KR101648628B1 - Method for prepation of cell spheroid using mesenchymal stem cells and cellular threapeutic agents or implants for treating liver diseases comprising cell spheroid - Google Patents

Abstract

The present invention relates to a method for producing an artificial liver using mesenchymal stem cells, and to the use of the same as a cell therapy agent and a transplantation body of the artificial liver.
The method of producing a cell aggregate according to the present invention and the cell aggregate produced thereby can securely perform the liver function safely through the vascularization process after the transplantation, There is no immune rejection reaction and thus it can be usefully used as a cell therapy agent and transplant for treating liver diseases.

Description

TECHNICAL FIELD [0001] The present invention relates to a method for preparing a cell aggregate using mesenchymal stem cells, and a method for preparing a cell therapeutic agent or a transplant for treating liver diseases, spheroid}

The present invention relates to a method for producing an artificial liver using mesenchymal stem cells, and to the use of the same as a cell therapy agent and a transplantation body of the artificial liver.

In recent years, tissue engineering (tissue engineering) has been attracting attention in order to restore function or regenerate organs or tissue itself to organs or tissues that have been lost or sick due to diseases or accidents. Particularly, interest in bio-artificial organs has been increased by studying culture conditions and factors for proliferation and differentiation of stem cells and undifferentiated cells into target cells.

For such morphological reconstruction of organs or tissues in tissue engineering, chemical synthesis such as polymers is used as a scaffold for cell proliferation, which is also referred to as a scaffold. It is preferable to sow the target cells on this scaffold and cultivate them in a suitable environment in vitro to proliferate and differentiate them into cells required or to implant the scaffold into the defective part of the organ or tissue to be regenerated, There have been reported many methods of regenerating the desired organs by proliferating the cells into a three-dimensional structure by proliferating and differentiating them into cells requiring the desired cells (Korean Patent Laid-Open No. 10-2012-0054603). However, even if a bio artificial organ is constructed by using a scaffold, the function of the organ can not be sufficiently exhibited, or it is difficult to construct the bio artificial organ itself regardless of the presence or absence of the scaffold.

On the other hand, research on the development of an artificial liver has been continuously carried out in order to treat liver damage which performs many functions among artificial organs. The artificial liver can be classified into abiotic artificial liver and biological artificial liver depending on existence of living body.

In the case of non-biological artificial liver, it was developed to eliminate the toxicity in the liver in case of liver failure. Typical non-biological artificial liver is MARS (Molecular Absorbents Recirculating System) which attaches albumin to the hollow fiber membrane, And a system for filtering toxic substances by using them. However, these abiotic artificial interactions have the side effect of removing not only toxic substances but also necessary substances.

Recently, the most commonly used biological artificial liver has been developed by mixing artificial materials and biomaterials, especially liver cells of living organisms. In the case of such a biological artificial liver, an animal or a human hepatocyte is inserted into a hollow fiber to pass the blood of the patient through dialysis. The most important part in the development of such a device is the development of the material and structure of the membrane, And cultivation of hepatocytes in an external environment. For example, a Japanese university has recently succeeded in producing mini-artificial liver from iPS cells (Takanori Takebe et al., Nature Vol.499 pp.481-485). However, there is a problem that iPS cells have not been proven to be safe because of the risk of cancer development as cells that are made by manipulating genes.

Under these circumstances, the present inventors have made intensive efforts to develop artificial liver capable of performing liver function properly through vascularization process after transplantation, while increasing the differentiation rate without genetic modification. As a result, The present inventors have completed the present invention by developing a stable and functional biological artificial liver by irradiating and irradiating with low output light.

It is an object of the present invention to provide a method for producing a mesenchymal stem cell comprising the steps of: dividing isolated mesenchymal stem cells into hepatic progenitor cells or hepatic cells; Dividing the isolated mesenchymal stem cells into endothelial progenitor cells or endothelial cells; And co-culturing the differentiated hepatocytes or hepatocytes, vascular endothelial progenitor cells or vascular endothelial cells with isolated mesenchymal stem cells, and co-culturing the cell aggregate.

Another object of the present invention is to provide a cell aggregate (spheroid) produced by the above production method.

It is still another object of the present invention to provide a cell therapy agent for treating liver disease comprising the cell aggregate (spheriod) or liver tissue formed therefrom.

It is another object of the present invention to provide an implant comprising the cell aggregate (spheriod) or liver tissue formed therefrom.

According to one aspect of the present invention, there is provided a method for producing a mesenchymal stem cell comprising the steps of: dividing isolated mesenchymal stem cells into hepatic progenitor cells or hepatic cells; Dividing the isolated mesenchymal stem cells into endothelial progenitor cells or endothelial cells; And co-culturing the differentiated hepatocytes or hepatocytes, vascular endothelial progenitor cells or vascular endothelial cells with isolated mesenchymal stem cells, and co-culturing the cell aggregate.

The method of manufacturing a spheroid of the present invention is a method of manufacturing a biological artificial liver, which can solve the problem of mechanical artificiality in which the mechanical device has a risk of inconvenience and instability. In addition, when the differentiation rate is increased by genetically modifying the cells, it is possible to produce an artificial liver capable of properly performing liver function through a vascularization process without transplantation because it is high in cancer development . In addition, we have for the first time demonstrated that the tissue formation ability of the cell aggregate can be greatly improved by irradiating stem cells with low-power light for a specific time during culturing.

The method of producing a cell cluster according to the present invention includes the step of differentiating the isolated mesenchymal stem cells into hepatic progenitor cells or hepatic cells.

In the present invention, the term "hepatocyte cell" includes all cells involved in the hepatocyte formation process except for the hepatocytes in which maturation has been completed. In the present invention, a method for differentiating mesenchymal stem cells into hepatocyte or hepatocyte is known in the art Lt; / RTI > As a specific example, differentiation can be induced by treating a substance known as a hepatocyte differentiation agent or culturing it in a medium containing them. Specific examples of such hepatocyte differentiation agents include epidermal growth factor, insulin, transforming growth factor (TGF) -α, TGF-beta, fibroblast growth factor (FGF) , Oncostatin M, OSM, interleukin (IL) -1, IL-6 in the presence of heparin, hepatocyte growth factor (HGF), dexamethazone, insulin-like growth factor IGF-II, heparin-binding growth factor (HBGF) -1, glucagon, corticoid, glucocorticoid, cortisol, cortisol, dexamethazone (US Patent No. 3,007,923) and derivatives thereof, prednisone, methylprednisone, hydrocortisone, triamcinolone (US Pat. No. 2,789,118 ) And induction thereof Although the like, without being limited thereto.

The kind and origin of mesenchymal stem cells used for differentiation into hepatocytes or hepatocytes in the present invention are not particularly limited. As a specific example, the mesenchymal stem cells of the present invention can be obtained from bone marrow, tissue, embryo, umbilical cord blood, blood or body fluids such as mammals including humans, and a method for obtaining mesenchymal stem cells from such mesenchymal stem cell sources Lt; RTI ID = 0.0 > mesenchymal < / RTI > stem cells can be obtained.

In addition, the method for preparing a cell cluster according to the present invention may further include the step of confirming whether or not the cells are differentiated into hepatic progenitor cells or hepatic cells.

Specifically, since the hepatocyte or hepatocyte differentiated from mesenchymal stem cells can express the specific marker associated therewith, it is possible to confirm whether the marker is differentiated depending on the expression level and expression level of a marker known in the art . Specific examples of the marker include, but are not limited to, GST-P, cytokeratin 19,? -Fetoprotein, albumin,? 1 -antitrypsin, transferrin, CK18 and AGPR. For detection of these markers, an antibody to these markers, or a gene-specific primer or probe that encodes a marker, can be used. When a marker-specific primer is used, a gene amplification method such as PCR can be used.

In one embodiment of the present invention, in order to differentiate the isolated mesenchymal stem cells into hepatocytes, the mesenchymal stem cells are cultured in Pretreatment Medium (Table 1) for 2 days in hepatocyte differentiation medium (Table 2) 7 days, and in hepatocyte maturation medium (Table 3) for 7 to 14 days (Fig. 3).

Subsequently, immunofluorescence staining was performed using an antibody against albumin (ALB) and alpha-fetoprotein (AFP), which are markers specific to hepatocytes, in order to confirm the differentiation into hepatocytes. As a result, it was confirmed that these markers were differentiated into hepatocytes by confirming the expression of these markers in the mesenchymal stem cells that underwent the differentiation process (FIG. 3).

As another step, the method for producing a cell cluster according to the present invention includes a step of differentiating the isolated mesenchymal stem cells into endothelial progenitor cells or endothelial cells.

In the present invention, "vascular endothelial cells" are also referred to as "endothelial cells ", and" vascular endothelial progenitor cells "include all cells involved in vascular endothelial cell formation, except for vascular endothelial cells that have undergone maturation.

In the present invention, the method of differentiating mesenchymal stem cells into endothelial progenitor cells or endothelial cells can also be carried out by a known method. As a specific example, a method of differentiating mesenchymal stem cells into vascular endothelial cells by treatment with vascular endothelial cell growth factor (VEGF) (Chinese Patent Publication No. 001546656), transcription factor activating vascular- (US Patent Application Publication No. 2004-0091468), a method of inducing differentiation into vascular endothelial cells using ELF-1 (US Patent Publication No. 2004-0091468), culturing embryonic stem cells in a scaffold and culturing vascular endothelial cells (Japanese Laid-Open Patent Application No. 2006-246770), a method of differentiating stem cells into vascular endothelial cells using the synthesized compounds (JP-A No. 2006-083095), cells overexpressing Notch ligand (Japanese Patent Laid-Open No. 2007-089536) and hepatocyte growth factor (HGF) were used to differentiate into endothelial progenitor cells (EPCs) A method of differentiating stem cells into vascular endothelial cells (U.S. Patent Application Publication No. 2007-0142279), a stent, an artificial blood vessel graft material and a heart valve material by coating with an antibody or an antibody component capable of binding or interacting with vascular progenitor cells, (US Patent Publication No. 2007-0055367), and methods of promoting adhesion, growth and differentiation to endothelial cells (US Patent Application No. 2007-0055367), and fully differentiated human fibroblasts by treatment with 5-azacytidine, a demethylation factor A method of differentiating into a vascular endothelial cell or a blood cell line (WO 2007-016037), but the present invention is not limited thereto.

The types and origins of mesenchymal stem cells used for differentiation into vascular endothelial progenitor cells or vascular endothelial cells in the present invention are not particularly limited. As a specific example, the mesenchymal stem cells of the present invention can be obtained from bone marrow, tissue, embryo, umbilical cord blood, blood or body fluids such as mammals including humans, and a method for obtaining mesenchymal stem cells from such mesenchymal stem cell sources Lt; RTI ID = 0.0 > mesenchymal < / RTI > stem cells can be obtained.

The present invention may further include a step of confirming whether or not the cells are differentiated into endothelial progenitor cells or endothelial cells.

Specifically, since vascular endothelial cells or vascular endothelial progenitor cells differentiated from mesenchymal stem cells can express specific markers associated therewith, it is possible to confirm whether or not they are differentiated according to the expression level and expression level of known markers . Specifically, CD34, eNOS, VCAM-1, VE-cadherin, VEGF-R2, and the like are known, but are not limited thereto. For detection of these markers, an antibody to these markers, or a gene-specific primer or probe that encodes a marker, can be used. When a marker-specific primer is used, a gene amplification method such as PCR can be used.

In one embodiment of the present invention, the isolated mesenchymal stem cells were cultured in DMEM-F12 medium supplemented with 50ng / ml of VEGF for 6 days to induce differentiation into vascular endothelial cells (Fig. 4).

The above steps can be performed simultaneously or sequentially, and the order of performing them is irrelevant. The rate of differentiation into target cells (hepatic progenitor cells or hepatocytes; vascular endothelial progenitor cells or vascular endothelial cells), and the like, those skilled in the art can appropriately determine these cells so that they can be co-cultured later.

As a further step, the method for preparing a cell cluster according to the present invention comprises co-culturing the differentiated precursor cells or hepatocytes with the mesenchymal stem cells separated from the vascular endothelial progenitor cells or vascular endothelial cells .

Liver progenitor cells or hepatocytes differentiated by the above method from mesenchymal stem cells; Vascular endothelial progenitor cells or vascular endothelial cells; And mesenchymal stem cells are mixed in one space and co-cultured to form a spheroid.

In the present invention, the hepatic progenitor cell or the hepatic cell and the endothelial progenitor cell or the endothelial cell may be differentiated from the same mesenchymal stem cell have.

Since mesenchymal stem cells of the same origin can be used for autologous cell transplantation, there is no fear of immune rejection reaction, and since the mesenchymal stem cells can be obtained by a single procedure and then differentiated into desired cells, This is advantageous in terms of ease of use.

In the present invention, the mixing ratio (cell number) of the hepatic progenitor cells or hepatocytes: vascular endothelial progenitor cells or vascular endothelial cells: mesenchymal stem cells may be 1: 0.2-0.7: 0.2-0.7, more preferably 1: 0.5 : 0.5 cell ratio and co-cultured.

Intercellular ratios can improve the functionality of the artificial liver. When the ratio of hepatocytes is low, there is a problem in that the efficiency of the liver is inferior in functional aspect. If the ratio of vascular endothelial cells is low, there is a problem that vascularization after in vivo implantation is difficult and artificial liver necrosis can occur. In addition, when the ratio of mesenchymal stem cells is low, there is a problem that the formation of the artificial interstices formed can be inhibited, so that cell aggregates can be formed most efficiently in the intercellular ratios.

In addition, the method may include irradiating low output light in the range of 1 to 30 mW / cm 2 at a wavelength of 630 to 700 nm during co-culture in the present invention, and the light may be irradiated for 5 to 30 minutes per day. In the embodiment of the present invention, light having a 10 mW / cm 2 output power of 660 nm wavelength was irradiated for 10 minutes per day.

When light is irradiated within 5 minutes per day, improvement of liver tissue formation ability by light irradiation is insufficient, and when irradiated for more than 30 minutes per day, there is no significant difference in liver tissue formation between the case of irradiation within 30 minutes and excessive cell stimulation Is rather inefficient.

In the present invention, the co-culture may be cultured in a hydrophobic culture container. Specifically, the culture container may be a culture container that is surface-treated with a polymer imparting hydrophobicity or a culture container made of the polymer. Examples of the hydrophobic polymer include polystyrene, polymethyl methacrylate (PMMA), polyethylene terephthalate (PET), polyvinyl chloride (PVC), polyethylene (PE), polypropylene (PP), polytetrafluoroethylene (PTFE), aliphatic polyester- (PDLA), poly (glycolic acid) (PGA), poly (caprolactone) (PCL), poly (hydroxyalkanoate), polydioxanone (PDS) , Polytrimethylene carbonate, derivatives thereof, and copolymers thereof, but is not limited thereto. In addition, the ECM proteins collagen, laminie, fibronec a culture container having a hydrophobic surface by coating with a surfactant or the like may be used.

Cells can be assembled by cell migration and cell-cell interaction if the cell is adhered to the culture vessel with appropriate force. Therefore, when cultured in a hydrophobic culture container, the cell adhesive force can have an appropriate adhesive strength that does not inhibit cell migration.

The cell aggregate formed by such a method can form liver tissue.

The cell aggregate produced by the production method of the present invention can form liver tissue to produce artificial liver. Liver precursor cells or hepatocytes differentiated from the mesenchymal stem cells; Vascular endothelial progenitor cells or vascular endothelial cells; And spheroids formed by mixing mesenchymal stem cells in one space and co-culturing can form liver tissue for a certain period of time.

In an embodiment of the present invention, 5 × 10 ⁵ cells of mesenchymal stem cells, 10 × 10 ⁵ cells of hepatocytes, and 5 × 10 ⁵ cells of vascular endothelial cells are mixed in a hydrophobic culture 24-well plate, . During the co-cultivation, the LEDs were irradiated at 10 mW / cm 2 with a low output power of 10 mW / cm 2 at a red wavelength band (660 nm) for 10 minutes every day. As a result, it was confirmed that a cell aggregate having a diameter of about 5 mm was formed after 48 hours, and a three - dimensional artificial liver tissue was formed after 4 days.

In another aspect for achieving the above object, the present invention provides a cell aggregate (spheroid) produced by the above production method.

In another aspect, the present invention provides a cell therapy agent for the treatment of liver disease comprising the cell aggregate (spheriod) or a liver tissue formed therefrom.

The cell aggregate obtained through the method for producing the cell aggregate of the present invention or the liver tissue formed therefrom can be used as a cell therapy agent by being transplanted into a subject suffering from liver disease including liver damage and hepatic disorder. The cell aggregate or liver tissue of the present invention greatly enhances the differentiation ability into hepatocytes through the formation of a stem cell biocompatible environment by interaction with peripheral cells in an animal model, It can be used as a cell therapy agent for the treatment of diseases and as an implantable material (artificial liver) which can support the liver function, and this technology can be used as a source technology for manufacturing the artificial liver which can replace the living body liver.

The liver disease may include, but is not limited to, liver function impairment, liver damage, hepatitis, hepatotoxicity, cholestasis, fatty liver, liver cirrhosis, liver ischemia, alcoholic liver disease, liver abscess, hepatic coma, liver atrophy and liver cancer.

The cell therapeutic agent according to the present invention can be injected into a living body of a subject in a state where the cells are cultured singly or on a film, for example, Lyndval et al. (1989, Arch. Neurol. 46: 615-31) or Douglas conchiolca Douglas Kondziolka, Pittsburgh, 1998). The preparation may contain a pharmaceutically acceptable carrier in addition to a three-dimensional cell cluster or liver tissue formed therefrom as an active ingredient. In the case of an injection, it may contain a preservative, an anhydrous agent, a solubilizing agent or a stabilizer, , A base, an excipient, a lubricant or a preservative in the case of a preparation for topical administration.

The cell therapeutic agent according to the present invention may be formulated into a unit dose form by using a pharmaceutically acceptable carrier and / or excipient according to a method which can be easily carried out by a person having ordinary skill in the art to which the present invention belongs. Or by intrusion into a multi-dose container. The pharmaceutically acceptable carrier to be contained in the cell treatment agent of the present invention is one usually used at the time of formulation, and includes lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia rubber, calcium phosphate, alginate, gelatin, calcium silicate , Microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methylcellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate or mineral oil, no. The cell therapy agent of the present invention may further include a lubricant, a wetting agent, an emulsifier, a suspending agent, a preservative, etc. in addition to the above components.

The cell therapeutic agent according to the present invention can be administered parenterally, intravenously, subcutaneously, intraperitoneally or topically. A suitable dose of the cell therapy agent of the present invention may be variously prescribed by such factors as the formulation method, administration method, age, body weight, sex, pathological condition, food, administration time, administration route, excretion rate and responsiveness of the patient . The dose of the cell therapy agent of the present invention is preferably 1 x 10 ⁶ to 3 x 10 ⁸ cells at a time, and can be usually administered once or several times.

In another aspect, the present invention provides an implant comprising the cell aggregate (spheriod) or liver tissue formed therefrom.

As used herein, the term "transplant" refers to a substance that can be implanted in a human or mammal, such as a scaffold for isolating a damaged site from the outside or allowing grafted cells or secreted therapeutic materials to stay. Such implants may include, without limitation, a variety of materials used in the art such as biodegradable synthetic polymers and natural materials as supports for tissue engineering. The implant according to the present invention may be configured such that a three-dimensional cell cluster of the present invention or a liver tissue formed therefrom is implanted in a support formed by molding a biodegradable polymer.

The biodegradable polymer is spontaneously and slowly decomposed in a living body for a certain period of time, and includes a polymer having at least one of biocompatibility, blood affinity, anticarcinogenic property, cell nutrition and intercellular matrix formation ability. Examples of such biodegradable polymers include, but are not limited to, fibrin, collagen, gelatin, chitosan, alginate, hyaluronic acid, dextran, polylactic acid, poly (glycolic acid), PGA, poly Polyglycolic acid (PLGA), poly-? - (caprolactone), polyanhydride, polyorthoesters, polyvinyl alcohol, polyethylene glycol, polyurethane, polyacrylic acid, poly- Poly (ethylene oxide) -poly (propylene oxide) -poly (ethylene oxide) copolymers, copolymers thereof, mixtures thereof, and the like.

In the implant according to the present invention, the biodegradable polymer may be contained in an amount of 5 to 99% by weight. If the content of the biodegradable polymer is less than 5% by weight, the molded body of the implant is not well formed, and the mechanical strength of the support is weakened. If the content of the biodegradable polymer is more than 99% by weight, This may cause difficulties.

The implants can be prepared by known methods such as solvent-casting and particle-leaching techniques, gas forming techniques, polymeric fibers made of non-woven fabric, A method of forming a biodegradable polymer by a method such as a fiber extrusion and fabric forming process, a thermally induced phase separation technique, an emulsion freeze drying method, a high pressure gas expansion, But is not limited thereto.

In one embodiment of the present invention, capillary formation and organization can be observed after transplantation of the cell aggregate prepared in the present invention or liver tissue formed therefrom into a mouse, and it is confirmed that it can be used as an implanted material (Figs. 12 and 13).

The method of producing a cell aggregate according to the present invention and the cell aggregate produced thereby can securely perform the liver function safely through the vascularization process after the transplantation, There is no immune rejection reaction and thus it can be usefully used as a cell therapy agent and transplant for treating liver diseases.

Brief Description of the Drawings Fig. 1 is a diagram showing the entire process of a method for producing a cell cluster of the present invention.
2 is a micrograph and a graph showing the results of verifying stem cells isolated from human adipose tissue in the present invention.
FIG. 3 is a graph showing the process of differentiating human adipose-derived stem cells into liver-like cells and the expression of albumin (ALB) and α-fetoprotein (AFP) in differentiated cells according to the present invention.
FIG. 4 is a graph showing the expression of CD31 and CD34 in cells differentiated from vascular endothelial cells from human adipose-derived stem cells in the present invention.
FIG. 5 is a diagram illustrating a process of forming a cell aggregate by co-culturing differentiated hepatocytes, vascular endothelial cells and mesenchymal stem cells according to the present invention.
FIG. 6 is a graph showing the difference in the ability to form cell aggregates when light irradiation is performed and when light irradiation is performed in the present invention.
FIG. 7 is a graph showing expression patterns of CD34, CD31 and hepatocyte marker albumin, A-fetoprotein, which are vascular endothelial cell markers in the artificial liver prepared in the present invention.
FIG. 8 is a view for observing collagen production through the artificial liver H & E staining method prepared in the present invention.
FIG. 9 is a view showing the artificial liver blood vessel cells prepared in the present invention. FIG.
FIG. 10 is a graph showing blood vessel formation and organization after transplantation of a mixture of artificial liver and matrigel prepared in the present invention into the subcutis of a mouse. FIG.
11 is a view showing the manufacturing process and the pre-clinical evaluation result of the artificial liver.
FIG. 12 is a view showing in vivo artificial organ vasculature through laser speckle contrast imaging.
Fig. 13 is a view showing whether the artificial liver is organized after transplantation in vivo.

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are for further illustrating the present invention, and the scope of the present invention is not limited by these examples.

Example 1: Isolation of human adipose-derived mesenchymal stem cells

Multidisciplinary stem cells were isolated from human subcutaneous adipose tissue. Normal human subcutaneous fat tissue was washed 3 times with PBS containing 2% penicillin / streptomycin to remove the contaminated blood and chopped with surgical scissors. The adipose tissue was immersed in a tissue lysis solution (serum-free DMEM + 1% BSA (w / v) + 0.3% collagenase type 1) prepared beforehand and stirred at 37 ° C for 2 hours and then centrifuged at 1,000 rpm for 5 minutes The supernatant and pellet were separated. The supernatant was discarded and the remaining pellet was collected, washed with PBS, and centrifuged at 1,000 rpm for 5 minutes to separate the supernatant. The separated supernatant was filtered with a 100 μm mesh to remove tissue debris and washed with PBS. Cells isolated from these were cultured in DMEM / F12 medium (Welgene) containing 10% FBS. Cells that were not adhered after 24 hours of culture were washed and washed with PBS and cultured in DMEM / F12 medium supplemented with 10% FBS every 2 days to obtain human subcutaneous adipose tissue-derived stem cells.

Multidisciplinary stem cells were isolated from human subcutaneous adipose tissue and were observed either by phase contrast microscopy (Nikon) or by immunofluorescence staining (B) or by flow cytometry (C). CD29, CD90, and CD105 were used as surface antigens for the identification of mesenchymal stem cells, and CD34, CD31, KDR (Flk1, ) And? -SMA, the expression patterns of these were analyzed using flow cytometry.

The recovered stem cells were washed several times with PBS, immersed in 4% paraformaldehyde solution, fixed at room temperature for 30 minutes, washed again with PBS, and immunostained. The immunological staining was performed by immersing the prepared stem cells in PBS containing the primary antibody, reacting overnight, washing with PBS three times, then treating the secondary antibody capable of binding to the primary antibody, Lt; / RTI > for 1 hour. After the reaction was terminated, the stem cells were washed 3 times with PBS and analyzed by fluorescence microscopy and FACS,

As a result, it was confirmed that the cells isolated from human subcutaneous adipose tissue were adipose-derived mesenchymal stem cells having mesenchymal stem cell phenotype (Fig. 2).

Example 2: Differentiation of Mesenchymal Stem Cells into Hepatocytes

In order to differentiate mesenchymal stem cells isolated in Example 1 into hepatocytes, the mesenchymal stem cells were cultured in Pretreatment Medium (Table 1) for 2 days, Hepatocyte Differentiation Medium (Table 2) for 7 days , And hepatocyte maturation medium (Table 3) for 7 to 14 days (Fig. 2).

Pretreatment medium Human Mesenchymal Stem Cell Hepatogenic Differentiation Basal Medium (Cat. No. HUXMA-03101) 100 mL EGF 20 μl bFGF 10 μl

Hepatocyte Differentiation Medium Human Mesenchymal Stem Cell Hepatogenic Differentiation Basal Medium (Cat. No. HUXMA-03101) 100 mL HGF (hepatocyte growth factor) 20 μl bFGF 10 μl Nicotinamide 100 μl

Hepatocyte Maturation Medium Human Mesenchymal Stem Cell Hepatogenic Differentiation Basal Medium (Cat. No. HUXMA-03101) 100 mL Oncostatin M 20 μl Dexamethasone 50 μl ITS + Premix -

Subsequently, immunofluorescence staining was performed using an antibody against albumin (ALB) and alpha-fetoprotein (AFP), which are markers specific to hepatocytes, in order to confirm the differentiation into hepatocytes. As a result, it was confirmed that these markers were differentiated into hepatocytes by confirming the expression of these markers in the mesenchymal stem cells that underwent the differentiation process (FIG. 3).

Example 3: Differentiation of mesenchymal stem cells into vascular endothelial cells

In order to differentiate the mesenchymal stem cells isolated in Example 1 into vascular endothelial cells, the mesenchymal stem cells were cultured to confluent 70% using growth medium (DMEM / F12 medium containing 10% FBS) , And cultured for 6 days using differentiation medium (DMEM / F12 medium containing 50ng / ml of VEGF).

In order to confirm the differentiation into vascular endothelial cells, CD31 and CD34 were used as surface antigens, and their expression patterns were analyzed by DAPI staining. As a result, these markers were expressed in the mesenchymal stem cells subjected to the differentiation process (Fig. 4). As shown in Fig.

Example 4: Formation of cell aggregate by co-culturing hepatocytes, vascular endothelial cells and mesenchymal stem cells

5 × 10 ⁵ cells of mesenchymal stem cells isolated in Example 1, 10 × 10 ⁵ cells of hepatocytes differentiated in Example 2, and 5 × 10 ⁵ cells of vascular endothelial cells differentiated in Example 3 were cultured in a hydrophobic culture container non-tissue culture-treated 24-well plate) and seeded and co-cultured. The culture medium was mixed with 1: 1 DMEM-F12 and Hepatocyte Differentiation Medium.

During the co - cultivation, low output light (output value: 10 mW / ㎠) of red wavelength band (660 nm) was irradiated for 10 minutes every day. As a result, a cell aggregate having a diameter of about 5 ㎜ was formed after 48 hours, Three days later, artificial liver was formed. On the other hand, it was confirmed that a cell aggregate was not formed in the control group cultured without light irradiation, or a spherical cell aggregate of a considerable size was not formed (FIG. 6).

Example 5: Immunological assay of three-dimensional cell aggregate

The three-dimensional cell aggregate structure formed in the above example was recovered, fixed at -70 ° C using an OCT compound, cut into a thickness of 4 μm using a microtome, the slice was fixed on a slide glass, and immunostaining was performed.

The recovered three-dimensional cell aggregate was physically broken up using a syringe, placed on a slide glass and adhered for 4 hours. The slide glass was washed several times with PBS, and immersed in 4% paraformaldehyde solution Immobilized at room temperature for 30 minutes, then washed again with PBS and immunostained.

The immunological staining was performed by immersing the slide glass prepared above in PBS containing various primary antibodies, reacting overnight, washing three times with PBS, then treating the secondary antibody capable of binding to the primary antibody, In a dark room for 1 hour. After the reaction was terminated, the slide glass was washed three times with PBS and mounted and analyzed by fluorescence microscopy.

As a result, it was confirmed that CD34, CD31, which is a vascular endothelial cell marker, and albumin and A-fetoprotein, which are hepatocyte markers, were well expressed, suggesting that artificial liver was well cultured like vascular endothelial cells and hepatocytes (Fig. 7).

Example 6: Increase in the extracellular matrix protein and the degree of organization of artificial liver

Collagen protein production was confirmed by H & E (Hematoxylin & Eosin) staining method to observe the increase of extracellular matrix protein and degree of organization of artificial liver through low power light irradiation.

As a result, it was confirmed that ECM protein collagen was stained blue between cells and cells, suggesting that the collagen expressed in the cells was well organized (FIG. 8).

On the other hand, in order to observe whether or not the vascularization was performed in the transplanted organs in vivo, it was observed using a laser speckle contrast imaging apparatus that the matrigel and the non-differentiated stem cell group were transparent, Capillary angiogenesis and organization were observed in the mixed group of stem cell spheroids and matrigel (Fig. 12).

As a result, it was confirmed that the artificial liver of the present invention can perform its function while engaging stably in the body through blood vessel formation and organization after transplantation.

Claims (11)

Dividing the isolated mesenchymal stem cells into hepatic progenitor cells or hepatic cells;
Dividing the isolated mesenchymal stem cells into endothelial progenitor cells or endothelial cells; And
The method comprising mixing the differentiated hepatic precursor cells or hepatocytes, vascular endothelial progenitor cells or vascular endothelial cells with isolated mesenchymal stem cells, co-culturing with light of 630 to 700 nm wavelength, ).
The method according to claim 1, wherein the hepatic progenitor cell or the hepatic cell and the endothelial progenitor cell or the endothelial cell are differentiated from the same mesenchymal stem cell ≪ / RTI >
The method according to claim 1, wherein the mixing ratio (cell number) of the hepatic progenitor cells or hepatocytes: vascular endothelial progenitor cells or vascular endothelial cells: mesenchymal stem cells is 1: 0.2-0.7: 0.2-0.7. ).
delete The method according to claim 1, wherein the light is irradiated for 5 to 30 minutes per day.
The method according to claim 1, wherein the co-culture is cultured in a culture vessel that is more hydrophobic than the culture solution.
The method according to claim 1, wherein the cell aggregate forms a liver tissue.
delete The method according to claim 1, wherein the spherical cell aggregate (spheriod) is usable as a cell therapy agent for the treatment of liver disease.
10. The method according to claim 9, wherein the liver disease is selected from the group consisting of hepatitis, hepatotoxicity, cholestasis, fatty liver, liver cirrhosis, liver ischemia, alcoholic liver disease, liver abscess, hepatic coma, liver atrophy and liver cancer.
A method for manufacturing an artificial liver comprising the step of mixing and culturing a spherical cell aggregate produced by the manufacturing method of claim 1 and a matrigel.

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