KR20210025460A - Extracelluar matrix derived from animal adipose and preservative solution of extracelluar matrix derived from animal adipose - Google Patents

Abstract

The present invention relates to an animal fat-derived extracellular matrix comprising: decellularized cells; and certain growth factors decellularized, thereby controlling the physiological activity of cells.

Description

Extracelluar matrix derived from animal adipose and preservative solution of extracelluar matrix derived from animal adipose}

The present invention relates to an animal fat-derived extracellular matrix and an animal fat-derived extracellular matrix preservation solution, and more particularly, to an animal fat-derived extracellular matrix having very excellent biocompatibility and physiological functions.

The extracelluar matrix is a tissue that is mainly responsible for structural support of animals, and plays a very important role as a structural role as a simple intercellular linker and as a regulatory factor for cell physiology such as division, differentiation, and death of cells. can do.

The extracellular matrix consists of connective tissue fibers, intangible components, and extracellular fluid.

Connective tissue fibers are formed in a triple helix structure, and collagen and elastin are the main components, providing strength and elasticity to the extracellular matrix.

The intangible component is a proteoglycan, in which glycosaminoglycans (GAGs) and a core protein are bound, as the main component, and because of the negative charge, it combines with a large amount of water to form a gel in the extracellular matrix.

These connective tissue fibers and intangible components help buffer the extracellular matrix.

The extracellular fluid contains ions, fat, glucose, and amino acids, so that cells survive and perform specific functions, and physiological factors such as protease and growth factors exist.

Growth factor is a generic term for a polypeptide that promotes various cell division or growth differentiation.

Several types have been discovered, functions are diverse, and they are known to be involved in the signaling system of cells.

In addition to nutrients that are the basic constituents of an organism, it is a material necessary for cell proliferation, proliferation and development of organisms, and is an essential element for cell growth and differentiation.

If it can be stored and properly supplied and provided with a physical environment that can be recognized by cells, the survival, growth, migration, and differentiation of cells can be greatly promoted.

In recent years, using the characteristics of the extracellular matrix, biological support materials derived from the extracellular matrix have been widely used in tissue engineering and regenerative medicine.

In particular, various biomaterials such as tissue repair agents and dressing bands using extracellular matrix have been introduced, but currently commercialized products use only some components of the extracellular matrix.

Various chemical and biological methods have been developed to extract the extracellular matrix, but the extracellular matrix containing the growth factor, which is the most important for the physiological activity of the cell in the process of extraction through animal adipose tissue, has not been disclosed. There is no bar.

Prior literature related to this includes a method for preparing a biocompatible porcine cartilage-derived extracellular matrix membrane with adjustable in vivo degradation rate and physical properties disclosed in Korean Patent No. 10-1772316 (announcement date: Aug. 29, 2017), and the extracellular matrix derived from porcine cartilage. There is an anti-adhesion composition containing as an active ingredient.

Korean Registered Patent Publication No. 10-1772316 (announced on August 29, 2017)

Accordingly, the present invention effectively extracts the extracellular matrix from animal fat, but increases biosafety and biocompatibility by decellularizing and degreasing, and containing a large amount of growth factors capable of regulating the physiological activity of cells, and preparation thereof. It is in providing a way.

The problem to be solved by the present invention is not limited to the problem(s) mentioned above, and another problem(s) not mentioned will be clearly understood by those skilled in the art from the following description.

In order to solve the above problems, according to an aspect of the present invention, the present invention provides an animal fat-derived extracellular matrix containing decellularized and degreased, and certain growth factors.

In addition, the decellularization and degreasing and the inclusion of growth factors may be performed through solvent extraction using a supercritical fluid.

In addition, solvent extraction using the supercritical fluid may increase biocompatibility by removing and sterilizing lipids remaining in the animal fat.

In addition, the animal fat may be lung adipose tissue extracted through liposuction.

In addition, the growth factors are AR, bFGF, b-NGF, EGF, EGFR, FGF-6, FGF-7, GCSF, GDNF, GM-CSF, HB-EGF, HGF, IGFB P-1, IGFB P-2, IGFB P-3, IGFB P-4, IGFB P-6, IGF-I, IGF-ISR, IGF-II, M-CSF, M-CSFR, NT-3, NT-4, PDGFRa, PDGFRb, PDGF-AA, PDGF-AB, PDGF-BB, PIGF, SCF, SCFR, TGF-a, TGF-b, TGF-b2, TGF-b3, VEGF, VEGFR2, VEGFR3 and VEGF-D.

In addition, the growth factor may be contained in 0.52 to 11.19 ng per 1 g of the extracellular matrix.

According to another aspect of the present invention, the present invention is decellularized and degreased through solvent extraction using a supercritical fluid, 0.1 to 5 parts by weight of an animal fat-derived extracellular matrix containing a certain growth factor; And it contains 95 to 99.9 parts by weight of the dispersion, it provides an animal fat-derived extracellular matrix preservation liquid, characterized in that the filtered.

In addition, the growth factors are AR, bFGF, b-NGF, EGF, EGFR, FGF-6, FGF-7, GCSF, GDNF, GM-CSF, HB-EGF, HGF, IGFB P-1, IGFB P-2, IGFB P-3, IGFB P-4, IGFB P-6, IGF-I, IGF-ISR, IGF-II, M-CSF, M-CSFR, NT-3, NT-4, PDGFRa, PDGFRb, PDGF-AA, Consisting of PDGF-AB, PDGF-BB, PIGF, SCF, SCFR, TGF-a, TGF-b, TGF-b2, TGF-b3, VEGF, VEGFR2, VEGFR3 and VEGF-D, filtered and per 1g of extracellular matrix It may contain 0.21 to 6.86 ng.

In addition, the growth factor may have a basic fibroblast growth factor (bFGF) content in the range of 40 to 50% through the filtration.

According to the present invention, biocompatibility is greatly increased by degreasing and decellularization, and an extracellular matrix capable of controlling the physiological activity of cells by containing growth factors and a method for producing the same is provided.

In addition, the extracellular matrix can be effectively obtained through a solvent extraction process using a supercritical fluid.

In addition, it is sterilized without acid, enzyme treatment, and radiation treatment, so that the biosafety of the extracellular matrix can be increased, and biocompatibility can be increased by decellularization and degreasing.

In addition, it provides an extracellular matrix in which growth factors are preserved among the components of the extracellular matrix, and it is possible to expect the regulation of cell physiological activity by adjusting the content of growth factors, and it is possible to quickly induce tissue regeneration.

In addition, since the structural characteristics of the extracellular matrix can be maintained as it is, a tissue structure mimetic that can be molded according to the shape of the implantation site can be easily manufactured.

In addition, it is possible to effectively extract and provide extracellular matrix by effectively treating human waste fat.

In addition, the preservation solution containing the extracellular matrix prevents contamination and can be stored for a long time, and the handling property is increased, so that the utility of the extracellular matrix can be greatly increased.

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 configuration of the invention described in the detailed description or claims of the present invention.

1 is a process flow diagram of a method for producing an animal fat-derived extracellular matrix according to an embodiment of the present invention.
2 is a process flow diagram of an animal fat-derived extracellular matrix preservation solution according to another embodiment of the present invention.
3 is an electrophoresis photograph according to the quantitative analysis of protein in an animal fat-derived extracellular matrix according to an embodiment of the present invention.
4 is a graph showing the amount of DNA remaining in the extracellular matrix derived from animal fat according to an embodiment of the present invention.
5 is a photograph of hematoxylin and eosin staining of an animal fat-derived extracellular matrix according to an embodiment of the present invention.
6 is a scanning electron micrograph of an animal fat-derived extracellular matrix according to an embodiment of the present invention.
7 is a photograph of an extracellular matrix sample, a 1 wt% dispersion, and an animal fat-derived extracellular matrix preservation solution prepared by filtration according to an embodiment of the present invention.
8 is a graph showing the result of an enzyme-linked immunosorbent assay based on VEGF of an animal fat-derived extracellular matrix according to an embodiment of the present invention.
9 is a graph showing the content of VEGF in a dispersion in which an animal fat-derived extracellular matrix is dispersed according to an embodiment.
10 is a graph showing DNA content before and after filtration of a dispersion in which an animal fat-derived extracellular matrix is dispersed according to an embodiment of the present invention.
11 is an image according to the results of the cytokine antibody array before and after filtration of an animal fat-derived extracellular matrix preservation solution according to an embodiment of the present invention.
12 shows the types and intensity of growth factors before and after filtration of an animal fat-derived extracellular matrix preservation solution according to an embodiment of the present invention.
13 shows the types and contents (ng/g) of growth factors before and after filtration of an animal fat-derived extracellular matrix preservation solution according to an embodiment of the present invention.

Hereinafter, preferred embodiments of the present invention will be described in detail with reference to the accompanying drawings.

Advantages and features of the present invention, and a method of achieving the same will become apparent with reference to the embodiments described below in detail together with the accompanying drawings.

However, the present invention is not limited by the embodiments disclosed below, but will be implemented in a variety of different forms, only these embodiments are intended to complete the disclosure of the present invention, and common knowledge in the technical field to which the present invention pertains. It is provided to completely inform the scope of the invention to those who have, and the invention is only defined by the scope of the claims.

In addition, in describing the present invention, when it is determined that related known technologies or the like may obscure the subject matter of the present invention, a detailed description thereof will be omitted.

The inventors of the present invention believe that conventional methods for extracting an extracellular matrix (ECM) from animal fat are mainly chemical treatment and biological treatment, as well as extracting and processing the extracellular matrix through radiation irradiation, thereby providing biosafety, biocompatible mechanical properties. However, the growth factor contained in the extracellular matrix has a problem that can be easily destroyed by chemical treatment, biological treatment, and radiation treatment, and the process uses a large amount of acids and enzymes, and radiation is used. Thus, it was confirmed that the risk of process control was large and the efficiency was greatly reduced, and thus it was not suitable for effectively obtaining an extracellular matrix.

On the other hand, solvent extraction using a supercritical fluid can be effectively extracted through a solvent in a supercritical state without physical change of the object to be extracted, thereby maintaining the mechanical performance properties of the object to be extracted. In particular, it is possible to maintain the mechanical performance properties of the object to be extracted under certain conditions. When fluid solvent extraction is applied, biostability and biocompatibility are greatly increased due to decellularization and degreasing, and the content of growth factor, which is a unique component of the extracellular matrix, is adjusted to create an extracellular matrix capable of controlling cell physiological activity. The present invention was completed by confirming that it can be obtained effectively.

Hereinafter, the present invention will be described in detail.

The animal fat-derived extracellular matrix according to the present invention is decellularized and degreased, and contains certain growth factors.

The animal fat may be pork fat, but it is preferable that it is lung fat extracted through liposuction.

Recently, a lot of liposuction for cosmetic plastic surgery has been performed, so it is possible to obtain a large amount of human-derived fat.However, in the case of extracting useful substances of stem cells and extracellular matrix in such liposuction, unnecessary components such as blood and lipids are contained. Since extracted human fat is difficult to utilize, it is possible to safely and effectively transplant the human body when decellularizing and degreasing through a solvent extraction process using a supercritical fluid to increase biostability and biocompatibility.

The animal fat is not limited as long as it contains extracellular matrix in the tissue.

The decellularization and degreasing may be performed through solvent extraction using a supercritical fluid.

The solvent extraction using the supercritical fluid can decellularize and degrease the animal adipose tissue by controlling the pressure, the flow rate introduced into the animal fat tissue to be extracted, the reaction process temperature and time during the production of the supercritical fluid solvent.

Solvent extraction using the supercritical fluid may increase biocompatibility by removing and sterilizing blood remaining in the animal fat.

Solvent extraction using the supercritical fluid increases the solubility of the supercritical fluid in animal fat by adding ethanol as a co-solvent, while effectively removing and sterilizing blood and contaminants remaining in animal fat.

Solvent extraction using the supercritical fluid extracts the extracellular matrix from animal fat, and may contain a certain growth factor in the extracellular matrix.

When the extracellular matrix contains growth factors, it is possible to control the physiological activity of cells, and when injected into human tissues in the form of tissue repair materials, it is possible to induce tissue regeneration quickly.

The growth factors include amphiregulin (AR), basic fibroblast growth factor (b-FGF), beta-nerve growth factor (b-NGF), and epithelial cell growth factor ( EGF, epidermal growth factor), epidermal growth factor receptor (EGFR), fibroblast growth factor-6 (FGF-6), fibroblast growth factor-6, FGF-7, fibroblast growth factor-7), granulocyte colony-stimulating factor (G-CSF), glial cell line-derived neurotrophic factor (GDNF), granulocyte phagocytic colony-stimulating factor (GM-CSF) , granulocyte-macrophage colony-stimulating factor), heparin-binding EGF-like growth factor (HB-EGF, heparin-binding EGF-like growth factor), hepatocyte growth factor (HGF), insulin-like growth factor-binding protein- 1 (IGFBP-1, insulin-like growth factor binding protein-1), insulin-like growth factor binding protein-2 (IGFBP-2, insulin-like growth factor binding protein-2), insulin-like growth factor binding protein-3 ( IGFBP-3, insulin-like growth factor binding protein-3), insulin-like growth factor binding protein-4 (IGFBP-4), insulin-like growth factor binding protein-6 (IGFBP- 6, insulin-like growth factor binding protein-6) , Insulin-like growth factor 1 (IGF-I), insulin-like growth factor insulin receptor (IGF-ISR), insulin-like growth factor-2 (IGF- II, insulin-like growth factor 2), macrophage colony-stimulating factor (M-CSF), macrophage colony-stimulating factor receptor (M-CSFR), neurotrophin- 3 (NT-3, neurotrophin-3), neurotrophin-4 (NT-4, neurotrophin-4), platelet-derived growth factor receptor-A (PDGFR A, platelet-derived growth factor receptor A), platelet-derived growth factor Receptor-B (PDGFRb, platelet-derived growth factor receptor B), platelet-derived growth factor-AA (PDGFAA, platelet-derived growth factor-AA), platelet-derived growth factor-AB (PDGF-AB, platelet-derived growth factor- AB), platelet-derived growth factor-BB (PDGF-BB, platelet-derived growth factor-BB), placental growth factor (PIGF), stem cell factor (SCF), stem cell growth factor Receptor (SCFR, stem cell factor receptor), transforming growth factor alpha (TGF-a), transforming growth factor beta 1 (TGF-b), transforming growth factor beta 1 (TGF-) b2, transforming growth factor beta 2), transforming growth factor beta 3 (TGF-b3, transforming growth factor beta 2) factor beta 3), vascular endothelial growth factor (VEGF), vascular endothelial growth factor-2 (VEGFR2, VEGF receptor-2), vascular endothelial growth factor-3 (VEGFR3, VEGF receptor-3), and It consists of vascular endothelial growth factor D (VEGF-D).

The extracellular matrix contains 40 kinds of growth factors excluding fibroblast growth factor-4 (FGF-4) among human growth factors through a solvent extraction process using a supercritical fluid.

The FGF-4 is removed in the process of decellularizing and degreasing animal fat by solvent extraction using a supercritical fluid, and the extracellular matrix according to an embodiment of the present invention is characterized in that the FGF-4 growth factor is excluded. do.

The growth factor may be contained in an amount of 0.52 to 11.19 ng per 1 g of the extracellular matrix.

The extracellular matrix obtained through the solvent extraction process using the supercritical fluid contains a certain amount or more of growth factor even after decellularization and degreasing, so that biocompatibility is greatly increased, as well as regulating the physiological activity of cells, and It can induce regeneration quickly.

When the animal fat is recovered through acid, enzyme treatment, and radiation treatment rather than solvent extraction using the supercritical fluid, it is difficult to recover all of the 40 kinds of growth factors. By decomposition, an extracellular matrix having an effective content within the above range cannot be obtained.

According to another aspect of the present invention, the present invention is decellularized and degreased through solvent extraction using a supercritical fluid, 0.1 to 5 parts by weight of an animal fat-derived extracellular matrix containing a certain growth factor; And it provides an animal fat-derived extracellular matrix preservation solution comprising 95 to 99.9 parts by weight of the dispersion, and filtered.

The extracellular matrix recovered by the solvent extraction method using a supercritical fluid can maintain mechanical properties, but it is not easy to manufacture various applications in a solid state.

When the extracellular matrix obtained through solvent extraction using the supercritical fluid is dispersed in a dispersion and filtered, biosafety and biocompatibility are increased due to the removal of high molecular weight proteins, and an extracellular matrix of 0.1 to 5 wt% or less is obtained. By dispersing in the dispersion, the handling property is increased and the usability is greatly increased.

When the extracellular matrix recovered by the solvent extraction method using a supercritical fluid is contained within the above range, it can be stored for a long time without changing the content of the growth factor in the extracellular matrix.

The growth factors are AR, bFGF, b-NGF, EGF, EGFR, FGF-6, FGF-7, GCSF, GDNF, GM-CSF, HB-EGF, HGF, IGFB P-1, IGFB P-2, IGFB P -3, IGFB P-4, IGFB P-6, IGF-I, IGF-ISR, IGF-II, M-CSF, M-CSFR, NT-3, NT-4, PDGFRa, PDGFRb, PDGF-AA, PDGF -AB, PDGF-BB, PIGF, SCF, SCFR, TGF-a, TGF-b, TGF-b2, TGF-b3, VEGF, VEGFR2, VEGFR3 and VEGF-D.

The animal fat-derived extracellular matrix preservation solution contains 40 kinds of growth factors excluding fibroblast growth factor-4 (FGF-4) among human growth factors.

The growth factor may be filtered and contained in an amount of 0.21 to 6.86 ng per 1 g of the extracellular matrix.

The animal fat-derived extracellular matrix preservation solution is filtered to adjust the content of growth factors.

The filtration may prevent contamination of the preservative by removing proteins in the extracellular matrix, and may contain growth factors within the above range.

The animal fat-derived extracellular matrix preservation solution has a reduced content of growth factors within the above range compared to the animal fat-derived extracellular matrix content, but can effectively preserve the extracellular matrix and greatly increases its utility.

The dispersion may be deionized water.

When the dispersion is selected as deionized water, contamination can be prevented when the extracellular matrix is dispersed, thereby increasing preservability.

The filtration may be filtered using a filter having a pore size of 0.2 to 0.45 µm.

By using a filter within the above range, the high molecular weight protein remaining in the extracellular matrix can be removed effectively to reduce the risk of contamination, the preservative can be sterilized, and the remaining DNA can be effectively removed.

If it is out of the above range, there is a concern that growth factors may be reduced in the preservation solution, or extracellular matrix other than proteins may be removed together, and there is a risk of infection and DNA remaining, thereby reducing biocompatibility.

The growth factor may have a basic fibroblast growth factor (bFGF) content in the range of 40 to 50% through the filtration.

The bFGF is included in or bound to a protein, and when the protein is removed by filtration in the preservation solution through the filtration, the content is reduced by filtering together.

According to another aspect of the present invention, the present invention provides a method for producing an extracellular matrix derived from animal fat.

1 is a process flow diagram of a method for producing an animal fat-derived extracellular matrix according to an embodiment of the present invention.

Referring to Figure 1, the method for producing an animal fat-derived extracellular matrix,

(1) washing and grinding animal fat to prepare adipose tissue;

(2) performing pretreatment by centrifuging the prepared adipose tissue to remove moisture and lipids;

(3) decellularizing and degreasing the adipose tissue pretreated by solvent extraction using a supercritical fluid to recover extracellular matrix; And

(4) identifying a growth factor in the extracellular matrix; includes.

First, the animal fat is washed and pulverized to prepare adipose tissue (S100).

The animal fat may be pork fat, but it is preferable that it is lung fat extracted through liposuction.

When the animal fat is waste fat derived from the human body, contaminants such as residual blood may be removed through washing, and the efficiency of centrifugation may be increased in a subsequent step in the case of pulverization.

The prepared adipose tissue is centrifuged and pretreated by removing moisture and lipids (S110).

Animal fat can be separated into water in the lower layer and lipid in the upper layer through the centrifugation. In the case of solvent extraction of the solid content in the middle layer by removing the water in the lower layer and the lipid in the upper layer, the solvent extraction process using a supercritical fluid The efficiency can be greatly increased.

The adipose tissue pretreated by solvent extraction using a supercritical fluid is decellularized and degreased to recover the extracellular matrix (S120).

Solvent extraction using the supercritical fluid may change the solvent extraction conditions in order to decellularize and degrease the adipose tissue.

In one embodiment of the present invention, the solvent extraction using the supercritical fluid is prepared as a supercritical fluid by pressurizing carbon dioxide at 200 to 600 bar, and the supercritical fluid at a flow rate of 18 to 70 mL/min, 30 to 35°C The extracellular matrix can be extracted by infiltrating the animal fat for 2 to 12 hours at.

The supercritical fluid produced by being pressurized within the above range can penetrate the cell wall of adipose tissue and dissolve the intracellular lipids to degrease, and when penetrating into the adipose tissue at a flow rate within the above range, while maintaining the mechanical properties of the extracellular matrix, Cells and can be degreased.

In the solvent extraction process using the supercritical fluid, contaminants remaining in the adipose tissue are removed once more, thereby increasing the contamination prevention and sterilization effect.

Growth factors are identified in the extracellular matrix (S130).

Decellularized and degreased extracellular matrix can be effectively obtained by the solvent extraction process using the supercritical fluid, and growth factors present in the extracellular fluid among the extracellular matrix can also be retained in a large amount.

Through the process of identifying the growth factors, an extracellular matrix containing a large amount of growth factors capable of controlling the physiological activity of cells and rapidly inducing tissue regeneration can be obtained.

Confirmation of the growth factor is the level of any one growth factor selected from the group consisting of VEGF, bFGF, IGF-1 and EGF among the growth factors by enzyme-linked immunosorbent assay to confirm the content of growth factors. I can.

The enzyme-linked immunosorbent assay is very preferable because it is possible to confirm the content of growth factors contained in the extracellular matrix based on any one growth factor selected from the group consisting of VEGF, bFGF, IGF-1, and EGF. As long as it is a method by which the content can be checked, it is not particularly limited.

Meanwhile, according to another aspect of the present invention, the present invention provides a method for preparing an animal fat-derived extracellular matrix preservation solution.

2 is a process flow diagram of an animal fat-derived extracellular matrix preservation solution according to another embodiment of the present invention.

Referring to Figure 2, the method for preparing an animal fat-derived extracellular matrix preservation solution

(a) washing and grinding animal fat to prepare adipose tissue;

(b) performing pretreatment by centrifuging the prepared adipose tissue to remove moisture and lipids;

(c) decellularizing and degreasing the adipose tissue pretreated by solvent extraction using a supercritical fluid to recover extracellular matrix;

(d) dispersing the recovered extracellular matrix in deionized water to prepare a dispersion;

(e) filtering the dispersion; And

(f) checking the content of the growth factor in the filtered dispersion.

First, the animal fat tissue is washed and pulverized to prepare adipose tissue (S200).

The prepared adipose tissue is centrifuged to remove moisture and lipids to pretreat (S210).

The adipose tissue pretreated by solvent extraction using a supercritical fluid is decellularized and degreased to recover the extracellular matrix (S220).

Since S200 to S220 are the same process as the method for producing an animal fat-derived extracellular matrix, a repeated description below will be omitted.

The recovered extracellular matrix is dispersed in deionized water to prepare a dispersion (S230).

When the dispersion is prepared by dispersing in the deionized water, subsequent filtration is easy, and the content of the growth factor can be easily checked to prepare an extracellular matrix preservation solution.

The dispersion can be dispersed at 15° C. at a pressure of 15,000 to 25,000 psi using a high pressure homogenizer.

When dispersed within the above range, the mechanical properties of the extracellular matrix can be maintained, and proteins remaining in the extracellular matrix can be eluted and dispersed into the dispersion.

In this case, dispersion may be repeated 1 to 5 times depending on the suspended state.

The dispersion is filtered (S240).

Since the dispersion is a suspension, there is a risk of contamination and infection because proteins of the extracellular matrix remain.

By removing the remaining protein through the filtration, contamination and infection of the preservative solution of the extracellular matrix can be prevented, thereby increasing biosafety, and handling and utility can also be increased.

The filtration may be performed by passing the dispersion through a syringe filter having a pore size of 0.2 to 0.45 µm.

By using the filter within the pore size, proteins remaining together with the extracellular matrix can be effectively removed, the preservation solution can be maintained in a sterile state, and remaining DNA can be effectively removed.

If it is out of the above range, the extracellular matrix content is reduced, so that growth factors are reduced or sterilization cannot be performed due to infection risk, and it is difficult to remove the remaining DNA.

Check the content of the growth factor in the filtered dispersion (S250).

Through the filtration, the growth factor may have a basic fibroblast growth factor (bFGF) content in the range of 40 to 50% through the filtration.

The bFGF is contained or bound to a protein, and when the protein is filtered and removed in the preservative through the filtration, the content is reduced by filtering together, so it can be confirmed that the protein in the preservative is removed by confirming the decrease in the content of bFGF. , An animal fat-derived extracellular matrix preservation solution with increased biosafety can be prepared.

Hereinafter, preferred examples are presented to aid in the understanding of the present invention, but the following examples are only illustrative of the present invention, and the scope of the present invention is not limited to the following examples.

Example 1. Supercritical fluid extraction extracellular matrix

In order to obtain extracellular matrix from animal fat through solvent extraction using supercritical fluid, the waste fat obtained during liposuction is treated to prevent contamination and transferred, and then the blood contained in the fat is completely washed away with the naked eye. Deionized water was sufficiently injected using a funnel, mixed, and allowed to stand for 20 minutes for washing. Washing was performed a total of 5 times.

1000 mL of washed fat is placed in a flask, dispersed at 50 rpm using a stirrer, and then passed through the ultrasonic treatment device at a constant flow rate using a pump (120 rpm pumping), so that ultrasonic waves can be uniformly transmitted to the entire fat. Made it possible.

The output of the ultrasonic wave was adjusted to 70% amplitude at 400 W, and the fat was crushed for 6 minutes to obtain adipose tissue.

The adipose tissue was maintained at 3° C. using a chiller during sonication.

The sonicated adipose tissue was transferred to a centrifuge, centrifuged at 4,000 rpm for 20 minutes, and the water in the lower layer and the lipid in the upper layer were separated and removed.

The solid content of the intermediate layer was recovered and placed in the reactor.

The reactor was provided with a carbon dioxide cylinder on one side, a cooler and a pump along a line through which carbon dioxide was discharged to pressurize carbon dioxide to prepare a solvent as a supercritical fluid.

One side of the carbon dioxide cylinder is provided with an ethanol tank as a common solvent, and a pump is provided in the line through which ethanol is discharged, and the ethanol is joined to the solvent, which is the supercritical fluid, to increase the solubility of the solvent, and to remove contaminants remaining in adipose tissue. It was sterilized and adjusted to exhibit a sterilization effect.

The solvent was extracted by introducing a solvent mixed with ethanol as a co-solvent and pressurized to 300 bar into the reactor at a flow rate of 20 mL/min at 32°C.

The extraction time was performed from 2 hours to 12 hours, and after the extraction was completed, the vent of the trap chamber provided in the reactor was opened to evaporate and remove the solvent to obtain an extracellular matrix as a white solid.

Experimental Example 1. Physical properties of extracellular matrix

Protein quantitative analysis (Bicinchonic Acid Assay; BCA) was performed to confirm the protein content in the extracellular matrix obtained by solvent extraction using a supercritical fluid.

According to the protein quantitative analysis method, the total amount of the injected protein was adjusted to 70 ug, and then electrophoresis was performed.

3 is an electrophoresis photograph according to the quantitative analysis of protein in an animal fat-derived extracellular matrix according to an embodiment of the present invention.

Referring to FIG. 3, from the left, the marker, the fat sample before extraction, and the two samples after extraction were maintained without any change in the band even after extraction.

When the same amount of protein was injected, the band was maintained, so it was confirmed that the protein was degreased after supercritical extraction, and the protein remained unchanged.

4 is a graph showing the amount of DNA remaining in the extracellular matrix derived from animal fat according to an embodiment of the present invention.

Extraction time DNA content (ng/mg) 2 hours 70.72211 6 hours 31.07875 12 hours 35.60731294

Table 1 shows the amount of DNA remaining in the extracellular matrix according to the solvent extraction time in the solvent extraction using the supercritical solvent of animal fat. 4 and Table 1, when supercritical extraction was performed for 2 hours, the DNA content was confirmed to be about 70 ng/mg, but when supercritical extraction was performed for 12 hours, the remaining amount of DNA was about 35 ng/mg. As shown, it was confirmed that the DNA content that can cause an immune response can be effectively reduced, and that the intracellular nucleus is destroyed and decellularized.

Meanwhile, hematoxylin and eosin staining was performed to confirm that the extracellular matrix obtained by solvent extraction using a supercritical fluid was decellularized.

5 is a photograph of hematoxylin and eosin staining of an animal fat-derived extracellular matrix according to an embodiment of the present invention.

5, in order to compare the effect of solvent extraction using a supercritical fluid, a stained picture of adipose tissue before supercritical extraction is shown on the left, and a stained picture of the extracellular matrix obtained after solvent extraction using a supercritical fluid is shown on the right. Indicated.

After solvent extraction using a supercritical fluid, the cell nucleus could not be observed in the extracellular matrix, and it was confirmed that the cell did not maintain its shape, thereby confirming that the cell was decellularized.

6 is a scanning electron micrograph of an animal fat-derived extracellular matrix according to an embodiment of the present invention.

Referring to FIG. 6, in order to confirm the change of cells in the solvent extraction process using a supercritical fluid, a cell picture of the adipose tissue before extraction is shown on the left on the left, and a scanning electron microscope picture of the tissue after extraction is shown on the right. Shown in.

After solvent extraction using a supercritical fluid, all cells were removed, and the fibrous shape was confirmed, confirming that the extracellular matrix was decellularized.

Example 2. Preparation of animal fat-derived extracellular matrix preservation solution

The extracellular matrix sample obtained in Example 1 was added to deionized water in an amount of 1 wt%, mixed, and dispersed at 15° C. at a pressure of 20,000 psi using a high-pressure homogenizer.

Dispersion was repeated 5 times to prepare a dispersion. The prepared dispersion was filtered using a syringe filter having a pore size of 0.2 μm to prepare a transparent animal fat-derived extracellular matrix preservation solution.

7 is a photograph of an extracellular matrix sample, a 1 wt% dispersion, and an animal fat-derived extracellular matrix preservation solution prepared by filtration according to an embodiment of the present invention.

Referring to FIG. 7, it was confirmed that an animal fat-derived extracellular matrix preservation solution in a transparent liquid state could be prepared by dispersing an extracellular matrix sample.

Experimental Example 2. Growth factor confirmation

Enzyme-linked immunosorbent assay (ELISA) was performed to confirm whether the extracellular matrix obtained according to Example 1 contained growth factors.

A 1 wt% dispersion prepared according to Example 2 was prepared, and a washing solution was added to each of the plate wells to be used for washing. The standard solution (Standard) and the sample (Sample) were put into each well and reacted at room temperature for 2 hours.

The unreacted standard solution and sample were washed with a washing solution.

Each well was injected with a detection antibody, incubated at room temperature for 2 hours, and washed.

Streptavidin-HRP was injected, incubated for 30 minutes at room temperature, and then washed again.

After injecting the TMB Solution, it was incubated until color development, and absorbance was measured at 450 nm wavelength.

8 is a graph showing the result of an enzyme-linked immunosorbent assay based on VEGF of an animal fat-derived extracellular matrix according to an embodiment of the present invention.

9 is a graph showing the content of VEGF in a dispersion in which an animal fat-derived extracellular matrix is dispersed according to an embodiment.

8 and 9, it was confirmed that the concentration of VEGF in the 1 wt% dispersion was 40.34 pg/ml, and the VEGF content in the extracellular matrix sample according to Example 1 was 4.03 ng/g. It was confirmed that the growth factor was present in the extracellular matrix sample obtained by solvent extraction using the critical fluid.

Experimental Example 3. Change of DNA content according to filtration

The DNA content of the preservative according to Example 2 was analyzed before and after filtration.

10 is a graph showing the DNA content before and after filtration of a dispersion in which an animal fat-derived extracellular matrix is dispersed according to an embodiment of the present invention.

Referring to Figure 10, it is reduced from 5.57 ng/μl before filtration to 0.13 ng/μl, and it is possible to further reduce residual DNA when the extracellular matrix obtained by the solvent extraction method using a supercritical fluid is dispersed and filtered. Confirmed.

Experimental Example 4. Confirmation of the type and content of growth factors

In order to confirm that the extracellular matrix according to Example 1 contains a large amount of various growth factors, analysis was performed using a Human Growth Factor Antibody Array membrane and Abcam kit.

The sample was processed according to the protocol of the kit, and diluted to 1/10 in order to control the concentration of protein present in the sample.

Meanwhile, in order to confirm the type and content of growth tensile contained in the animal fat-derived extracellular matrix preservation solution according to Example 2, it was analyzed with a cytokine antibody array.

A 1 wt% dispersion (Control) and a filter solution were prepared by diluting 1/10, and the membrane (Membrane) was washed with a washing buffer and then blocked at room temperature for 30 minutes.

The diluted analysis target solution was applied to a membrane, and after incubation at 4° C. for 24 hours, the analysis target solution was removed with an aspirator.

The remaining analysis target solution was washed with a buffer solution, biotin-conjugated anti-cytokine was injected at room temperature, and after incubation for 2 hours, the liquid was removed and the residue was washed.

HRP-Conjugated Streptavidin was injected at room temperature, and after incubation for 2 hours, the liquid was removed and the residue was washed.

Within 5 minutes after the detection buffer was applied, a photograph was taken with a chemiluminescence detection camera and analyzed using the ImageJ Program.

11 is an image according to the results of the cytokine antibody array before and after filtration of an animal fat-derived extracellular matrix preservation solution according to an embodiment of the present invention.

Using the image J program, the intensity of the black point in FIG. 10 was measured.

The intensity of the signal for the antibody in the image measured through the cytokine array was measured to determine the relative value of how much the corresponding substance was contained in the sample.

Based on the relative value of VEGF, which is a positive control, the relative value of the remaining growth factors detected was converted and calculated.

12 shows the types and intensity of growth factors before and after filtration of an animal fat-derived extracellular matrix preservation solution according to an embodiment of the present invention.

11 and 12, a total of 40 growth factors can be identified in the extracellular matrix obtained through solvent extraction using a supercritical fluid, and the growth factor content decreases after filtration of the preservative containing the extracellular matrix. It was confirmed.

13 shows the types and contents (ng/g) of growth factors before and after filtration of an animal fat-derived extracellular matrix preservation solution according to an embodiment of the present invention.

13, it was confirmed that the extracellular matrix according to Example 1 contains a total of 40 growth factors out of 41 human growth factors, and FGF-4 is removed in the solvent extraction process using a supercritical fluid. .

It was confirmed that the decellularization and degreasing effect of the solvent extraction process using the supercritical fluid had an effect on the content of the growth factor, and the cell whose content and type were adjusted according to the extraction conditions of the solvent extraction process using the supercritical fluid. It was confirmed that external substrates could be obtained.

On the other hand, it was confirmed that the animal fat-derived extracellular matrix preservative solution prepared according to Example 2 was 0.21 to 6.86 ng per 1 g of extracellular matrix, and it was confirmed that the growth factor was decreased by filtration, and the content of the growth factor in the preservative solution could be controlled .

In addition, it was confirmed that the content of bFGF (Basic fibroblast growth factor) was reduced in the range of 40 to 50% through the filtration process among growth factors. Since it is removed through filtration, it was confirmed that the protein in the preservative can be removed.

This indicates that the extracellular matrix in the preservative solution is removed to reduce the risk of infection and contamination to increase biosafety, can be sterilized in the filtration process, and the remaining DNA can be removed. It was confirmed that the handling and storage properties of the preservation solution can be increased.

The inventors of the present invention note that a large amount of human fat is discarded according to the recently increased liposuction, and while seeking a way to utilize it, the extraction conditions were adjusted by using a solvent extraction method using a supercritical fluid in adipose tissue. In this case, it was confirmed that the extracellular matrix with increased biosafety and biocompatibility can be obtained by decellularizing and degreasing while maintaining the mechanical properties of the extracellular matrix.

In particular, in the solvent extraction using a supercritical fluid, it was confirmed that a large amount of growth factors remained in the extracellular matrix, thereby controlling the physiological activity of cells and inducing rapid tissue regeneration. By confirming the feature that the growth factor is excluded, it was confirmed that the extracellular matrix and the extracellular matrix different in the composition of the growth factor can be effectively extracted through conventional chemical and biological treatment.

Until now, specific examples of the animal fat-derived extracellular matrix and the animal fat-derived extracellular matrix preservation solution according to the present invention have been described, but it is obvious that various implementation modifications are possible within the limit not departing from the scope of the present invention.

Therefore, the scope of the present invention should not be defined by being limited to the described embodiments, and should be defined by the claims and equivalents as well as the claims to be described later.

That is, it should be understood that the above-described embodiments are illustrative in all respects and not limiting, and the scope of the present invention is indicated by the claims to be described later rather than the detailed description, and the meaning and scope of the claims and All changes or modified forms derived from the equivalent concept should be construed as being included in the scope of the present invention.

Claims (4)

Decellularized and defatted, as an animal fat-derived extracellular matrix containing growth factors;
The decellularization and degreasing and the inclusion of growth factors are performed through solvent extraction using a supercritical fluid;
Solvent extraction using the supercritical fluid is prepared as a supercritical fluid by pressurizing carbon dioxide at 200 to 600 bar;
Animal fat-derived extracellular matrix, characterized in that the supercritical fluid is treated for 2 to 12 hours at a flow rate of 18 to 70 mL/min to extract the extracellular matrix.
The method of claim 1,
The animal fat is
Animal fat-derived extracellular matrix, characterized in that the lung fat extracted through liposuction.
The method of claim 1,
The growth factor is
AR, bFGF, b-NGF, EGF, EGFR, FGF-6, FGF-7, GCSF, GDNF, GM-CSF, HB-EGF, HGF, IGFB P-1, IGFB P-2, IGFB P-3, IGFB P-4, IGFB P-6, IGF-I, IGF-ISR, IGF-II, M-CSF, M-CSFR, NT-3, NT-4, PDGFRa, PDGFRb, PDGF-AA, PDGF-AB, PDGF -BB, PIGF, SCF, SCFR, TGF-a, TGF-b, TGF-b2, TGF-b3, VEGF, VEGFR2, VEGFR3 and VEGF-D, characterized in that consisting of animal fat-derived extracellular matrix.
The method of claim 3,
The growth factor is
Animal fat-derived extracellular matrix, characterized in that contained in 0.52 to 11.19 ng per 1 g of the extracellular matrix.

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