, was captured per bead. Figure 3 ).

[0098] Example 5. Secretion of paracrine factors by cell-hydrogel beads

[0099] Mesenchymal stem cells are known to have anti-inflammatory effects, anti-apoptotic effects, and cell proliferation promoting effects through paracrine effects.

[0100] Therefore, after adding DMEM culture medium to the cell-hydrogel beads cultured-washed-frozen-thawed according to Example 2 and culturing for 72 hours, the culture supernatant was taken and then the enzyme was added to the remaining cell-hydrogel beads to dissolve the fibrin glue to obtain the dissolution solution to analyze the factors secreted from the cell-hydrogel beads.

[0101] Furthermore, in order to create a microenvironment (inflammatory environment) at the lesion site, the cells were treated with interleukin 1β (IL-1β), and the culture supernatant and the eluate were obtained and analyzed in the same manner.

[0102] The results are as follows Figure 4 As shown in Figure 2, it was confirmed that although prostaglandin E2 (PGE2), which is known to be a representative inflammatory regulatory factor secreted from mesenchymal stem cells, is not secreted in normal environments, its secretion is promoted in inflammatory environments ( Figure 4 ).

[0103] Furthermore, it was confirmed that among the representative growth factors secreted from mesenchymal stem cells, hepatocyte growth factor and vascular endothelial growth factor (VEGF), which exhibit anti-inflammatory, anti-apoptotic, cell proliferation-promoting, and angiogenesis-promoting effects, are secreted similarly in both normal and inflammatory environments, whereas collagen type II, the main structural protein of articular cartilage, which is slightly secreted in normal environments, is secreted in large amounts in inflammatory environments ( Figure 4 ).

[0104] Therefore, it was confirmed that when the cell-hydrogel beads of the present invention are administered to a diseased environment, paracrine factors can be slowly released to exhibit a significant therapeutic effect.

[0105] Example 6. Animal Experiment

[0106] After 30 cell-hydrogel beads cultured-washed-frozen-thawed according to Example 2 were subcutaneously injected at two sites on the left and right sides of mice, the volume change within 7 days was measured.

[0107] The results are as follows Figure 5 As shown, it was confirmed that after subcutaneous injection, stem cells are not easy to disappear or die, and the paracrine effect of stem cells continues to be exerted. The stem cells are slowly released as the hydrogel decomposes.

[0108] Example 7. Chondrocyte apoptosis inhibition ability of cell-hydrogel beads

[0109] Suppression of osteoarthritis, a representative musculoskeletal disease, which is further aggravated by apoptosis of chondrocytes Therefore, the ability of the cell-hydrogel beads prepared according to Example 2 to inhibit apoptosis of chondrocytes was confirmed.

[0110] Specifically, human chondrocytes were seeded and attached to a 48-well plate, and then the cell-hydrogel beads prepared according to Example 2 or the hydrogel beads without cells were added and co-cultured for 24 hours. Then, t-butyl hydroperoxide (tBOOH) was used at concentrations of 100 μM, 200 μM, and 400 μM for 16 hours to induce oxidative apoptosis of human chondrocytes. After that, water soluble tetrazolium salt (WST-1) was added and cultured for about 3 hours, and the absorbance was measured to determine the number of living cells.

[0111] The results are as follows Figure 6 As shown in Table 2, it was confirmed that the cell-hydrogel beads inhibited the apoptosis of chondrocytes due to oxidative stress (Table 2 and Figure 6 ).

[0112] Table 2

[0113]

[0114] Example 8. Anti-inflammatory macrophage differentiation-promoting ability of cell-hydrogel beads

[0115] After human monocytes were treated with phorbol 12-myristate 13-acetate (PMA) for 24 hours to induce differentiation into macrophages, the cells-hydrogel beads prepared according to Example 2 were added and cultured for another 48 hours. After that, the supernatant was collected and the amount of tumor necrosis factor-α (TNF-α) secreted by activated M1 macrophages with pro-inflammatory properties and interleukin-10 (IL-10) secreted by activated M2 macrophages with anti-inflammatory properties was measured using enzyme-linked immunosorbent assay (ELISA).

[0116] The results are as follows Figure 7 As shown in the figure, when phorbol 12-myristate 13-acetate (PMA) was added to human monocytes, when the amount of secreted tumor necrosis factor-α was 1.00, if cell-hydrogel beads were added thereto, it decreased to 0.73. On the contrary, when phorbol 12-myristate 13-acetate was added, when the amount of secreted interleukin-10 was 1.00, if cell-hydrogel beads were added thereto, it increased to 1.61. Thus, it was confirmed that the cell-hydrogel beads had the effect of inhibiting inflammatory response ( Figure 7 ).

[0117] Industrial Applicability

[0118] The present invention relates to a composition comprising an injectable mesenchymal stem cell-hydrogel and a preparation method thereof. Specifically, the injectable mesenchymal stem cell-hydrogel composition prepared by the method of the present invention has the following advantages: stem cells are attached to the scaffold of the hydrogel beads, so the stem cells are not easy to disappear or apoptosis after injection, and the paracrine effect of the stem cells is continuously exerted. The stem cells are slowly released as the hydrogel decomposes, thereby increasing the survival rate. In addition, the composition can be prepared into an injectable dosage form without using a protease treatment, so that healthy cells can be used without damaging the cell membrane. At the same time, the cryopreservation solution can be easily removed from the mesenchymal stem cell-hydrogel beads after freezing and thawing, so that it can be used usefully.

Claims

1. A cell-hydrogel bead composition, characterized in that: Contains cells and hydrogel, In bead form, The above 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, and the fibrin glue contains fibrinogen at a concentration of 1.8 mg / mL to 90 mg / mL, The cell-hydrogel bead composition maintains the bead morphology after freezing and thawing compared with that before freezing.

2. The cell-hydrogel bead composition according to claim 1, characterized in that The diameter of the cell-hydrogel beads is 1 mm or more and 4 mm or less.

3. A pharmaceutical composition for preventing or treating musculoskeletal diseases, fistula diseases or inflammatory diseases, characterized in that: The composition according to claim 1 or 2 is contained as an active ingredient.

4. The pharmaceutical composition for preventing or treating musculoskeletal diseases, fistula diseases or inflammatory diseases according to claim 3, characterized in that: The musculoskeletal disease is one selected from the group consisting of bone disease, muscle weakness, and ligament and tendon injuries.

5. The pharmaceutical composition for preventing or treating musculoskeletal diseases, fistula diseases or inflammatory diseases according to claim 3, characterized in that: The above musculoskeletal diseases are joint trauma or degenerative diseases.

6. The pharmaceutical composition for preventing or treating musculoskeletal diseases, fistula diseases or inflammatory diseases according to claim 3, characterized in that: The above-mentioned musculoskeletal disease is one selected from the group consisting of carpal tunnel syndrome, radial tunnel syndrome, ulnar tunnel syndrome, Raynaud's syndrome, median nerve recurrent branch entrapment syndrome, turtle neck syndrome, myofascial pain syndrome, shoulder impingement syndrome, degenerative arthritis, degenerative lumbar disease and spondylolisthesis.

7. The pharmaceutical composition for preventing or treating musculoskeletal diseases, fistula diseases or inflammatory diseases according to claim 3, characterized in that: The above-mentioned musculoskeletal disease is one selected from the group consisting of myositis, tendinitis, bursitis, tenosynovitis, lateral epicondylitis, medial epicondylitis, lumbar strain, radiculopathy, cervical spondylotic radiculopathy, lumbar disc herniation and spondylolisthesis caused by nerve damage in joints.

8. The pharmaceutical composition for preventing or treating musculoskeletal diseases, fistula diseases or inflammatory diseases according to claim 3, characterized in that: The musculoskeletal disease is one selected from the group consisting of wrist vibration syndrome, wrist tendinitis, olecranon bursitis, adhesive capsulitis, white finger disease, trigger finger, ganglion cyst and urinary incontinence.

9. The pharmaceutical composition for preventing or treating musculoskeletal diseases, fistula diseases or inflammatory diseases according to claim 3, characterized in that: The inflammatory disease is one selected from the group consisting of fascial diseases, chronic inflammatory diseases caused by viral or bacterial infections, fibromyalgia, fungal infections, burns, wounds caused by surgical or dental operations, multiple sclerosis and cystic fibrosis.

10. The pharmaceutical composition for preventing or treating musculoskeletal diseases, fistula diseases or inflammatory diseases according to claim 3, characterized in that: The inflammatory disease is one selected from the group consisting of inflammatory bowel disease, inflammatory collagen vascular disease, chronic obstructive pulmonary disease, chronic fatigue syndrome, arthritis, dermatitis and amyotrophic lateral sclerosis.

11. The pharmaceutical composition for preventing or treating musculoskeletal diseases, fistula diseases or inflammatory diseases according to claim 3, characterized in that: The above-mentioned inflammatory disease is one selected from the group consisting of lupus erythematosus, lupus erythematosus nephritis, colitis, sinusitis, rhinitis, conjunctivitis, glomerulonephritis, encephalitis, myocarditis, endocarditis, pericarditis, uveitis, Hashimoto's thyroiditis, inflammatory bowel disease, osteoarthritis, undifferentiated joint disease, inflammatory osteolytic scleroderma, Graves' disease, psoriasis, leprosy, Lyme disease, Borrelia spirocheteosis, tuberculosis, sarcoidosis, Parkinson's disease, malnutrition bullous epidermolysis, rheumatic fever, osteoporosis, diabetic foot ulcer, type I diabetes, type II diabetes, asthma, sepsis, septic shock, pulmonary fibrosis, atherosclerosis, syphilis, macular degeneration, irritable bowel syndrome, Sjögren's syndrome and chronic fatigue immune dysfunction syndrome.

12. The pharmaceutical composition for preventing or treating musculoskeletal diseases, fistula diseases or inflammatory diseases according to claim 3, characterized in that: The above-mentioned inflammatory disease is one selected from the group consisting of myalgic encephalomyelitis, atopic dermatitis, ulcerative colitis, rheumatoid arthritis, systemic lupus erythematosus, pernio lupus erythematosus, tuberculous lupus erythematosus, undifferentiated spondyloarthropathy, reactive arthritis, ulcerative skin disease, neurogenic Borrelia spirocheteosis, Crohn's disease and low back pain.

13. An injection, characterized in that: Comprising the composition according to claim 1 or 2.

14. The injection according to claim 13, characterized in that: The injection is administered by one method selected from the group consisting of transdermal injection, subcutaneous injection, intramuscular injection, submucosal injection and intraperitoneal injection.

15. A method for preparing a cell-hydrogel bead composition, characterized in that: include: Step (a), culturing cells; Step (b), mixing the cultured cells with a hydrogel solution to form cell-hydrogel beads; as well as Step (c), culturing the cell-hydrogel beads, The above 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, and the fibrin glue contains fibrinogen at a concentration of 1.8 mg / mL to 90 mg / mL, The cell-hydrogel bead composition maintains the bead morphology after freezing and thawing compared with that before freezing.

16. The method for preparing the cell-hydrogel bead composition according to claim 15, characterized in that: The diameter of the cell-hydrogel beads is 1 mm or more and 4 mm or less.

17. The method for preparing the cell-hydrogel bead composition according to claim 15, characterized in that: After culturing the cell-hydrogel beads in step (c), the method further includes freezing and thawing steps.

18. The method for preparing the cell-hydrogel bead composition according to claim 15, characterized in that: After step (c), the method further comprises step (d) of filling the cell-hydrogel bead composition into a syringe.

19. Use of a cell-hydrogel bead composition in the preparation of a pharmaceutical composition for preventing or treating musculoskeletal diseases, fistula diseases or inflammatory diseases, characterized in that: The cell-hydrogel composition comprises cells and hydrogel in the form of beads. The above 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, and the fibrin glue contains fibrinogen at a concentration of 1.8 mg / mL to 90 mg / mL, The cell-hydrogel bead composition maintains the bead morphology after freezing and thawing compared with that before freezing.

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