KR101389849B1 - Method for culture of mesenchymal stem cell from gastrointestinal tissue, nerve tissue, or blood vessels and Uses Therefor - Google Patents

Abstract

The present invention relates to a method for culturing mesenchymal stem cells, mesenchymal stem cells obtained by the culturing method, and a cell therapeutic agent using the mesenchymal stem cells. And incorporating one or more tissues selected from the group consisting of vascular outer membrane tissue into the hydrogel and culturing the same. And recovering the mesenchymal stem cells grown and moved into the hydrogel from one or more tissues selected from the group consisting of subgastric mucosal tissue, peripheral nerve epithelial tissue and vascular epithelial tissue by decomposing only hydrogel. It provides a culture method, mesenchymal stem cells cultured by the culture method, the mesenchymal stem cells of the present invention is excellent in self-replicating ability and multi-differentiation ability, and is very useful as a cell therapy for tissue or organ regeneration.

Description

Method for culture of mesenchymal stem cell from gastrointestinal tissue, nerve tissue, or blood vessels and Uses Therefor}

The present invention relates to a method for isolating and culturing mesenchymal stem cells from gastrointestinal submucosa, peripheral nerve epithelial tissue or vascular epithelial tissue, and mesenchymal stem cells cultured by the method.

Hematopoietic stem cells are the most studied of adult stem cells, and recent studies on mesenchymal stem cells (Mesenchymal stem cells) is also vigorous.

In 1999, Pittenger et al. Found that mesenchymal stem cells isolated from bone marrow have the ability to self renewal and differentiate into bone, cartilage and fat. This experiment revealed that adult mesenchymal stem cells could be manipulated in the laboratory, and the concept of stem cell therapy could be introduced. After that, bone marrow mesenchymal stem cells can be differentiated into hepatocytes, cardiomyocytes, neurons and brain cells, and adult stem cells can be differentiated into all kinds of cells by reactivating genes. Therefore, attempts to replace all the damaged organs using adult stem cells have been actively attempted. Currently, fractures, tendon damage, cartilage damage, hepatic failure, myocardial infarction, nerve damage, brain damage, stroke, and Parkinson's disease , Active research is underway for the treatment of other neurodegenerative diseases (Horwitz, EM et al., Nat. Med. 5: 309-313, 1999; Pittenger, MF et al., Circ.Res. 95: 9- 20, 2004).

Mesenchymal stem cells are derived from bone marrow, cord blood, periosteum, synoviium, fat, etc., as well as the ability to differentiate into cells and tissues of mesodermal origin. It is a multipotentcy stem cell that can differentiate into cells and tissues of ectoderm origin such as cells and cardiomyocytes. Unlike hematopoietic stem cells, mesenchymal stem cells have the advantage that they can be cultured and amplified in vitro, which is very likely as a cell therapy. Mesenchymal stem cells can be differentiated into bone cells, chondrocytes, meniscus cells, cardiomyocytes, neurons, etc. and their ability to reconstruct the defective tissues has been proved experimentally or clinically. Clinical trials are being used as cell therapies to rebuild the function of organs and organs. In other words, mesenchymal stem cells have been developed as a cell therapy to treat osteoporosis, fractures, arthritis, myocardial infarction, congestive heart failure, degenerative and traumatic brain diseases.

As a cell therapy, mesenchymal stem cells are required to be separated from tissues in animals and human bodies and then amplified and cultured in vitro. As a cell therapy for application to patients, mesenchymal stem cells require at least 10 million cells. Bone marrow has been the most used tissue source for isolating mesenchymal stem cells, but mesenchymal stem cell density in bone marrow tissue is known to exist in only 1 mesenchymal stem cell out of 1 billion hematopoietic cells. It is also known that the number of mesenchymal stem cells in bone marrow decreases rapidly in inverse proportion to age. In the middle-aged patients, at least 100 c.c. of bone marrow tissue should be collected from the bone marrow to prepare mesenchymal stem cell therapy, and the harvesting process carries the risk of requiring general anesthesia and invasive surgery. In addition, due to the scarcity of mesenchymal stem cells in the bone marrow has been a disadvantage that the amplification culture is not as easy as the number of effective dose as a cell therapy.

As a result, researches to discover tissue sources other than bone marrow that can separate mesenchymal stem cells have been actively conducted. Mesenchymal stem cells are also found in the periosteum, synovial membrane, fat, skeletal muscle, heart, skin, umbilical cord blood, umbilical cord, amniotic membrane, and placenta. It turns out to exist. However, the collection of these tissues is accompanied by invasive techniques and risks, and also requires the development of a technique for separating high-purity mesenchymal stem cells from these tissues. Stem cells are also present in the umbilical cord blood, but in the case of patients who do not store umbilical cord blood at birth, there is a problem in that autologous cell therapeutics cannot be prepared.

Meanwhile, two methods described below have been widely applied to separate mesenchymal stem cells from conventional adult humans. First, tissues that separate dissociated single cell populations from solid tissues by treating the periosteum, synovial membrane, and adipose tissue of adult with histolytic enzymes such as collagenase, dispase, or trypsin. Single cells were amplified by monolayer culture through a tissue dissociation step. Since the cells that do not adhere to the culture vessel is removed, the method of separating and culturing the mesenchymal stem cells through the process of culturing only the cells attached to the culture vessel has been widely used. In the second method, as in the first method, single dissociated cells obtained after tissue dissociation of solid tissues are separated, and then, using a marker such as CD34 or CD29, only the cells expressing the marker are immunized. Purification using a pharmacological method, and then amplification by monolayer culture method has been proposed.

However, the conventional method for separating and culturing mesenchymal stem cells described above has presented some problems. First, the tissue dissociation process is essential in order to separate single dissociated cells from solid tissue. The tissue dissociation process shows a large variation in the number of single cells obtained according to the type, titer, reaction time, reaction temperature, and state of tissue used, and the damage of cells during tissue dissociation is inevitable. Presented. As a result, the organizational dissociation process is difficult to standardize and the inefficiency of low separation yield is suggested. Second, the single dissociated cells obtained after the tissue dissociation process contain all the cells with various heterogeneous characteristics that make up the organ or tissue of origin. Single dissociated cells isolated from the tissues include all vascular endothelial cells, vascular smooth muscle cells, adipocytes, blood cells, and fibroblasts in addition to mesenchymal stem cells. In order to selectively separate the mesenchymal stem cells from the heterogeneous single dissociated cells, an immunological purification method using a marker specifically expressed only in the mesenchymal stem cells is required. However, a fundamental problem arises that markers that are specific and consistently expressed only in mesenchymal stem cells are not known at present.

Submucosal tissues of the gastrointestinal tract, outer membrane tissues of peripheral nerves, and outer membrane tissues of blood vessels are constituents of extracellular matrix and moisture, and are widely distributed along all solid organs of humans and mammals. There is no known result of mesenchymal stem cells in submucosal tissue, peripheral nerve tissue, and blood vessel outer membrane tissue.

Accordingly, the present invention is to provide a method for separating and culturing mesenchymal stem cells of high purity from a single process from the gastrointestinal submucosa, peripheral nerve epithelial tissue and vascular epithelial tissue of a human adult. The present invention does not use histolytic enzymes to selectively separate mesenchymal stem cells present in the gastrointestinal tract, peripheral nerves and blood vessels of human adults, and is stable and efficient without destroying mesenchymal stem cell microspheres in the tissue. The purpose of this study was to propose a method of isolation and culture of neural crest stem cells.

The present invention comprises the steps of culturing by incorporating one or more tissues selected from the group consisting of submucosal tissue, peripheral nerve epithelial tissue and vascular epithelial tissue into a hydrogel; And recovering the mesenchymal stem cells grown and moved into the hydrogel from one or more tissues selected from the group consisting of subgastric mucosal tissue, peripheral nerve epithelial tissue and vascular epithelial tissue by decomposing only hydrogel. To provide a culture method.

In addition, the present invention is to provide a stem cell obtained by the culture method, and a cell therapy comprising the same.

The present invention comprises the steps of culturing by incorporating one or more tissues selected from the group consisting of submucosal tissue, peripheral nerve epithelial tissue and vascular epithelial tissue into a hydrogel; And recovering the mesenchymal stem cells grown and moved into the hydrogel from one or more tissues selected from the group consisting of subgastric mucosal tissue, peripheral nerve epithelial tissue and vascular epithelial tissue by decomposing only hydrogel. Provide a culture method.

As an example of the method for culturing the mesenchymal stem cells, 1) a first step of incorporating at least one tissue selected from the group consisting of gastrointestinal submucosa, peripheral nerve epithelial tissue and vascular epithelial tissue into fibrin hydrogel; 2) a second step of three-dimensional organ culture in a shaker at a speed of 5 to 30 rpm by adding the growth culture solution; And 3) at least one tissue selected from the group consisting of urokinase, streptokinase, and plasmin to decompose only fibrin hydrogels, thereby at least one tissue selected from the group consisting of gastrointestinal submucosa, peripheral nerve epithelium and vascular epithelial tissue. Recovering at least one tissue selected from the group consisting of mesenchymal stem cells and gastrointestinal submucosa, peripheral nerve epithelial tissue, and vascular epithelial tissue grown and moved from the hydrogel to the hydrogel; And 4) amplifying the recovered mesenchymal stem cells by monolayer culture.

The gastrointestinal submucosa may be one or more tissues selected from the group consisting of gastric submucosa, small intestine submucosa and colonic submucosa.

The peripheral nerve epithelial tissue may be one or more tissues selected from the group consisting of upper and lower peripheral nerve epithelial tissue and lower peripheral nerve epithelial tissue.

The vascular epithelial tissue may be one or more tissues selected from the group consisting of venous outer membrane tissue, aortic outer membrane tissue, middle vein outer membrane tissue, and middle arterial outer membrane tissue.

The hydrogel is collagen, gelatin, chondroitin, hyaluronic acid, alginic acid, matrigel, chitosan, chitosan, peptide. It may be one or more selected from the group consisting of fibrin (fibrin), polyglycolic acid (PGA), polylactic acid (PLA), polyethylene glycol (PEG) and polyacrylamide (polyacrylamide).

The hydrogel can be degraded through one or more enzymes selected from the group consisting of collagenase, gelatinase, urokinase, streptokinase, TPA (tissue plasminogen activator), plasmin and hyaluronidase. have.

The mesenchymal stem cells (i) immunological properties positive to at least one marker group selected from the group consisting of CD29, CD44, CD73, CD90, CD105 and STRO-1; (Ii) immunological properties negative to at least one marker group selected from the group consisting of flk-1, CD14, CD34, and CD45; And (iii) immunological properties positive for CD140b, CD146, and one or more marker groups selected from the group consisting of α-smooth muscle actin.

One or more tissues selected from the group consisting of the recovered gastrointestinal submucosa, peripheral nerve epithelial tissue and vascular epithelial tissue may be repeatedly cultured by incorporating into the hydrogel of the first step.

Method for culturing the mesenchymal stem cells, for example, 1) one or more tissues selected from the group consisting of submucosal tissue, peripheral nerve epithelial tissue and vascular epithelial tissue in a mixture mixture of thrombin and fibrinogen 25 ℃ to 40 Incorporating into the fibrin hydrogel by polymerizing at 10 ° C. for 10 to 180 minutes; 2) adding three-dimensional organ culture in a shaker at a speed of 5 to 30 rpm for 3 to 28 days at 25 ° C. to 40 ° C. by adding a growth culture solution; 3) 0.5 to 4 times (volume ratio) of urokinase of hydrogel was treated for 10 to 180 minutes at 20 ° C to 40 ° C to decompose the hydrogel only, and in the group consisting of subgastrointestinal mucosa, peripheral nerve epithelial tissue and vascular epithelial tissue. Recovering one or more tissues selected from the group consisting of mesenchymal stem cells and gastrointestinal submucosa, peripheral nerve epithelial tissue and vascular epithelial tissue grown and moved into the hydrogel from the selected one or more tissues; And 4) amplifying the mesenchymal stem cells recovered from the hydrogel by a monolayer culture method. Wherein the cultured mesenchymal stem cells comprise (i) immunological properties positive to at least one marker group selected from the group consisting of CD29, CD44, CD73, CD90, CD105 and STRO-1; (Ii) immunological properties negative to at least one marker group selected from the group consisting of flk-1, CD14, CD34, and CD45; And (iii) immunological properties positive for one or more marker groups selected from the group consisting of CD140b, CD146, and α-smooth muscle actin; mesenchymal stems having one or more immunological characteristics selected from the group consisting of: It provides a method for culturing cells.

The present invention is a mesenchymal stem cell obtained by the culture method, the mesenchymal stem cells are (i) immunological positive to at least one marker group selected from the group consisting of CD29, CD44, CD73, CD90, CD105 and STRO-1 characteristic; (Ii) immunological properties negative to at least one marker group selected from the group consisting of flk-1, CD14, CD34, and CD45; And (iii) immunological properties positive for one or more marker groups selected from the group consisting of CD140b, CD146, and α-smooth muscle actin; mesenchymal stems having one or more immunological characteristics selected from the group consisting of: Provide the cells.

The mesenchymal stem cells may have differentiation ability into one or more cells selected from the group consisting of osteoblasts, adipocytes, chondrocytes, vascular endothelial cells, neurons and skeletal muscle cells.

The present invention comprises a cell therapeutic agent of at least one cell selected from the group consisting of osteoblasts, adipocytes, chondrocytes, vascular endothelial cells, neurons and skeletal muscle cells, including the mesenchymal stem cells or differentiated cells thereof as an active ingredient. to provide.

The mesenchymal stem cells may be mixed with the hydrogel.

The cell therapy agent may further include one or more selected from the group consisting of anti-inflammatory agents, stem cell mobilization factors and vascular growth inducers.

The present invention provides mesenchymal stem cells excellent in the multipotent and self-replicating ability separated from the submucosal tissues of the gastrointestinal tract, peripheral nerve tissues and blood vessels of the outer membrane tissues, which produce bio-new drugs such as cell and histological therapeutics. In addition to its high industrial applicability, it can be used for basic cellular and molecular biological studies of adult stem cells to help identify key cellular signaling pathways involved in the study of autologous proliferation and differentiation.

Figure 1 is a photograph showing the submucosal tissue, neuroepiderm, and vascular epidermal tissue collected from the gastrointestinal tract, peripheral nerves, blood vessels (Scale bar = 1 cm; IS, intestinal submucosa; BV, blood vessels; PN, peripheral nerve tissue).
FIG. 2 is a phase contrast micrograph of cultured gastrointestinal submucosa, peripheral nerve epithelium, and vascular epithelium by hydrogel-supported three-dimensional organ culture. Tissue In the picture, fusiform cells (arrows) move and grow into fibrin (Fibrin) (Scale bar = 100㎛.ACT-IS, gastrointestinal submucosa; ACT-BV, vascular outer membrane tissue ACT-PN, outer membrane tissue of peripheral blood vessels).
FIG. 3 shows the morphological and immunological characteristics of cells grown into the hydrogels of the submucosal tissues of the gastrointestinal tract, peripheral nerve tissues of the peripheral nerves, or blood vessels after incorporation into the fibrin hydrogels during the three-dimensional culture. Figure (Scale bar = 100 μm, ACT-IS, gastrointestinal submucosa; ACT-BV, vascular outer membrane tissue; ACT-PN, peripheral vascular outer membrane tissue).
Figure 4 is a monolayer culture environment of the recovered cells after selectively recovering the cells grown and moved from the subgastrointestinal submucosa, peripheral nerve epithelial tissue, or vascular epithelial tissue into the hydrogel after the hydrogel-supported three-dimensional organ culture. Figure of growth characteristics in (ACT-IS, gastrointestinal submucosa; ACT-BV, vascular outer membrane tissue; ACT-PN, peripheral vascular outer membrane tissue).
Figure 5 shows the high colony capacity, that is, self-replicating ability of the cells grown by diluting the cells grown by migration into the hydrogel, and transplanted into the hydrogel from the gastrointestinal submucosa, peripheral nerve epithelial tissue, or vascular epidermal tissue. (ACT-IS, submucosal tissue of the gastrointestinal tract; ACT-BV, vascular outer membrane tissue; ACT-PN, peripheral vascular outer membrane tissue).
FIG. 6 shows that cells migrated and grown from subgastrointestinal submucosa, peripheral nerve epithelial tissue, or vascular epithelial tissue into the hydrogel form a greater number of colonies when compared to human dermal fibroblasts (HDF). (*, P <0.01. ACT-IS, submucosal tissue of the gastrointestinal tract; ACT-BV, vascular outer membrane tissue; ACT-PN, peripheral vascular outer membrane tissue).
7 is a diagram showing the result that cells that have migrated and grown from hydrogastrointestinal submucosa, peripheral nerve epithelial tissue, or vascular epithelial tissue into hydrogels can be passaged more than 10 times in a monolayer culture environment (ACT-IS, gastrointestinal submucosa). ACT-BV, vascular outer membrane tissue; ACT-PN, peripheral vascular outer membrane tissue).
Figure 8 is a photograph showing the laboratory multi-differentiation ability of cells derived from peripheral nerve epithelial tissue and submucosa of the gastrointestinal tract, the mesenchymal stem cells migrating in the gastrointestinal submucosa or peripheral nerve epithelial tissue is osteoblasts (calcein staining) and fat Figure showing the ability to differentiate into cells (sudan black staining) (Scale bar = 100 μm, ACT-IS, gastrointestinal submucosa; ACT-PN, peripheral vascular outer membrane tissue).
Figure 9 is a photograph showing the laboratory differentiation of cells derived from the outer membrane tissue of blood vessels, the mesenchymal stem cells migrated and grown in the outer membrane tissue of blood vessels are osteoblasts (above A, alizarin red staining) and adipocytes (under A, oil red O staining) (Scale bar = 100 μm). Induced differentiation of cells derived from the outer membrane of blood vessels into osteoblasts expresses osteoblast-specific mRNAs such as bone sialoprotein, osteocalcin and osteopontin.Induced differentiation into adipocytes, leptin, lipoprotein lipase and PPAR. It is an electrophoretic diagram showing that-is expressed (B). In addition, when inducing differentiation of vascular endothelial cells from vascular epithelial tissues, they form capillary structures (top), uptake acetylated low density lipoprotein (bottom, left), and CD31, a vascular endothelial cell-specific marker. It is a figure which shows (below, right) to express (C).
Figure 10 is a diagram showing the ability to form bone tissue in vivo of the gastrointestinal submucosa-derived mesenchymal stem cells (IS-MSC) and peripheral nerve epithelial tissue-derived mesenchymal stem cells (PN-MSC). Bone tissue (*) is formed by the transplanted mesenchymal stem cells, and osteoblasts (arrows) are surrounded by the surroundings. These osteoblasts are positive for human-specific mitochondrial antibodies (Anti-hMT), indicating that bone is formed by the transplanted cells.
11 is a diagram showing the ability to form adipose tissue in vivo of the gastrointestinal submucosa-derived mesenchymal stem cells (IS-MSC) and peripheral nerve epithelial tissue-derived mesenchymal stem cells (PN-MSC). Adipose tissue (A, D, arrows) and adipocytes (B, E, oil red O staining) were formed by the transplanted mesenchymal stem cells, and these adipocytes were positively transplanted to CM-DiI (C, F). Adipose tissue is formed by one cell.

The present invention comprises the steps of culturing by incorporating one or more tissues selected from the group consisting of submucosal tissue, peripheral nerve epithelial tissue and vascular epithelial tissue into a hydrogel; And recovering the mesenchymal stem cells grown and moved into the hydrogel from one or more tissues selected from the group consisting of subgastric mucosal tissue, peripheral nerve epithelial tissue and vascular epithelial tissue by decomposing only hydrogel. It provides a culture method, mesenchymal stem cells cultured by the culture method and a cell therapy using the same.

Hereinafter, the present invention will be described in detail.

The present invention comprises the steps of culturing by incorporating one or more tissues selected from the group consisting of submucosal tissue, peripheral nerve epithelial tissue and vascular epithelial tissue into a hydrogel; And recovering the mesenchymal stem cells grown and moved into the hydrogel from one or more tissues selected from the group consisting of subgastric mucosal tissue, peripheral nerve epithelial tissue and vascular epithelial tissue by decomposing only hydrogel. Provide a culture method.

In one embodiment of the present invention, the method for culturing the mesenchymal stem cells includes the steps of 1) incorporating one or more tissues selected from the group consisting of submucosal tissue, peripheral nerve epithelial tissue and vascular epithelial tissue into fibrin hydrogel; 2) a second step of three-dimensional organ culture in a shaker at a speed of 5 to 30 rpm by adding the growth culture solution; And 3) at least one tissue selected from the group consisting of urokinase, streptokinase, and plasmin to decompose only fibrin hydrogels, thereby at least one tissue selected from the group consisting of gastrointestinal submucosa, peripheral nerve epithelium and vascular epithelial tissue. Recovering at least one tissue selected from the group consisting of mesenchymal stem cells and gastrointestinal submucosa, peripheral nerve epithelial tissue, and vascular epithelial tissue grown and moved from the hydrogel to the hydrogel; And 4) amplifying the recovered mesenchymal stem cells by monolayer culture.

The gastrointestinal submucosa is not particularly limited, but preferably, one or more tissues selected from the group consisting of gastric submucosa, small intestine submucosa and colon submucosa.

For example, the gastrointestinal submucosal tissue may be divided into tissues distributed in the submucosal layer from the stomach, small intestine, and large intestine to remove fat, nerves, and blood clots.

The vascular envelope membrane is not particularly limited, but may be preferably one or more tissues selected from the group consisting of venous outer membrane tissue, aortic outer membrane tissue, midvenous outer membrane tissue, and middle arterial envelope tissue.

For example, tissues that are distributed on the outer wall of blood vessels may be used to remove fat, nerves, and blood clots.

The peripheral nerve epithelial tissue is not particularly limited, but may preferably be one or more tissues selected from the group consisting of upper and lower peripheral nerve tissue and lower peripheral nerve outer tissue.

For example, tissues that are distributed on the outer wall of nerves may be used to remove fat, nerves, and blood clots.

The tissue is hydrogel-supported in vitro at least one tissue selected from the group consisting of submucosal tissue of the gastrointestinal tract, peripheral nerve membrane and blood vessel envelope in a state in which the structure in vivo is preserved without pretreatment lyase. Dimensional organ culture.

The hydrogel may refer to a three-dimensional network structure formed by crosslinking hydrophilic polymers by covalent or non-covalent bonds. Due to the hydrophilicity of the constituent material, it absorbs and swells a large amount of water in an aqueous solution and in an aqueous environment, but does not dissolve by crosslinking structure. In addition, the hydrogel of the present invention is preferably a phase transitional hydrogel that can be converted into a sol and a gel after being in solution for hydrogel-supported three-dimensional organ culture. It can be easily mixed with solid organs in solution and can be incorporated into three-dimensional hydrogels physically and completely as they transition into sols and gels. That is, in the liquid phase, the polymer is phase-transformed into the gel through polymerization and cross-linkage, which can control the phase transition into the gel by controlling the polymerase, cross-linking enzyme, and temperature.

Hydrogel of the present invention is not particularly limited, but collagen (collagen), gelatin (gelatin), chondroitin (chondroitin), hyaluronic acid (hyaluronic acid), alginic acid (alginic acid), metrigel (MatrigelTM), chitosan (chitosan), peptide (peptide), fibrin (fibrin), polyglycolic acid (PGA), polylactic acid (PLA), polyethylene glycol (PEG) and polyacrylamide (polyacrylamide) may be one or more selected, preferably May be a hydrogel composed of an in vitro extracellular matrix-derived polymer such as collagen, fibrin, metrigel, gelatin.

By incorporating three-dimensionally into the hydrogel of the present invention, the hydrogel provides safe physical support to the submucosal tissue of the gastrointestinal tract, peripheral nerve tissue and vascular epidermal tissue, and at the same time submucosal tissue of the gastrointestinal tract and peripheral nerve tissue. And the mesenchymal stem cells present in the outer membrane tissue of the blood vessels can provide the extracellular matrix function to move and grow into the hydrogel at the same time.

In one embodiment of the present invention, one or more tissues selected from the group consisting of submucosal tissues of the gastrointestinal tract, outer membrane tissues of the peripheral nerves, and outer membrane tissues of blood vessels are placed in a mixed solution of thrombin and fibrinogen and mixed at 10 to 180 at 25 ° C to 40 ° C. By polymerizing for a minute, the tissue can be incorporated three-dimensionally into the fibrin hydrogel.

The growth medium of the present invention (culture media) may mean a medium capable of inducing the growth of mesenchymal stem cells in vitro, and may include all the usual medium used for mammalian cell culture. have. As an example of the present invention, a commercially prepared cell culture medium DMEM, RPMI 1640, F-10, F-12, a-Minimal Essential Medium (a-MEM), Glasgow's Minimal Essential Medium, Iscove's Modified Dulbecco's Medium can be used.

The culture medium may further include growth factors capable of mobilizing mesenchymal stem cells and promoting growth, and these growth factors may include serum, that is, serum of animals including humans, basic fibroblastic growth factor (bFGF), and VEGF. (vascular endothelial growth factor), insulin (Insulin), epidermal growth gactor (EGF), leukemia inhibitory factor (LIF), insulin-like growth factor (IGF), platelet-derived growth factor (PDGF), stem cell factor (SCF) And the like. In addition, the medium may include antibiotics such as penicillin, streptomycin, gentamicin and the like.

In one embodiment of the present invention, the growth medium may be composed of DMEM: HamsF12 (1: 1) mixture, fetal calf serum, EGF, bFGF, IGF, gentamicin, more specifically 70 to 95 (vol / vol)% DMEM : Hams F12 (1: 1) mixture, 5-15 (vol / vol)% Fetal Bovine Serum, 5-50 ng / ml EGF, 0.5-10 ng / ml bFGF, 5-50 ng / ml IGF, 5-50 mg / ml gentamycin.

As an example of the present invention, the growth medium is added to the culture vessel containing the submucosal tissue of the gastrointestinal tract, peripheral nerve membrane or blood vessel outer membrane tissue embedded into the hydrogel to be cultured for 5 to 28 days at 25 ℃ to 40 ℃ Can be.

Conventionally, in the three-dimensional culture of organ sections, a method of culturing by exposing organ sections in air to enhance oxygen and nutrient exchange, and a method of dynamically culturing in an orbital shaker, The method of culturing organ sections by exposing them to air is not suitable for long-term cultivation because of limited nutrient supply. In addition, the dynamic culture has a problem in that organ sections exposed to culture medium by shear vortex are damaged by shearing.

However, when organ cultured in the orbital shaker in the state of being embedded in the hydrogel of the present invention, myocardial tissue is embedded in the hydrogel so that it may not be damaged by shearing and supply sufficient oxygen and nutrients through shaking. It can accelerate the growth of cardiac precursor cells.

Shaking speed is not particularly limited, but may be preferably 5 to 30 rpm speed. When the shaking speed is lower than the above, supply of oxygen and nutrients may not be sufficient, and when higher, the shearing damage of myocardial sections may occur and the stability of cardiac progenitor cell culture in the hydrogel may be impaired.

To recover the mesenchymal stem cells migrated / grown into the hydrogel after the hydrogel-supported three-dimensional organ culture step, only the hydrogel may be selectively degraded.

Degradation of the hydrogel can be achieved by adding an enzyme that selectively degrades the hydrogel constituent polymer to the culture.

The hydrogel constituent polymerase is not particularly limited, but collagenase, gelatinase, eurokinase, streptokinase, TPA (tissue plasminogen activator), plasmin, and hyaluronidase ( hyaluronidase) may include one or more selected from the group consisting of.

For example, in the case of 3-dimensional organ culture of the tissue of the present invention using collagen hydrogel, gelatin hydrogel, or matrigel, hydrogel may be selectively added by adding collagenase or gelatinase into the culture medium. Fibrin hydrogel can be decomposed, and when fibrin hydrogel is used, fibrin hydrogel can be added to the culture medium by selecting one or more from eurokinase, streptokinase, TPA (tissue plasminogen activator) and plasmin (plasmin). It can be selectively decomposed, hyaluronic acid hydrogel can be selectively decomposed only hydrogel using hyaluronidase (hyaluronidase).

After addition of a degrading enzyme that selectively decomposes the hydrogel constituent polymer into the culture medium, the hydrogel is selectively decomposed when reacted at 20 ° C. to 40 ° C. for 10 minutes to 180 minutes, but the submucosal tissue of the gastrointestinal tract in the hydrogel and peripheral nerves At least one selected from the group consisting of mesenchymal stem cells and submucosal tissues of the gastrointestinal tract, perimucosal tissues of the gastrointestinal tract, and outer membrane tissues of the blood vessels Tissue can be isolated and recovered from the hydrogel without damage.

The amplification culture method of the mesenchymal stem cells recovered from the hydrogel is not particularly limited, but preferably by using a monolayer culture method, high yields of mesenchymal stem cells can be obtained.

In one example of the present invention, when the mesenchymal stem cells recovered from the hydrogel are seeded in the culture vessel, and occupy 60 to 90% of the surface area of the culture vessel, the cells are treated with trypsin-EDTA and separated from the culture vessel. Subcultures can be taken out and seeded again in new culture vessels. The mesenchymal stem cells recovered from the hydrogel through 15 or more passages in this monolayer culture method can be amplified and cultured 100 times or more.

In addition, one or more tissues selected from the group consisting of submucosal tissue of the gastrointestinal tract, peripheral nerve tissue and blood vessel outer tissue recovered by selectively decomposing only the hydrogel are re-incorporated into the hydrogel and repeatedly three-dimensional organ culture (organ It is possible to isolate and culture the mesenchymal stem cells repeatedly by performing a culture) and to maintain the structure of the mesenchymal stem cells intact because the hydrogel is selectively decomposed when the culture is completed and recovered.

More than 2 million mesenchymal stems from 1 g of submucosal tissue of the gastrointestinal tract, peripheral outer membrane tissue, and / or outer membrane tissue of blood vessels after 14 days of hydrogel-supported three-dimensional organ culture. Cells could be recovered and at least 95% of the mesenchymal stem cells separated and recovered from the hydrogel showed viability.

In addition, the mesenchymal stem cells obtained by the above culture method (i) immunological properties positive to at least one marker group selected from the group consisting of CD29, CD44, CD73, CD90, CD105 and STRO-1; (Ii) immunological properties negative to at least one marker group selected from the group consisting of flk-1, CD14, CD34, and CD45; And (iii) an immunological property positive for one or more marker groups selected from the group consisting of CD140b, CD146, and α-smooth muscle actin; and may comprise one or more immunological properties selected from the group consisting of: Excellent self-replicating ability and multi-differentiation ability to differentiate into bone, cartilage, fat, tendon, blood vessels, fibroblasts, muscle cells, nerve cells and the like.

In one preferred embodiment of the present invention, the method for culturing the mesenchymal stem cells 1) one or more tissues selected from the group consisting of submucosal tissue, peripheral nerve epithelial tissue and vascular epithelial tissue into a mixture of thrombin and fibrinogen 25 Incorporating into the fibrin hydrogel by subjecting the polymerization reaction at 10 ° C. to 40 ° C. for 10 to 180 minutes; 2) adding a culture broth to hydrogel-supported three-dimensional organ culture in a shaker at a speed of 5 to 30 rpm for 3 to 28 days at 25 ° C. to 40 ° C .; 3) 0.5 to 4 times (volume ratio) urokinase of hydrogel was treated for 10 to 180 minutes at 20 ° C to 40 ° C to decompose the hydrogel, and in the group consisting of gastrointestinal submucosa, peripheral nerve epithelial tissue and vascular epithelial tissue Recovering the mesenchymal stem cells grown and moved from the selected one or more tissues into the hydrogel; And 4) amplifying the mesenchymal stem cells recovered from the hydrogel by a monolayer culture method. Comprising: (i) immunological properties positive to at least one marker group selected from the group consisting of CD29, CD44, CD73, CD90, CD105 and STRO-1; (Ii) immunological properties negative to at least one marker group selected from the group consisting of flk-1, CD14, CD34, and CD45; And (iii) immunological properties positive for one or more marker groups selected from the group consisting of CD140b, CD146, and α-smooth muscle actin; mesenchymal stems having one or more immunological characteristics selected from the group consisting of: It can be by the culture method of the cell.

The present invention is a mesenchymal stem cell obtained by the culture method, the mesenchymal stem cells are (i) immunological positive to at least one marker group selected from the group consisting of CD29, CD44, CD73, CD90, CD105 and STRO-1 characteristic; (Ii) immunological properties negative to at least one marker group selected from the group consisting of flk-1, CD14, CD34, and CD45; And (iii) immunological properties positive for one or more marker groups selected from the group consisting of CD140b, CD146, and α-smooth muscle actin; mesenchymal stems having one or more immunological characteristics selected from the group consisting of: Provide the cells.

The mesenchymal stem cells can be passaged without progress of aging up to 1 to 15 times and may have a cell division capacity of 110 to 140 times.

The mesenchymal stem cells are excellent in self-replicating ability and multi-differentiation ability, as a method of inducing differentiation from the mesenchymal stem cells to a variety of cells, may be by a method known in the art, serum from differentiation-inducing medium Or differentiation can be induced by removing serum replacements or by adding components that promote differentiation.

In one embodiment of the present invention, as a medium for inducing differentiation of mesenchymal stem cells into adipocytes, 3-isobutyl-1-methylxanthine, indomethacin, dexamethasone (dexamethasone), insulin, DMEM containing fetal bovine serum may be used, preferably 0.1 to 1 mM 3-isobutyl-1-methylxanthine, 60 to 100 μM indomethacin, 0.5 to 2 μM dexamethasone, DMEM medium containing 1-10 μg / ml insulin, 1-20% fetal bovine serum can be used. The differentiation induction period in the medium is not particularly limited, but may be 5 to 30 days, preferably 10 to 20 days.

In one embodiment of the present invention, as a medium for inducing differentiation of mesenchymal stem cells into osteoblasts, dexamethasone, ascorbate-2-phosphate, (beta) -glycerophosphate (β-glycerophosphate) ), And α-MEM comprising fetal calf serum, preferably 0.1 to 2 μM dexamethasone, 30 to 80 μM ascorbate-2-phosphate, 5 to 15 mM (beta) -glycerophosphate, 1 Α-MEM comprising 20% fetal calf serum.

In one example of the present invention, a micromass culture method may be used to induce differentiation of mesenchymal stem cells into chondrocytes. At this time, as a medium for inducing differentiation, DMEM medium containing a transforming growth factor- (beta) 1, ascorbate-2-phosphate, insulin, and fetal bovine serum may be used. Preferably 1 to 10 μg / ml insulin, 5 to 20 ng / ml transforming growth factor-β1, 30 to 80 μM ascorbate-2-phosphate, 0.1 to 2% DMEM medium containing fetal serum may be used.

In one embodiment of the present invention, after seeding the cells on fibrin hydrogel to induce differentiation of vascular endothelial cells, vascular endothelial growth factor (VEGF), bFGF, EGF, dimethyl methyl sulfoxide ( DMSO) can be used.

In one embodiment of the present invention, after seeding the mesenchymal stem cells in the culture vessel to a density of 100% to induce differentiation of skeletal muscle cells, transforming growth factor- (beta) 1 (transforming growth factor-β1) and 10% DMEM containing fetal bovine serum can be used to induce differentiation into skeletal muscle cells.

The present invention comprises a cell therapeutic agent of at least one cell selected from the group consisting of osteoblasts, adipocytes, chondrocytes, vascular endothelial cells, neurons and skeletal muscle cells, including the mesenchymal stem cells or differentiated cells thereof as an active ingredient. to provide.

In the present invention, the mesenchymal stem cell-derived cell therapeutic agent is a biopharmaceutical (Korea Food and Drug Administration and US FDA regulations) used for the purpose of treatment, diagnosis, and prevention with cells and tissues prepared through isolation, culture, and special manipulation from humans. In order to restore the function of a cell or tissue, a drug used for the purpose of treatment, diagnosis, and prevention through a series of actions, such as amplifying, selecting, or changing the biological characteristics of a living autologous or allogeneic cell in vitro. May be referred to. Cell therapy agents are largely classified into somatic cell therapy and stem cell therapy according to the degree of differentiation of cells, and the present invention particularly relates to stem cell therapy.

Stem cells of the present invention can be used directly in methods of treatment, including methods of regenerating and restoring tissue cells, and methods of differentiating such cells that can occur in vivo. Stem cells and differentiated cells may be used to regenerate and / or form damaged tissues, and may also be applied when organs are not damaged, such as when they do not occur normally. Thus, tissue cell regeneration of the present invention should be broadly interpreted to encompass both methods of growth or improvement of tissue.

In addition, the present invention may be included in the scope of the present invention when a genetic modification is applied to the mesenchymal stem cells or their differentiated cells as well as the mesenchymal stem cells or the differentiated cells thereof.

Mesenchymal stem cells of the present invention and cells differentiated therefrom may be genetically engineered with specific properties and then used for gene therapy.

In one embodiment of the present invention, there is provided a cell therapy agent for bone tissue regeneration or treatment comprising mesenchymal stem cells capable of differentiating into osteoblasts or mesenchymal stem cells differentiated into osteoblasts by the differentiation method as an active ingredient.

The cell therapeutic agent for bone tissue regeneration may be used for bone tissue disease or malformation. For example, bone metastatic lesions or primary tumors caused by tumors such as breast cancer or prostate cancer metastasized to bone may be applied to bone. Osteoporosis, rheumatoid or degenerative arthritis, periodontal disease, inflammatory alveolar bone resorption disease, inflammatory bone resorption disease and Paguet's disease when bones that occur after radiation or surgical resection are treated to treat the resulting tumor diseases) and the like, but the scope of the present invention is not limited thereto.

In one embodiment of the present invention, there is provided a cell therapy agent for adipose tissue regeneration comprising mesenchymal stem cells capable of differentiating into adipocytes or mesenchymal stem cells induced to differentiate into adipocytes by the differentiation method as an active ingredient. . The cell therapy agent for adipose tissue regeneration may be included in the scope of the present invention even when used for soft tissue defect-related diseases and cosmetic and cosmetic purposes.

In one embodiment of the present invention, there is provided a cell therapy for cartilage tissue regeneration comprising mesenchymal stem cells that can be differentiated into chondrocytes or mesenchymal stem cells differentiated into chondrocytes by the differentiation method as an active ingredient. The cell therapy agent for cartilage tissue regeneration may be used for cartilage-related diseases or malformations, and for example, it may be used for degenerative arthritis, rheumatoid arthritis, cartilage damage due to trauma, but the scope of the present invention is not limited thereto.

 In one embodiment of the present invention, there is provided a cell therapy agent for vascular regeneration comprising mesenchymal stem cells differentiated into vascular endothelial cells or mesenchymal stem cells differentiated into vascular endothelial cells by the differentiation method as an active ingredient.

The angiogenesis or therapeutic cell therapy may be used for ischemic diseases, and may be used for the treatment, alleviation, or prevention of any disease or disorder that can be alleviated, treated, or prevented through angiogenesis. For example, the cell therapy agent for vascular tissue treatment may be used for cardiovascular disease, cerebrovascular disease, ischemic disease, and the like, but the scope of the present invention is not limited thereto. In addition, it may be used in burns, wounds, lacerations, skin ulcers, bedsores, diabetic ulcers, which are diseases caused by lack or destruction of blood vessels, but the scope of the present invention is not limited thereto.

In one embodiment of the present invention, there is provided a therapeutic agent for muscle tissue regeneration comprising mesenchymal stem cells capable of differentiating into skeletal muscle cells or mesenchymal stem cells induced to differentiate into skeletal muscle cells by the differentiation method as an active ingredient.

Cell therapy for muscle tissue regeneration may be applied to skeletal muscle defects, neurological skeletal muscle atrophy, dysplastic skeletal muscle disease due to accidents and trauma, but the scope of the present invention is not limited thereto.

The mesenchymal stem cell-derived cell therapy of the present invention may be administered by a method suitable for the purpose of treatment, and may be delivered to a patient by local or systemic administration. For example, the administration method may be intravenous injection, subcutaneous injection, intramuscular injection, intraperitoneal injection, or the like, but is not limited thereto.

The cell therapy may be in the form of a general cell therapy known in the art (Remington's Pharmaceutical Science; Mack Publishing Company, Easton PA), for example, may be prepared in the form of injections, and surgical Can be directly implanted into damaged tissues and organs, and can be administered intravenously, wherein the administered cell therapy can also migrate to the damaged tissues or organs.

The therapeutic composition comprising the mesenchymal stem cells of the present invention or cells differentiated from the mesenchymal stem cells as an active ingredient may be injected into the patient's body in a culture alone or in a carrier.

The carrier is not particularly limited in its kind, but may preferably be a hydrogel. The hydrogel can control the decomposition rate in vivo by controlling the concentration of the constituent polymer. The hydrogel can prevent the disadvantage of being lost into the blood during cell injection, and can also reduce the cell damage by inflammatory cells and enzymes in the damaged area.

The cell therapy agent may include one or more diluents, but the diluent is not particularly limited, but may include a saline solution, a phosphate buffered saline (PBS), a buffer solution such as HBSS (Hank's balanced salt solution), plasma or blood components, and the like. In addition to the diluent, it may further include a lubricant, a humectant, a sweetener, a flavoring agent, an emulsifier, a suspending agent, a preservative, and the like.

Suitable dosages of the cell therapy products of the present invention may be prescribed in various ways depending on factors such as the formulation method, mode of administration, age, weight, sex, morbidity, food, time of administration, route of administration, rate of excretion and response to the patient. Can be. On the other hand, the dosage of the cell therapeutic agent of the present invention is preferably 1 to 50 million cells once, and is usually administered once or twice. However, their dose can be increased or decreased depending on the weight, age, sex, and severity of the patient.

The cell therapy may further include one or more selected from the group consisting of anti-inflammatory agents, stem cell mobilization factors and vascular growth inducers.

The anti-inflammatory agent can be used to protect the progenitor cells transplanted with hydrogel from excessive inflammatory reactions, and induce cell recruitment and vascular growth by inducing mobilization and vascular growth of stem cells in the myocardium, including stem cell recruitment factors or vascular growth inducers. The effect can be increased.

The anti-inflammatory agent is not particularly limited, but may include, for example, one or more selected from the group consisting of a COX inhibitor, an ACE inhibitor, salicylate, and dexamethasone.

The stem cell mobilization factor is not particularly limited, but may include one or more selected from the group consisting of, for example, IGF, bFGF, PDGF, and EGF.

The vascular growth inducer is not particularly limited, but may include, for example, one or more selected from the group consisting of EGF, PDGF, VEGF, ECGF, and angiogenin.

Hereinafter, the present invention will be described in more detail by way of examples. However, the following examples are illustrative of the present invention, and the content of the present invention is not limited by the examples.

<Examples>

Example 1 Isolation of Gastrointestinal Mesenteric Tissue, Peripheral Neural Outer Tissue, or Vascular Outer Tissue and Incorporation into Fibrin Hydrogel

After approval from Inje University School of Medicine ethics committee, the outer membrane of the calf was selectively isolated from the outer wall, and the submucosa of the gastrointestinal tract was selectively separated from the stomach, small intestine, and large intestine, and from the calf artery and vein. Outer membrane tissues distributed on the outer wall of blood vessels were selectively isolated. Observation under a microscope to remove adipose tissue, nerves, blood clots, etc. in the tissue (Fig. 1). The isolated tissues were washed 5 times with DPBS for 5 minutes, cut from 1 to 5 mm ^{3 in} size, and then washed twice for 5 minutes in DMEM containing 1 NIH U / ml of thrombin (Sigma, Louis, MO). . The tissue was dispersed by mixing with a DMEM solution containing 10 ml of 1 NIH U / ml concentration thrombin per gm. After the fibrinogen solution in which fibrinogen (Green Cross, Suwon, Korea) was dissolved at the same amount of DMEM in the same amount of DMEM into the thrombin solution was mixed. The gastrointestinal mucosa, peripheral nerve epithelial tissue, or vascular epithelial tissue were made to contain 100 to 250 mg of submucosal tissue, peripheral nerve epithelial tissue, or vascular epithelial tissue per ml of thrombin and fibrinogen mixed solution. The thrombin and fibrinogen mixed solution including the gastrointestinal submucosal tissue, peripheral nerve epithelial tissue, or vascular epithelial tissue is transferred to a 100-mm culture vessel before the formation of the hydrogel, followed by 2 at 37 ° C. in a wet chamber. The polymerization process took place over time.

Fibrinogen is polymerized by thrombin, and the solution forms a solid fibrin hydrogel. As a result, the gastrointestinal mucosa, peripheral nerve membrane tissue, or vascular outer membrane tissue are three-dimensionally infiltrated into the fibrin hydrogel. It became.

Example 2 Hydrogel-supported Three-Dimensional Organ Culture of Gastrointestinal Submucosa, Peripheral Neuroe and Tissue

As in Example 1, the gastrointestinal submucosa, peripheral nerve epithelial tissue, or vascular epithelial tissue were completely incorporated into the hydrogel. Then, the growth culture solution was added to the culture dish. Growth cultures consisted of 10% fetal bovine serum (FBS; Invitrogen) and 10µg / ml gentamicin (Yuhan, Seoul, Korea) in 90% DMEM: HamsF12 (1: 1) culture (Invitrogen). The culture vessel containing the gastrointestinal submucosa, peripheral nerve epithelial tissue, or vascular epithelial tissue was placed in an orbital shaker at a speed of 15 rpm and placed in a humidified chamber for 14 days at 37 ° C. (organ culture method). The growth culture was replaced with fresh growth culture every two days.

As shown in FIG. 2, migration of cells into the hydrogels was observed from 12 h of culture from all tissues separated from the gastrointestinal tract, nerves, or blood vessels through a hydrogel-supported three-dimensional organ culture method. On the 6th day of culture, many areas in the hydrogel were occupied by the cells grown from the gastrointestinal submucosa, peripheral nerve epithelial tissue, or vascular epithelial tissue, and the cell density also increased as the culture period increased (FIG. 2).

Example 3 Immunosuperficial Properties of Mesenchymal Stem Cells Grown and Moved into Hydrogel from Gastrointestinal Submucosal Tissue, Peripheral Nerve Tissue, or Vascular Tissue

Subcutaneous gastrointestinal submucosa, peripheral nerve epithelial tissue, or vascular epithelial tissue were embedded in fibrin hydrogel, followed by organ culture for 2 weeks in vitro, followed by fixation with 2% neutral formalin for 2 hours. The tissues, cells, and hydrogels were incorporated into paraffins, paraffin blocks were prepared, and the location and morphological characteristics of the cultured cells were determined by hematoxylin-eosin staining. In addition, immunohistochemical staining was performed to characterize the immunosurface characteristics of cells that migrated and grown during in vitro culture. The primary antibodies used were monoclonal anti-human CD73, CD90, CD105, CD140b, CD146, alpha-smooth muscle actin (SMA). Expression was assessed using the EnVisionTM system (DAKO) and 3, 3-diaminobenzidine (3, 3-diaminobenzidine, DAB, Sigma).

As shown in FIG. 3, the cells grown and migrated into the hydrogel uniformly expressed at least 95% of CD73, CD90, and CD105 antibodies, which were the mesenchymal stem cell markers, and uniformly expressed the CD140b, CD146, and SMA markers. Expression (FIG. 3). 40% of cells migrated and grown into the hydrogel expressed CD146, whereas at least 95% of the cells expressed CD140b and SMA.

Example 4 In Vitro Amplification by Selective Isolation and Single-layer Culture of Mesenchymal Stem Cells Grown into Hydrogel

After organ culture of the gastrointestinal submucosa, peripheral nerve epithelial tissue, or vascular epithelial tissue in the fibrin hydrogel by the method described in Example 2, the culture vessel was washed three times for 10 minutes with DPBS. After washing, DMEM containing 1,000 unit / ml urokinase and 20% calf serum (CS) per 1 ml of hydrogel was added. The culture dish was then reacted for 30 minutes to 2 hours in a 37 ° C. wet chamber on an orbital shaker at 15 rpm. After the fibrin hydrogel is decomposed, the 25 ml transfer pipette is repeatedly used to promote release of the grown cells into the fibrin hydrogel through inhalation and release, and then the cells dissociated into the solution are transferred to conical tubes ( conical tube) and centrifuged at 100 xg force for 10 minutes. After removing the supernatant, the sunk cells were dispersed in growth culture. Thereafter, cells recovered from fibrin hydrogels were amplified and cultured by conventional monolayer culture. Cells recovered from the hydrogel were seeded in 2000 water per cm ² of the culture vessel. When the seeded cells occupy 80% of the culture vessel area, trypsin-EDTA (Invitrogen) was removed from the culture dish and seeded at 2,000 cells / cm ² concentration in a new culture vessel and subcultured.

After urokinase administration, the fibrin hydrogels loosened and began to degrade. Cells migrated and grown into the fibrin hydrogel were released from the hydrogel after urokinase treatment and dissociated into the reaction solution. As a result of trypan blue exclusion assay, more than 95% of the cells dissociated from the hydrogel survived. Dissociated cells also adhered to the plastic culture dish within 10 minutes when seeded in a new plastic culture vessel. These cells showed the same morphological characteristics of the spindle shape as typical mesenchymal stem cells (FIG. 4).

Example 5 Self-replicating and Proliferating Ability of Mesenchymal Stem Cells Recovered from Fibrin Hydrogels

After urokinase treatment as in Example 4, cells that were migrated and grown into the fibrin hydrogel were selectively recovered from the gastrointestinal submucosa, peripheral nerve epithelial tissue, or vascular epithelial tissue. In vitro amplification capacity of cells isolated in a monolayer culture environment was evaluated by analysis of colony forming unit-fibroblast (CFU-F) and population doubling time (PDT). CFU-F was formed by seeding the cells recovered from the hydrogel in a 60-mm culture dish with a density of 5 cells / cm ² and incubated for 11 days after adding the growth culture solution. The culture vessel was then fixed with 2% buffered formalin and stained with 10% crystal violet (LabChem Inc., Pittsburgh, PA) for 5 minutes. The dyeing vessel was imaged with a digital CCD camera, and the number of CFU-Fs formed was calculated using an image analysis program (Image J, NIH, Bethesda, MD). Fibroblasts (HDFs) isolated from human dermal dermis were used as controls. In order to measure PDT of cells recovered from the hydrogel, 2 ㅧ 10 ³ cells were seeded in 48-multiwell culture vessels and then cultured. Cells on day 1 and day 5 were lysed with CelLytic ^™ solution (Sigma), and the DNA content in the lysed samples was measured using PicoGreen dsDNA fluorescent dye (Invitrogen). Fluorescence intensities were measured at a wavelength of 485 nm emission and 540 nm excitation using a fluorescent microplate reader (SynergyTM HT; Bio-Tek Instruments, Neufahrn, Germany). In order to convert the measured fluorescence intensity into cell number, a standard curve was obtained using adipose stem cells isolated from adipose tissue, and the fluorescence intensity in the sample was converted into cell number using this standard curve. PDT was calculated according to the following formula. PDT = [(days in exponential phase) / ((logN2-logN1) / log2)] where N1 is the initial cell number of the exponential growth phase and N2 is the cell number at the end of the exponential growth phase.

Fibrin hydrogel migrated cells in the gastrointestinal submucosa, peripheral nerve epithelial tissue, or vascular epithelial tissue showed fusiform shapes in a monolayer culture environment irrespective of the origin of the tissue (FIG. 4). CFU-F was able to form in 20% of cells grown in gastrointestinal submucosa, peripheral nerve epithelial tissue, or vascular epithelial tissue (Figs. 5 and 6), and there was no difference in self-replicating ability according to tissue origin. (FIG. 6). Cells grown in gastrointestinal submucosa, peripheral nerve epithelial tissue, or vascular epithelial tissue were able to be passaged up to 15 times and were able to divide cells about 120 times. From 4th to 10th subculture, PDT grew relatively fast from 24.2 hours to 46.7 hours. However, when passaged more than 15 times, the cells began to age with longer PDT (FIG. 7).

Example 6 Immunosuperficial Properties of Mesenchymal Stem Cells Derived from Submucosal Tissue, Peripheral Neural Tissue, or Vascular Outer Tissue

After amplification by the monolayer culture method of Example 4, immunosurface characteristics were analyzed using flow cytometry on the third passaged cells. One hundred thousand cells were reacted with primary or isotype-matched control antibodies for 30 minutes. Primary antibodies are anti-human antibodies bound with phycoerythrin (PE) or fluorescein isothiocyanate (FITC), CD14, CD29, CD34, CD44, CD45, CD73, CD90, CD133, STRO-1, MHC-I and MHC-II were used. All primary and control antibodies combined with the fluorescent material were purchased from BD Bioscience (San Jose, Calif.). Flk-1 (R & D Systems, Minneapolis, MN), CD105 (R & D Systems), c-kit (DAKO, Glosrup, Denmark), PDGFR-β (CD140b; Abcam, Cambridge, MA), CD146 (Abcam), α-smooth muscle actin (SMA; DAKO) were reacted with 100,000 cells for 30 minutes and then with FITC-bound secondary antibody for 30 minutes. After the reaction was completed, the cells were washed three times with DPBS and fixed for 5 minutes in 2% paraformaldehyde. Positive rates for each antibody were analyzed using FACSCalibur (Becton Dickinson, San Jose, Calif.), And expression rates were analyzed in at least 10,000 cells.

As shown in Table 1, cells from gastrointestinal submucosa, peripheral nerve epithelial tissue, or vascular epithelial tissue were positive for mesenchymal stem cell markers such as CD29, CD44, CD73, CD90, and CD105 in at least 95%. In addition, the cells expressed STRO-1, one of the mesenchymal stem cell markers, at about 45%. HLA-A, B, C, which is an I-antigen marker HLA-A, B, C, was positively expressed in 95% or more, but HLA-DR, a type II MHC marker, was negative, and the result was gastrointestinal submucosa, peripheral nerve epithelial tissue, Or mesenchymal stem cells derived from vascular epidermal tissue showed immunosurface characteristics that allogeneic transplantation is possible. In addition, markers such as CD34 and KDR and CD14, CD34, CD45, c-kit, and CD133 were negative for mesenchymal stem cells derived from gastrointestinal submucosa, peripheral nerve epithelial tissue, or vascular epithelial tissue. It was confirmed that there was no contamination with the cells. In addition, mesenchymal stem cells derived from gastrointestinal submucosal tissue, peripheral nerve epithelial tissue, or vascular epidermal tissue expressed perivascular markers such as CD140b, CD146, and SMA.

Example 7 Multipotential Capability of Mesenchymal Stem Cells of Gastrointestinal Submucosa, Peripheral Nerve Tissue, or Vascular Tissue

After amplification by the monolayer culture method of Example 4, differentiation into laboratory adipocytes, osteoblasts, and vascular endothelial cells was induced in the passaged third cells. Differentiation into adipocytes was seeded in a 6-multiwell cell culture vessel 300,000 cells, 0.5 mM 3-isobutyl-1-methylxanthine, 80 μM indomethacin, 1 μM dexamethasone, 5 μg Differentiation was induced into adipocytes by culturing cells for 14 days in DMEM containing / ml insulin, 10% calf serum. Intracellular lipid accumulation was confirmed by Oil Red O (Sigma) or Sudan Black staining. In addition, the expression of adipocyte-specific mRNA was determined by electrophoresis after reverse-transcriptase polymerase chain reaction (RT-PCR).

Differentiation into osteoblasts was performed by seeding 300,000 mesenchymal stem cells derived from subgastrointestinal submucosa, peripheral nerve epithelium, or vascular epidermal tissue into a 6-multiwell cell culture vessel, followed by 1 μM dexamethasone and 50 μM ascorbate-2-. Differentiation was induced by incubation for 2 weeks in a-MEM containing phosphate, 10 mM β-glycerophosphate, 10% calf serum. Differentiation into osteoblasts was determined by mineral deposition of extracellular matrix by staining with Alizarin Red (Sigma) or calcein (calcein, Sigma). In addition, osteoblast-specific mRNA expression was determined by electrophoresis after reverse-transcriptase polymerase chain reaction (RT-PCR).

Differentiation into vascular endothelial cells was performed by seeding 100,000 mesenchymal stem cells derived from gastrointestinal submucosa into a 6-multiwell cell culture vessel coated with a fibrin gel, followed by 1 μM dexamethasone, 50 μM ascorbate-2-phosphate, Differentiation was induced by incubating for 1 week in DMEM containing 10 ng / ml vascular endothelial growth factor and 1% calf serum. Differentiation into vascular endothelial cells was determined by capillary network formation, Acetylated-low density lipoprotein uptake and CD31 expression.

Irrespective of the tissue of origin, all the cells migrated and grown into the hydrogel were able to confirm their ability to differentiate into adipocytes (FIGS. 8 and 9). After induction of differentiation into adipocytes, the cells migrated and grown into hydrogels were uniformly differentiated into adipocytes. Lipid accumulation was observed in the cytoplasm from the 5 th day of induction. Accumulation was observed (FIG. 8, FIG. 9A). In addition, after induction of differentiation into adipocytes, the cells were confirmed to express the adipocyte specific mRNAs leptin, lipoprotein lipase, PPAR-gamma (Fig. 8B).

Cells induced differentiation into osteoblasts was changed to a cubic epithelial shape, and it was confirmed that minerals were deposited on Alizarin Red (FIG. 9A) or calcein (calcein) staining (FIG. 8). . In addition, osteoblast-specific mRNAs such as bone sialoprotein, osteocalcin, and osteopontin mRNAs were prominently increased in the cells induced differentiation into osteoblasts (Fig. 8B).

The gastrointestinal submucosa-derived cells were induced to differentiate into vascular endothelial cells using VEGF and fibrin gel, and from day 1 of differentiation, cells formed a capillary-like network (Fig. 9C), and CD31, a vascular endothelial cell-specific marker, was expressed. And had the ability to absorb acetylated low density lipoproteins (FIG. 9C).

Example 8 Tissue Regeneration Capability of Mesenchymal Stem Cells from Gastrointestinal Submucosa and Peripheral Neural Outer Tissue

After amplification by the monolayer culture of Example 4, 2 million passaged mesenchymal stem cells were mixed with 50 mg hydroxyapatite-tricalcium phosphate particles (Zimmer Inc., Warsaw, IN). It was. It was inserted between the muscles of the lumbar region adjacent to nude mice (Balb / c nu / nu, Charles River Lab., Seoul, Korea; n = 5). Four weeks after transplantation, the transplants were harvested and histologically determined for ectopic bone formation in the implants.

In addition, 2 million mesenchymal stem cells were mixed with fibrin hydrogel and injected into the subcutaneous layer of the nippled mice to determine the adipose tissue regeneration capacity of the gastrointestinal submucosa and peripheral nerve epithelial-derived mesenchymal stem cells. After 4 weeks of infusion, the tissues were harvested and histologically identified. To identify the differentiation of mesenchymal stem cells injected into nude mice into adipocytes in vivo, they were labeled and transplanted with CM-DiI (Molecular Probes). Analyzes were performed using specific mitochondrial antibodies.

Heterogeneous osteoblasts (arrows) surrounded by gastrointestinal mucosa-derived mesenchymal stem cells (IS-MSC) and peripheral nerve outer membrane-derived mesenchymal stem cells (PN-MSC) injected into the muscle with hydroxyapatite Bone tissue (*) was formed (FIG. 10). Osteoblasts surrounding ectopic bone tissue were positive for human-specific mitochondrial antibodies, and it was confirmed that human mesenchymal stem cells transplanted with hydroxyapatite differentiated into osteoblasts to form bone tissue. In addition, white adipose tissue (A, D, arrows) forms on gastrointestinal mucosa-derived mesenchymal stem cells (IS-MSC) and peripheral nerve epithelial-derived mesenchymal stem cells (PN-MSC) transplanted into the subcutaneous layer with fibrin hydrogel. By histological examination, the deposition of fat (B, E) into these cells confirmed the differentiation of adipocytes, and these mesenchymal stem cells transplanted with fibrin hydrogel positively for CM-DiI. It was confirmed that the cells differentiate into adipocytes to form adipose tissue (FIG. 11).

Claims (14)

Immersing one or more tissues selected from the group consisting of gastrointestinal submucosa, peripheral nerve epithelial tissue and vascular epithelial tissue into a hydrogel and culturing the same; And
Culturing mesenchymal stem cells, comprising: recovering the mesenchymal stem cells grown and moved into the hydrogel from one or more tissues selected from the group consisting of subgastrointestinal submucosa, peripheral nerve epithelial tissue and vascular epithelial tissue by degrading hydrogel only. Way. The method of claim 1,
1) a first step of incorporating at least one tissue selected from the group consisting of gastrointestinal submucosa, peripheral nerve epithelial tissue and vascular epithelial tissue into the fibrin hydrogel;
2) a second step of three-dimensional organ culture in a shaker at a speed of 5 to 30 rpm by adding the growth culture solution; And
3) by treating at least one degrading enzyme selected from the group consisting of urokinase, streptokinase and plasmin to decompose only fibrin hydrogel, and from at least one tissue selected from the group consisting of gastrointestinal submucosa, peripheral nerve epithelial tissue and vascular epithelial tissue. Recovering one or more tissues selected from the group consisting of mesenchymal stem cells and gastrointestinal submucosa, peripheral nerve epithelial tissue, and vascular epithelial tissue grown into a hydrogel; And
4) a fourth step of amplifying the recovered mesenchymal stem cells by monolayer culture method;
Culture method of mesenchymal stem cells comprising a. The method of claim 1,
The gastrointestinal submucosa is at least one tissue selected from the group consisting of gastric submucosa, small intestine submucosa and colonic submucosa, mesenchymal stem cell culture method. The method of claim 1,
The peripheral nerve epithelial tissue is one or more tissues selected from the group consisting of upper and lower peripheral nerve epithelial tissue and lower peripheral nerve outer membrane tissue, mesenchymal stem cell culture method. The method of claim 1,
The vascular epithelial tissue is one or more tissues selected from the group consisting of aorta outer membrane tissue, aortic outer membrane tissue, middle vein outer membrane tissue and middle arterial outer membrane tissue, mesenchymal stem cell culture method. 3. The method according to claim 1 or 2,
The hydrogels are collagen, gelatin, chondroitin, hyaluronic acid, alginic acid, matrigel, chitosan, chitosan, and peptide. Fibrin (fibrin), polyglycolic acid (PGA), PLA (polylactic acid), PEG (polyethylene glycol) and one or more selected from the group consisting of polyacrylamide (polyacrylamide), a method for culturing mesenchymal stem cells. 3. The method according to claim 1 or 2,
The hydrogel is degraded through one or more enzymes selected from the group consisting of collagenase, gelatinase, urokinase, streptokinase, TPA (tissue plasminogen activator), plasmin and hyaluronidase, Method for culturing mesenchymal stem cells. The method of claim 1,
The mesenchymal stem cells (i) immunological properties positive to at least one marker group selected from the group consisting of CD29, CD44, CD73, CD90, CD105 and STRO-1; (Ii) immunological properties negative to at least one marker group selected from the group consisting of flk-1, CD14, CD34, and CD45; And (iii) immunological properties positive for one or more marker groups selected from the group consisting of CD140b, CD146, and α-smooth muscle actin; mesenchymal with one or more immunological properties selected from the group consisting of Stem cell culture method. 3. The method of claim 2,
A method of culturing mesenchymal stem cells, wherein the at least one tissue selected from the group consisting of gastrointestinal submucosa, peripheral nerve epithelial tissue and vascular epithelial tissue recovered in the third step is immersed in the hydrogel of the first stage. delete delete delete delete delete

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