KR102563616B1 - Soluble extracellular matrix derived from stem cells and preparing method thereof - Google Patents

Abstract

The present invention provides a cell culture step of culturing human-derived adipose stem cells to prepare a cell culture, a decellularization step of decellularizing the cell culture to obtain an extracellular matrix, and washing and grinding the decellularized extracellular matrix. It provides a method for producing a water-soluble extracellular matrix comprising the steps of water-solubilizing the pulverized extracellular matrix and dialysis of the water-soluble extracellular matrix.
The water-soluble extracellular matrix prepared by the production method of the present invention is non-cytotoxic, has an effect of increasing cell proliferation, and can be formulated into an injection, so it can be usefully used as a therapeutic agent for tissue regeneration.

Description

Soluble extracellular matrix derived from stem cells and preparing method thereof}

The present invention relates to a stem cell-derived water-soluble extracellular matrix and a method for preparing the same.

Extracellular matrix (ECM) is the remaining part of tissue other than cells. It fills the gaps between cells to physically support tissues, thereby creating an environment in which the original structure of living tissues can be maintained. It is a collection of polymers. Since this extracellular matrix not only plays a structural role as a simple cell-to-cell connection but also plays a very important role as a regulator of various cell physiology such as cell division, differentiation and death, many studies have been conducted to utilize the extracellular matrix for tissue regeneration. is in progress However, the main problems are that it is difficult to secure raw materials to use the extracellular matrix as a tissue regeneration treatment, and that the extracellular matrix is mostly polymer and difficult to develop as an injection because of its low water solubility.

A commonly used extracellular matrix is an animal tissue-derived extracellular matrix. A lot of research is being conducted to use an extracellular matrix obtained by decellularizing the same animal organ as the damaged organ as a therapeutic agent for organ regeneration. For example, VentriGel (Ventrix Inc), an extracellular matrix obtained by decellularizing pig hearts, is being clinically studied as a treatment for myocardial infarction. However, because animal-derived extracellular matrix is not allogeneic, there is a high possibility of immune rejection and problems with animal virus infection.

In order to solve the problem of heterologous origin, many studies on allogeneic extracellular matrix are being conducted. In the case of L&C Bio, a domestic company, extracellular matrix extracted from human corpses is currently being used for regeneration of damaged tissues. However, there are ethical issues and difficulties in securing raw materials for mass production. In order to solve these problems, a human cell culture-based extracellular matrix production method has been proposed as an alternative. If a cell culture system is secured, it is easy to secure raw materials for mass production, and immunogenicity and ethical problems can be avoided. The most suitable cells for the cell culture system are stem cells. Stem cells are cells with self-renewal and multipotency, and include embryonic stem cells (ESC), induced pluripotent stem cells (iPSC), and adult stem cells. cells) can be broadly classified. Embryonic stem cells and induced pluripotent stem cells have pluripotency that can differentiate into all cells, so they are attractive for use in rare and intractable diseases, but embryonic stem cells are difficult to use due to ethical problems, and induced pluripotent stem cells There is a problem of tumorigenicity because it overexpresses cancer-related genes for manufacturing induced pluripotent stem cells. Adult stem cells exist in human tissues, and are typically present in bone marrow, fat, teeth, and umbilical cord blood. It is known that adult stem cells can avoid the ethical problems of embryonic stem cells and are free from the tumorigenicity of induced pluripotent stem cells. Therefore, adult stem cells are the most suitable stem cells for extracellular matrix production.

Under this background, the present inventors have completed the present invention by developing a method for producing an extracellular matrix and a method for soluble the extracellular matrix using an adult stem cell-based cell culture system.

The problem to be solved by the present invention is to provide a water-soluble extracellular matrix for tissue regeneration and a method for preparing the same.

In order to achieve the above technical problem, in one aspect of the present invention, a cell culture step of producing a cell culture by culturing human-derived adipose stem cells, decellularization to obtain a decellularized extracellular matrix by decellularizing the cell culture Preparation of a water-soluble extracellular matrix comprising steps of washing and grinding the decellularized extracellular matrix, water-solubilizing the pulverized extracellular matrix, and dialysis-purifying the soluble extracellular matrix. A method is provided.

In one embodiment, the cell culture may be a cell sheet.

In one embodiment, the decellularized extracellular matrix may be an extracellular matrix sheet (ECM sheet).

In one embodiment, the decellularization step may be decellularized by treatment with a surfactant.

In one embodiment, the surfactant is Triton X-100 (Triton X-100), Triton X-114 (Triton X-114), nonyl phenoxypolyethoxyethanol (nonyl phenoxypolyethoxylethanol, NP-40), polyoxyethylene Polyoxyethylene lauryl ether (Brij-35), Polyethylene glycol hexadecyl ether (Brij-58), Tween-20, Tween-80, Octyl-b -Octyl-b-Glucoside, Octylthioglucoside (OTG), 3-((3-cholamidopropyl)dimethylammonio)-1-propanesulfonate (3-((3-cholamidopropyl) dimethylammonio)-1-propanesulfonate, CHAPS), 3-((3-cholamidopropyl)dimethylammonio)-2-hydroxy-1-propanesulfonate (3-((3-cholamidopropyl)dimethylammonio)-2- It may be one or more surfactants selected from the group consisting of hydroxy-1-propanesulfonate (CHAPSO) and sodium dodecyl sulfate (SDS).

In one embodiment, the grinding may be freeze grinding.

In one embodiment, the water-solubilization step includes denaturing the pulverized extracellular matrix by treating it with an acid, freeze-drying the denatured extracellular matrix, and using a surfactant in the lyophilized extracellular matrix. It may include the step of processing.

In one embodiment, the acid may be one or more acids selected from the group consisting of acetic acid and hydrochloric acid.

In one embodiment, the surfactant is Triton X-100 (Triton X-100), Triton X-114 (Triton X-114), nonyl phenoxypolyethoxyethanol (nonyl phenoxypolyethoxylethanol, NP-40), polyoxyethylene Polyoxyethylene lauryl ether (Brij-35), Polyethylene glycol hexadecyl ether (Brij-58), Tween-20, Tween-80, Octyl-b -Octyl-b-Glucoside, Octylthioglucoside (OTG), 3-((3-cholamidopropyl)dimethylammonio)-1-propanesulfonate (3-((3-cholamidopropyl) dimethylammonio)-1-propanesulfonate, CHAPS), 3-((3-cholamidopropyl)dimethylammonio)-2-hydroxy-1-propanesulfonate (3-((3-cholamidopropyl)dimethylammonio)-2- It may be one or more surfactants selected from the group consisting of hydroxy-1-propanesulfonate (CHAPSO) and sodium dodecyl sulfate (SDS).

In one embodiment, the dialysis in the purification step may be electrodialysis.

In another aspect of the present invention, a pharmaceutical composition for tissue regeneration comprising the water-soluble extracellular matrix prepared by the method for preparing the water-soluble extracellular matrix is provided.

In one embodiment, the pharmaceutical composition for tissue regeneration may be an injection.

In one embodiment, the pharmaceutical composition for tissue regeneration may be for the treatment of ischemic disease.

In one embodiment, the ischemic disease may be cardiac angina pectoris, myocardial infarction, cerebral infarction or foot ulcer.

Unlike conventional methods using enzymes, the method for preparing a water-soluble extracellular matrix according to the present invention can minimize damage to the extracellular matrix because decellularization and water-solubilization are possible using surfactants without using enzymes, and protein enzymes It is possible to avoid additional purification difficulties that occur during use, so that a high purity extracellular matrix can be secured, and impurities that take a long time in general dialysis, such as electrolytes and surfactants, can be quickly removed using electrodialysis. In addition, the water-soluble extracellular matrix prepared by the above extracellular matrix preparation method was confirmed to have no cytotoxicity and efficacy in cell proliferation in cell experiments, and since it can be manufactured as an injection formulation, it can be locally injected into damaged tissue to regenerate tissue. It can be used as a therapeutic agent for

The effects of the present invention are not limited to the above effects, and should be understood to include all effects that can be inferred from the description of the present invention or the configuration of the invention described in the claims.

1 shows a method for producing a water-soluble extracellular matrix according to an embodiment of the present invention.
FIG. 2 is a photograph of the results of each step in the process of decellularization, freeze-grinding, freeze-drying, and water-solubilization in the method for producing a water-soluble extracellular matrix according to an embodiment of the present invention.
3 is a fluorescence staining result of a sample without decellularization.
4 is a fluorescence staining result of the sample after the decellularization step.
5 is an electron microscope image of a sample without decellularization at (a) 300x magnification and (b) 1000x magnification.
6 is an electron microscope image of the sample after the decellularization step at (a) 10,000x magnification and (b) 30,000x magnification.
Figure 7 is a result of confirming the amount of DNA remaining in the sample before and after the decellularization step, (a) a stained image of cell nuclei and (b) a result of quantitative analysis of the concentration of DNA.
Figure 8 shows the concentration of SDS performed in hADSC (0 μg / ml, 0.5 μg / ml, 2.5 μg / ml, 5 μg / ml, 10 μg / ml, 20 μg / ml, 50 μg / ml and 100 μg / ml ) It is a graph of (a) microscopic image and (b) survival rate by concentration of the cytotoxicity evaluation result.
Figure 9 is by SDS concentration (0 μg / ml, 0.5 μg / ml, 1 μg / ml, 3 μg / ml, 5 μg / ml, 10 μg / ml, 20 μg / ml and 100 μg / ml performed in HDF) ) It is a graph of (a) microscopic image and (b) survival rate by concentration of the cytotoxicity evaluation result.
Figure 10 is prepared for SDS quantification (a) by SDS concentration (0.15 μg / ml, 0.3 μg / ml, 0.6 μg / ml, 1.25 μg / ml, 2.5 μg / ml, 5 μg / ml, 10 μg / ml and 20 μg/ml) and (b) a standard curve obtained by measuring the absorbance of the sample for each concentration of SDS.
11 is a result of SDS measurement before and after dialysis of a water-soluble extracellular matrix prepared by a method for preparing a water-soluble extracellular matrix according to an embodiment of the present invention.
12 is a result of cytotoxicity evaluation of a water-soluble extracellular matrix prepared by a method for preparing a water-soluble extracellular matrix according to an embodiment of the present invention.
13 is a result of cell proliferation evaluation of a water-soluble extracellular matrix prepared by a method for preparing a water-soluble extracellular matrix according to an embodiment of the present invention.
14 is a result of measuring the protein concentration of the water-soluble extracellular matrix prepared by the method for preparing the water-soluble extracellular matrix according to an embodiment of the present invention.
15 is a result of analyzing the molecular weight aspects of proteins of the water-soluble extracellular matrix prepared by the method for preparing the water-soluble extracellular matrix according to an embodiment of the present invention.

The present invention provides a cell culture step of culturing human-derived adipose stem cells to prepare a cell culture, a decellularization step of obtaining a decellularized extracellular matrix by decellularizing the cell culture, and washing the decellularized extracellular matrix. and crushing, a water-solubilization step of water-solubilizing the pulverized extracellular matrix, and a purification step of dialysis of the water-soluble extracellular matrix.

In the present invention, "water solubilization" means a process of preparing a water-soluble form to change an injectable formulation into an injectable formulation.

In the present invention, "stem cell" is a cell that forms the basis of a cell or tissue constituting an individual, and its characteristic is that it can self-renew by repeating division, and can differentiate into cells with specific functions depending on the environment. It refers to cells that have the ability to differentiate. It is produced in all tissues during fetal development, and even in adulthood, it is found in some tissues where cells are actively replaced, such as bone marrow and epithelial tissues. Stem cells include totipotent stem cells, which are formed when the fertilized egg begins to divide for the first time, and pluripotent stem cells in the inner membrane of the blastocyst, which are formed by continued division, according to the types of cells capable of differentiation. They are also classified as multipotent stem cells that exist in mature tissues and organs. At this time, pluripotent stem cells are cells that can differentiate only into cells specific to tissues and organs that contain these cells, and maintain the homeostasis of adult tissues as well as the growth and development of each tissue and organ in the fetal, neonatal, and adult stages. It is involved in the maintenance and function of inducing regeneration in case of tissue damage. These tissue-specific pluripotent cells are collectively referred to as adult stem cells.

Mesenchymal stem cells, which are classified as adult stem cells, are cells that are in the limelight as a material for regenerative medicine and can be collected from tissues such as bone marrow, umbilical cord blood, adipose tissue, and umbilical cord. It has the ability to differentiate into cells constituting various human tissues such as cells, bone cells, chondrocytes, nerve cells, and cardiomyocytes. In the present invention, adipose stem cells isolated from human adipose tissue were used, but are not limited thereto.

In the present invention, "cell culture" is used as a meaning including materials produced from cultured cells and cultured cells themselves. Substances produced from the cultured cells refer to all substances produced by cells while culturing cells, such as extracellular matrix, growth factors, exosomes, and mRNA.

In the present invention, the adipose stem cells can be cultured to produce a cell culture. The cell culture may be cultured in a conventional manner, for example, it may be cultured by attaching cells in a culture dish or cultured in suspension, but is not limited thereto. Preferably, the cells may be attached and cultured.

When cells are attached to a culture dish and cultured, the cells proliferate and an extracellular matrix is formed, and the cells are naturally formed in a sheet form. This is called a cell sheet. The cell sheet may be recovered using a physical recovery method or a special culture dish. The physical recovery method may use a tool such as a scraper. The special culture dish may be a culture dish coated with a temperature-sensitive polymer material, but is not limited thereto. Since the temperature-sensitive polymer material undergoes reversible swelling and contraction according to temperature change, the cell sheet can be easily and quickly recovered without damaging the cells.

The cell sheet may be recovered from the culture dish before the decellularization step, but since it is easy to perform the decellularization step and wash it in a form attached to the culture dish, the decellularization step may be performed without recovering the cell sheet.

In the present invention, the decellularization step may utilize enzymes or surfactants to destroy cell walls. Since the enzyme is a protein, it is difficult to remove it from the extracellular matrix, which is a protein, and since it may damage the extracellular matrix, a surfactant may be preferably used.

The surfactant is Triton X-100, Triton X-114, nonyl phenoxypolyethoxylethanol (NP-40), polyoxyethylene lauryl ether lauryl ether, Brij-35), polyethylene glycol hexadecyl ether, Brij-58, Tween-20, Tween-80, Octyl-b-glucoside b-Glucoside), Octylthioglucoside (OTG), 3-((3-cholamidopropyl)dimethylammonio)-1-propanesulfonate (3-((3-cholamidopropyl) dimethylammonio)-1- propanesulfonate, CHAPS), 3-((3-cholamidopropyl)dimethylammonio)-2-hydroxy-1-propanesulfonate , CHAPSO) and sodium dodecyl sulfate (SDS), but is not limited thereto. Preferably, SDS can be used.

When the cell sheet is treated with the surfactant, a decellularized extracellular matrix from which cells are removed can be obtained. Cell cultures can be disrupted prior to treatment with the surfactant. Preferably, in order to simplify the manufacturing method, the cell culture may be decellularized by treating the cell culture with a surfactant without crushing the cell culture. When the cell sheet is decellularized, an extracellular matrix sheet (ECM sheet) can be prepared.

Since the decellularized extracellular matrix contains impurities such as DNA and RNA derived from cells, it may be washed to remove them. The washed decellularized extracellular matrix can be ground. It may be pulverized by a conventional method, and for example, homogenizing pulverization and freeze pulverization may be used, but is not limited thereto. Preferably, freeze-grinding may be performed to minimize damage to the extracellular matrix.

In the present invention, the water-solubilization step includes denaturing the pulverized extracellular matrix by treating it with an acid, freeze-drying the denatured extracellular matrix, and treating the freeze-dried extracellular matrix with a surfactant. steps may be included.

The acid may be one or more acids selected from the group consisting of acetic acid and hydrochloric acid.

The surfactant is Triton X-100, Triton X-114, nonyl phenoxypolyethoxylethanol (NP-40), polyoxyethylene lauryl ether lauryl ether, Brij-35), polyethylene glycol hexadecyl ether, Brij-58, Tween-20, Tween-80, Octyl-b-glucoside b-Glucoside), Octylthioglucoside (OTG), 3-((3-cholamidopropyl)dimethylammonio)-1-propanesulfonate (3-((3-cholamidopropyl) dimethylammonio)-1- propanesulfonate, CHAPS), 3-((3-cholamidopropyl)dimethylammonio)-2-hydroxy-1-propanesulfonate , CHAPSO) and sodium dodecyl sulfate (SDS), but is not limited thereto. Preferably, SDS can be used. Enzyme treatment is also possible without treatment with a surfactant, but in the case of enzymes, specific conditions for enzyme activity must be maintained, a long reaction time is required, and it is difficult to separate the enzyme from the extracellular matrix after aqueous solution.

While treating the surfactant, a substance that decomposes bonds between proteins may be additionally treated. For example, beta-mercaptoethanol or DTT (1,4-dithiothreitol) may be treated. Preferably, DTT capable of breaking down disulfide bonds in proteins may be added.

In the present invention, the purification step may be purified in a conventional manner for protein purification. For example, solvent precipitation (precipitation of the protein fraction using acetone, ethanol, etc.), dialysis, gel filtration, ion exchange, column chromatography such as reverse phase column chromatography, and ultrafiltration may be applied alone or in combination to perform purification. It can be purified, preferably through dialysis.

The dialysis may use a conventional method for protein purification. Preferably, it may be electrodialysis suitable for removing a substance having an electric charge in order to remove the residual surfactant after the aqueous solution step. Electrodialysis refers to a technique in which dialysis and electrolysis are combined and can be used for the separation or concentration of ions in a solution, which is based on the selective electron transfer of ions through a semi-permeable membrane.

The basic electrodialysis principle consists of an electrolytic cell consisting of a pair of electrodes immersed in an electrolyte for the conduction of ions connected to a DC generator. The electrode connected to the anode of the DC generator is called the anode, and the electrode connected to the cathode is called the cathode. The electrolyte solution also supports current flow due to the movement of negatively and positively charged ions towards the anode and cathode, respectively. Membranes used in electrodialysis are basically sheets of porous ion exchange resins with negative or positive charges and are therefore referred to as cationic or anionic membranes, respectively. Ion exchanger membranes are usually made of polystyrene with appropriate functional groups crosslinked with divinylbenzene (eg, sulfonic acid or quaternary ammonium groups for cationic or anionic membranes, respectively). As the electrolyte, sodium chloride or sodium acetate, sodium propionate or the like can be used. Next, an electrodialysis stack is assembled in such a way that the anionic and cationic membranes are parallel in a filter press between two electrode blocks, and the stream undergoing ion depletion is ion enriched. ) is well separated from the proceeding stream. A solution in which ion deficiency proceeds is called a dilute solution, and a solution in which ion excess proceeds is called a concentrate. The heart of the electrodialysis process is the membrane stack, which consists of several anion and cation exchange membranes separated by spacers and placed between two electrodes. By applying a direct electric current, anions and cations are transported through the membrane towards the electrodes creating desalinated diluate and concentrated streams.

The electrodialysis can be used without limitation as long as the dialysis membrane can remove (remove by dialysis) the surfactant used in the step of water-solubilizing the extracellular matrix, and typically has a molecular weight cut off (MWCO) of 0.1 to 500 kDa. A dialysis tube may be used. Preferably, the molecular weight cut off (MWCO) may be 0.5 to 100 kDa, more preferably 1 to 50 kDa, and most preferably 5 to 20 kDa, but is not limited thereto.

The present invention provides a pharmaceutical composition for tissue regeneration comprising the water-soluble extracellular matrix prepared by the method for preparing the water-soluble extracellular matrix.

The pharmaceutical composition for tissue regeneration may be provided as a pharmaceutical composition including one or more pharmaceutically acceptable carriers, excipients or diluents.

The "pharmaceutically acceptable" means that it exhibits non-toxic properties to cells or humans exposed to the composition.

The pharmaceutical composition for tissue regeneration may be formulated for parenteral administration. The preparation for parenteral administration may be a sterilized aqueous solution, a non-aqueous solvent, a suspension, an emulsion, a lyophilized preparation, and an injection. Preferably, it may be an injection that is easy to administer locally to a damaged tissue.

The pharmaceutical composition for tissue regeneration may be administered in combination with stem cells in order to increase the effect of tissue regeneration. The mixed administration means that the pharmaceutical composition of the present invention and the stem cells are administered simultaneously or with a time difference, independently or independently, for the purpose of preventing, treating, reducing or alleviating symptoms of a disease. The mixed administration may be used together with the combined administration.

The pharmaceutical composition for tissue regeneration can be used for the treatment of ischemic diseases. The ischemic disease may be cardiac angina, myocardial infarction, cerebral infarction, or foot ulcer.

Hereinafter, the present invention will be described in more detail through examples and test examples. However, the following examples and test examples are intended to illustrate the present invention, and the scope of the present invention is not limited thereto.

<Example: Preparation of water-soluble extracellular matrix from human adipose stem cells>

1. Human adipose stem cell culture

Human adipose tissue-derived mesenchymal stem cells (hADSC) were commercially purchased from Thermo Fisher and used. The hADSC culture medium contained FBS (Gibco), ascorbic acid (Sigma), basic fibroblast growth factor (bFGF, R&D system), and insulin-like growth factor 1 (Insulin-like growth factor 1) in DMEM (Gibco). growth factor-1; IGF-1, R&D system), epidermal growth factor (EGF, R&D system), hydrocortisone (Sigma), and antibiotics were added and used. The hADSCs were inoculated at 5 X 10 ³ cells/cm ² and cultured at 37° C. under 5% CO 2 humid conditions, and subculture was performed every 3-4 days. Stem cells used in the test were passage number 5.

2. Preparation of extracellular matrix

To produce an extracellular matrix, hADSC were inoculated into a culture dish at 1 X 10 ⁴ cells/cm ² , and DMEM-low glucose (Gibco) was added with 10% FBS, 50 μg/ml ascorbic acid and 10 ng After culturing for 3 days in a medium containing /ml bFGF, the medium was replaced with a medium containing 10% FBS and 50 μg/ml ascorbic acid in DMEM-low glucose and cultured for 11 days. After washing with distilled water for decellularization of the cultured cells, they were treated with 0.1% SDS for 30 minutes at room temperature and then washed with distilled water for 24 hours. The decellularized sample was obtained using a scraper, cryo-milled, treated with 1M acetic acid at 4 degrees for 48 hours, and then lyophilized. After lyophilization, the dry weight was measured for water solubilization of the sample, and the final concentration was set to 3 mg/ml, followed by reaction in a 10 mM DTT and 5% SDS solution at 98 degrees for 2 hours. The sample was centrifuged at 2000 rpm for 5 minutes to obtain a supernatant, and then electrodialysis was performed at a voltage of 50 V using a dialysis tube having a molecular weight cutoff (MWCO) of 10 kDa for 3 days, followed by dialysis using distilled water for 3 days. day was carried out. Dialysis fluid was changed daily. The final sample after dialysis was lyophilized and used for further experiments.

The process is summarized in FIG. 1, and pictures of the results of each step from the decellularization process to the water solubilization process are shown in FIG.

<Experimental Example 1: Confirmation of extracellular matrix protein and decellularization through fluorescence staining>

In the above example, in the hADSC culture step for preparing extracellular matrix, hADSCs were cultured for 14 days, and then the samples before and after decellularization were treated with 4% paraformaldehyde (PFA) at room temperature for 20 minutes and fixed. The fixed samples were washed with PBS and antibodies to fibronectin (Fibronectin, ThermoFisher scientific), collagen type 1 (Collagen type 1, ThermoFisher scientific), elastin (abcam), and laminin (Laminin, abcam) at 4°C. After 16 hours of reaction, each component present in the sample was stained using Alexa488 and Alexa594 (ThermoFisher scientific) as secondary antibodies. Additionally, cell nuclei were stained using DAPI (4',6-diamidino-2-phenylindole, sigma). The staining results before decellularization are shown in FIG. 3 , and the staining results after decellularization are shown in FIG. 4 . No cell nuclei were identified in the sample after decellularization. Through this, it was confirmed that the decellularization was well performed.

<Experimental Example 2: Structural observation of extracellular matrix>

For structural observation of the extracellular matrix before and after decellularization, hADSCs were inoculated at 1 X 10 ⁴ cells/cm ² and cultured for 14 days. It was fixed with PFA and then dried. The dried sample was coated with platinum-palladium and observed using SEM. The imaging result of the sample before decellularization is shown in FIG. 5 , and the imaging result of the sample after decellularization is shown in FIG. 6 .

<Experimental Example 3: DNA Residual Amount Verification>

In order to confirm the decellularization, the residual amount of DNA in the sample before decellularization and after decellularization was analyzed. After each sample was lyophilized, DNA was extracted manually using a DNA extraction kit (PureLink genomic DNA mini kit, Invitrogen), and quantitative analysis was performed using molecular analysis equipment (spectramax M2, Molecular devices) capable of measuring DNA concentration. . The results are shown in FIG. 7 . No DNA was measured in the samples after decellularization. Through this, it was confirmed that decellularization was well performed.

<Experimental Example 4: Toxicity evaluation of SDS>

To evaluate the toxicity of SDS, experiments were performed on hADSC and human dermal fibroblast (HDF). HDF was also purchased and used commercially from ThermoFisher scientific. hADSCs were cultured in the same manner as in the above example, and HDFs were cultured using DMEM supplemented with FBS and antibiotics, and passage number 10 was used in the experiment.

hADSCs were treated with SDS concentrations of 0 μg/ml, 0.5 μg/ml, 2.5 μg/ml, 5 μg/ml, 10 μg/ml, 20 μg/ml, 50 μg/ml and 100 μg/ml for 24 hours. . Thereafter, WST-8 solution (dojindo, Tokyo, Japan; also known as cell counting kit-8, CCK8) was added to the cells, reacted for 1 hour, and absorbance was measured at a wavelength of 450 nm using a spectrophotometer. The results are shown in FIG. 8 .

HDFs were treated with SDS concentrations of 0 μg/ml, 0.5 μg/ml, 1 μg/ml, 3 μg/ml, 5 μg/ml, 10 μg/ml, 20 μg/ml and 100 μg/ml for 24 hours. . Thereafter, WST-8 solution was added to the cells, reacted for 1 hour, and absorbance was measured at a wavelength of 450 nm using a spectrophotometer. The results are shown in FIG. 9 .

Cytotoxicity was not confirmed in hADSC and HDF up to 20 μg/ml of SDS. Therefore, an experiment to confirm whether the residual SDS after purification was within 20 μg/ml, which is a non-cytotoxic concentration, was conducted in Experimental Example 5 below.

<Experimental Example 5: Analysis of residual SDS>

Since SDS treated in the decellularization and water-solubilization steps is cytotoxic, residual SDS was confirmed after purification. First, in order to create a standard curve sample for quantification, SDS was prepared at 0.15 μg/ml, 0.3 μg/ml, 0.6 μg/ml, 1.25 μg/ml, 2.5 μg/ml, 5 μg/ml, 10 μg/ml and 20 μg/ml. After dissolving ml in water, absorbance was measured at a wavelength of 650 nm using a spectrophotometer. The results are shown in FIG. 10 .

In order to check the residual SDS before and after dialysis, 10 μl of methylene blue 1% (w / v) was added to 900 μl of the sample before and after dialysis, followed by 400 μl of chloroform. Then, after vortexing for 30 seconds, centrifugation was performed at 14000 rpm for 10 minutes. After carefully obtaining the lower layer of the sample in which layer separation occurred, the absorbance was measured at a wavelength of 650 nm using a spectrophotometer to confirm the remaining amount of SDS. The results are shown in FIG. 11 .

The amount of residual SDS measured in the sample after dialysis was 0.52 μg/ml, which was not a concentration in the range showing cytotoxicity.

<Experimental Example 6: Evaluation of cytotoxicity of water-soluble extracellular matrix>

In order to confirm the cytotoxicity of the water-soluble extracellular matrix of Example, cytotoxicity was evaluated in HDF. HDFs were inoculated into a culture dish at 1 X 10 ⁴ cells/cm ² and cultured for 24 hours in serum-free DMEM medium. After washing the cultured cells with PBS, the extracellular matrix was treated with serum-free DMEM at various concentrations (0.5 mg/ml, 1 mg/ml, 3 mg/ml, and 5 mg/ml) for 24 hours. Thereafter, WST-8 solution (dojindo, Tokyo, Japan; also known as cell counting kit-8, CCK8) was added to the cells, reacted for 1 hour, and absorbance was measured at a wavelength of 450 nm using a spectrophotometer. The results are shown in FIG. 12 . It was confirmed that there was no cytotoxicity in all concentration ranges.

<Experimental Example 7: Evaluation of Cell Proliferation of Water-Soluble Extracellular Matrix>

In order to confirm the effect of the water-soluble extracellular matrix of Example on cell proliferation, cell proliferation was evaluated in HDF. HDF was inoculated into a culture dish at 1 X 10 ⁴ cells/cm ² , and extracellular matrix was added to serum-containing DMEM at various concentrations (0.5 mg/ml, 1 mg/ml, 3 mg/ml, 5 mg/ml). It was prepared, treated with cells, and cultured for 2 days. After adding the WST-8 solution to the cultured cells and reacting for 1 hour, the absorbance was measured at a wavelength of 450 nm using a spectrophotometer. The results are shown in FIG. 13 .

<Experimental Example 8: Protein analysis of water-soluble extracellular matrix>

For the protein analysis of the water-soluble extracellular matrix of Example, the dry weight of the lyophilized extracellular matrix was measured and dissolved in PBS at concentrations of 3 mg/ml, 1 mg/ml, and 0.5 mg/ml to prepare samples. The samples were quantitatively analyzed for protein through a DC protein assay (Bio-rad) kit. For the analysis method, reagents were prepared according to the method provided by the manufacturer, reacted with the sample, and measured at a wavelength of 750 nm. A standard curve was established by dissolving gelatin in PBS. The quantitative analysis results are shown in FIG. 14 . The measured protein concentration was similar to the concentration set by dry weight, confirming that the water-soluble extracellular matrix of Example was composed of protein.

In order to confirm the molecular weight of the protein, 5x sample buffer (5x lane marker reducing sample buffer, thermofisher) was added to the lyophilized extracellular matrix and boiled at 98 degrees for 5 minutes to prepare a sample. As a control, cell lysate obtained by washing the cell sheet with distilled water (DW) before decellularization, freeze-drying, and treating with RIPA buffer (Biosesang) was used. The prepared samples were loaded onto mini-protean TGX Gels (bio-rad), subjected to electrophoresis, and then stained with InstantBlue coomassie protein stain (Abcam) to determine molecular weight. The appearance of the protein was confirmed. The results are shown in FIG. 15 . As a result of the analysis, it was confirmed that the water-soluble extracellular matrix was mainly composed of proteins of a specific size.

In order to additionally analyze the protein components, LC/MS analysis was performed. After dissolving the lyophilized extracellular matrix in 2% SDS and 100 mM TEAB (triethylammonium bicarbonate), 200 μg of the sample was treated with 10 mM DTT and 25 mM IAA (iodacetamide), followed by an alkylation reaction. Thereafter, a sample was prepared by treating the sample with trypsin to convert it into a peptide, vacuum drying, and dissolving with 0.1% TFA.

The prepared samples were analyzed using a Thermo Dionex Ultimate 3000 UPLC system and a Thermo Q-Exactive HF-X MS. Proteins were identified from mass spectrometry spectral data using the human protein sequence (UP000005640) provided by the UniProt database and Proteome discoverer. Up to two missed cleavages were allowed, and cysteine alkylation (+57.0214 Da) and methionine oxidation (+15.9949 Da) were considered as peptide modifications.

As a result of LC/MS analysis, it was confirmed that 22 collagens, 30 glycoproteins, 30 proteoglycans, and 8 other extracellular matrix proteins were present. Specific components are listed in Table 1 below.

division protein gene Collagen Collagen alpha-3(VI) chain COL6A3 Collagen alpha-1(XII) chain COL12A1 Collagen alpha-1(VI) chain COL6A1 Collagen alpha-2(VI) chain COL6A2 Collagen alpha-2(I) chain COL1A2 Collagen alpha-1(I) chain COL1A1 Collagen alpha-1(XIV) chain COL14A1 Collagen alpha-1(V) chain COL5A1 Collagen alpha-6(VI) chain COL6A6 Collagen alpha-1(III) chain COL3A1 Collagen alpha-2(V) chain COL5A2 Collagen alpha-1(VIII) chain COL8A1 Collagen alpha-1(XVIII) chain COL18A1 Collagen alpha-1(XI) chain COL11A1 Collagen alpha-1(XVI) chain COL16A1 Collagen alpha-1(XV) chain COL15A1 Collagen alpha-3(V) chain COL5A3 Collagen alpha-1(X) chain COL10A1 Collagen alpha-2(VIII) chain COL8A2 Collagen alpha-1(XXI) chain COL21A1 Collagen alpha-1(IV) chain COL4A1 Collagen alpha-1(VII) chain COL7A1 glycoprotein Fibronectin FN1 Tenascin TNC Thrombospondin-1 THBS1 Fibrillin-1 FBN1 Thrombospondin-2 THBS2 Fibrillin-2 FBN2 SPARC SPARC Metalloproteinase inhibitor 1 TIMP1 Cartilage oligomeric matrix protein COMP Basigin BSG Endoglin ENG Chitinase-3-like protein 1 CHI3L1 Nidogen-2 NID2 Dermatopontin DPT Teneurin-3 TENM3 Zinc-alpha-2-glycoprotein AZGP1 EGF-containing fibulin-like extracellular matrix protein 1 EFEMP1 Trophoblast glycoprotein TPBG Lysosome-associated membrane glycoprotein 2 LAMP2 Tenascin-X TNXB Thy-1 membrane glycoprotein THY1 Vitronectin VTN Lysosome-associated membrane glycoprotein 1 LAMP1 Transmembrane glycoprotein NMB GPNMB Glycoprotein integral membrane protein 1 GINM1 Microfibril-associated glycoprotein 4 MFAP4 Nuclear pore glycoprotein p62 NUP62 Laminin subunit beta-1 LAMB1 Laminin subunit gamma-1 LAMC1 Laminin subunit beta-2 LAMB2 proteo
glycan Keratin, type II cytoskeletal 1 KRT1 Keratin, type I cytoskeletal 9 KRT9 Keratin, type I cytoskeletal 10 KRT10 Keratin, type II cytoskeletal 2 epidermal KRT2 Keratin, type II cytoskeletal 6B KRT6B Keratin, type II cytoskeletal 6C KRT6C Keratin, type II cytoskeletal 6A KRT6A Keratin, type I cytoskeletal 16 KRT16 Keratin, type II cytoskeletal 5 KRT5 Keratin, type I cytoskeletal 14 KRT14 Keratin, type II cytoskeletal 79 KRT79 Keratin, type II cytoskeletal 75 KRT75 Keratin, type II cytoskeletal 4 KRT4 Keratin, type II cytoskeletal 80 KRT80 Keratin, type II cytoskeletal 7 KRT7 Keratin, type II cytoskeletal 8 KRT8 Keratin, type II cytoskeletal 3 KRT3 Keratin, type II cytoskeletal 2 oral KRT76 Keratin, type I cytoskeletal 15 KRT15 Keratin, type I cytoskeletal 13 KRT13 Keratin, type I cytoskeletal 17 KRT17 Keratin, type I cytoskeletal 19 KRT19 Basement membrane-specific heparan sulfate proteoglycan core protein HSPG2 Biglycan BGN Decorin DCN Versican core protein VCAN Lumican LUM Glypican-4 GPC4 Testican-1 SPOCK1 Hyaluronan and proteoglycan link protein 1 HAPLN1 etc
extracellular matrix
protein Transforming growth factor-beta-induced protein ig-h3 TGFBI Periostin POSTN Fibulin-2 FBLN2 Vimentin VIM Fibulin-1 FBLN1 Extracellular matrix protein 1 ECM1 Fibulin-5 FBLN5 EGF-containing fibulin-like extracellular matrix protein 2 EFEMP2

The above description of the present invention is for illustrative purposes, and those skilled in the art can understand that it can be easily modified into other specific forms without changing the technical spirit or essential features of the present invention. will be. Therefore, the embodiments described above should be understood as illustrative in all respects and not limiting. For example, each component described as a single type may be implemented in a distributed manner, and similarly, components described as distributed may be implemented in a combined form.

The scope of the present invention is indicated by the following claims, and all changes or modifications derived from the meaning and scope of the claims and equivalent concepts should be interpreted as being included in the scope of the present invention.

Claims (14)

A cell culture step of culturing human-derived adipose stem cells to prepare a cell culture product;
A decellularization step of obtaining a decellularized extracellular matrix by treating the cell culture with a surfactant;
washing and grinding the decellularized extracellular matrix;
denaturing the pulverized extracellular matrix by treating it with an acid;
freeze-drying the denatured extracellular matrix;
A water-solubilization step of treating the lyophilized extracellular matrix with a surfactant to make it water-soluble; and
A purification step of electrodialysis of the solubilized extracellular matrix; including,
The cell culture is a cell sheet,
The surfactant in the decellularization step is SDS,
The decellularized extracellular matrix is an extracellular matrix sheet,
The grinding is freeze grinding,
the acid is acetic acid,
The surfactant in the water solubilization step is SDS,
The surfactant in the water solubilization step is treated like DTT,
The water solubilization step is carried out at a temperature of 95 ° C,
The electrodialysis method of producing a water-soluble extracellular matrix, characterized in that using a dialysis membrane having a cut-off molecular weight of 5 to 20 kDa.
delete delete delete delete delete delete delete delete delete A pharmaceutical composition for tissue regeneration comprising the water-soluble extracellular matrix prepared by the method of claim 1.
The pharmaceutical composition for tissue regeneration according to claim 11, wherein the pharmaceutical composition for tissue regeneration is an injection.
The pharmaceutical composition for tissue regeneration according to claim 11, wherein the pharmaceutical composition for tissue regeneration is used for the treatment of ischemic disease.
The pharmaceutical composition for tissue regeneration according to claim 13, wherein the ischemic disease is cardiac angina, myocardial infarction, cerebral infarction or foot ulcer.