KR102015368B1 - Method of decellularized scaffold for tissue regeneration using oil and composition of tissue decellularization - Google Patents

Abstract

The present invention relates to a method for preparing a decellular support for tissue regeneration using tissue and a tissue decellularization treatment composition, specifically, the present invention comprises the steps of (a) preparing a tissue for decellularization target; (b) The tissue to be decellularized is added to a fixed solution consisting of acrylamide, bis-acrylamide, azo-initiator, and Phosphate-buffered saline (PBS). Penetrating the fixative into the solution; (c) adding oil to the tissue in which the fixative has penetrated, and polymerizing the reaction at 37 ° C. to 45 ° C. for 3 to 4 hours in a vacuum state to prepare a tissue polymerized polymer; And (d) adding the tissue polymer prepared in step (c) to the surfactant solution and performing electrophoresis or physical agitation, and a tissue for decellularization of tissue. It relates to a decellularized treatment composition.

Description

Method of decellularized scaffold for tissue regeneration using oil and composition for tissue decellularization {Method of decellularized scaffold for tissue regeneration using oil and composition of tissue decellularization}

The present invention relates to a method for preparing a decellular support for tissue regeneration using oil and a tissue decellularized treatment composition.

The human body's ability to regenerate itself is very limited and its ability to regenerate itself is very limited because of its subtle tissue structure. The human body possesses the possibility of regeneration in case of tissue damage by the function of stem cells, but it is caused by the reason of accident, disease, aging, etc., which is beyond the limit of regeneration function. Recently, as the aging society enters an increasing number of diseases and accidents, organ tissues of living bodies face limitations of regeneration, and the necessity for regeneration of organ tissues is rapidly increasing. In addition, there is an increasing demand for the development of technologies that can effectively replace or transplant biological tissues.In the early days, attempts have been made to develop biocompatible materials and organ transplants from artificial substitutes. It is developing into biotissue engineering with integrated application.

Tissue engineering is the area of regenerative medicine, from cells to artificial organs, based on the study of biological and engineering technologies from biological materials to materials that can help restore tissues and organs. It is recognized as one of the important technologies in the field. Various methods have been researched to achieve the purpose of restoring, maintaining and improving the function of the human body by understanding the correlation between the structure and the function of living tissues and by making and transplanting the living substitutes. In particular, one of the core technologies of tissue engineering is to create a support or scaffold to support the growth of cells, and the support plays a very important role in biotissue engineering.

The support plays an important role in the growth of cells sown within the porous structure and cells migrated from around the tissue. Most cells in the body are adherent cells that grow by attaching. If there is no place to attach, the cells cannot grow and die. Thus, the support must provide a suitable environment for cell attachment, differentiation, growth and cell migration. Such supports may be made of various materials, and researches are being actively conducted to develop supports for tissue regeneration using natural materials, synthetic polymers, bio ceramics, and polymer-ceramic composites.

Meanwhile, among various tissues in the human body, brain tissue has a higher cell density than other tissues in the body, and the mechanical strength of the tissue is soft. Therefore, when the cells are removed by applying a general decellularization method for preparing a support, extracellular matrix Damage easily appears, and it is difficult to maintain the shape intact, and there is a problem of low reproducibility.

Therefore, in order to solve the problem by performing various experiments such as treatment material, treatment concentration, treatment temperature, treatment time, etc. in order to find a decellularization method suitable for brain tissue, efforts have been made. Development is inadequate.

Republic of Korea Patent Publication No. 10-2017-0078460

Therefore, the present inventors applied tissue clearing technology to prepare a decellularized support of a hydrogel-fixed tissue using oil in the process of manufacturing a decellularized support for tissue regeneration and differentiation, the brain tissue that could not be conventionally prepared It has been confirmed that the decellular support of soft tissues such as these can be successfully prepared, and in particular, proteins and extracellular matrix existing in tissues can be preserved in an intact form without damage. The present invention has been completed by confirming that shorter time can be used to prepare a more efficient decellular support.

Accordingly, an object of the present invention is to provide a method for preparing a decellular support for intact tissue regeneration without damaging the tissue to soft tissues such as brain tissue.

It is another object of the present invention to provide a tissue decell-treated composition for decellularization of soft tissues such as brain tissue.

In order to achieve the above object, the present invention comprises the steps of (a) preparing a tissue of a decellularized subject; (b) The tissue to be decellularized is added to a fixed solution consisting of acrylamide, bis-acrylamide, azo-initiator, and Phosphate-buffered saline (PBS). Penetrating the fixative into the solution; (c) adding oil to the tissue in which the fixative has penetrated, and polymerizing the reaction at 37 ° C. to 45 ° C. for 3 to 4 hours in a vacuum state to prepare a tissue polymerized polymer; And (d) adding the tissue polymer prepared in step (c) to a surfactant solution and performing electrophoresis or physical agitation, the method for preparing a decellular support for tissue regeneration.

In one embodiment of the present invention, the tissue may be brain tissue or pancreatic tissue.

In one embodiment of the present invention, the fixed solution is 8 to 12 wt% acrylamide, 0.1 to 0.2 wt% bis-acrylamide and 0.2 to 0.3 wt% azo-initiator ( azo-initiator).

In one embodiment of the present invention, the penetrating the fixed solution to the tissue in the step (b) may be performed by adding the tissue to the fixed solution and stirring for 10-14 hours at a temperature of 4 ~ 6 ℃.

In one embodiment of the present invention, the oil in step (c) may be corn oil, olive oil or mineral oil.

In one embodiment of the present invention, the oil in step (c) may be to use in an amount of 8 ~ 12ml relative to the tissue weight (g).

In one embodiment of the present invention, the surfactant solution in the step (d) may be 3 ~ 5wt% sodium dodecyl sulfate (SDS) solution.

In one embodiment of the present invention, the electrophoresis in the step (d) is to perform electrophoresis for 10-14 hours at 1.5 kPa, the physical stirring is for 2 to 3 days at 200 ~ 300rpm at 37 ℃ It may be to stir with a stirrer.

In addition, the present invention includes acrylamide, bis-acrylamide, azo-initiator, PBS (Phosphate-buffered saline) and oil, tissue for decellularization of the tissue Provide a decellularized treatment composition.

In one embodiment of the present invention, the acrylamide (acrylamide) is contained in an amount of 8 to 12 wt%, the bis-acrylamide is contained in an amount of 0.1 to 0.2wt%, azo- relative to the total weight of the composition The initiator (azo-initiator) may be contained in 0.2 to 0.3 wt%.

In one embodiment of the present invention, the type of oil is not limited to this, corn oil, olive oil or mineral oil may be used.

The present invention relates to a method for preparing a decellularized scaffold for tissue regeneration using tissue and a tissue decellularized treatment composition, wherein the gel is formed using oil in the process of preparing a decellularized scaffold for tissue regeneration and differentiation by applying tissue clearing technology. When a decellularized scaffold of immobilized tissue is prepared, it is possible to successfully prepare a decellularized scaffold of soft tissue such as brain tissue, which cannot be conventionally prepared, and to preserve proteins and extracellular matrix existing in the tissue without damage. And, it is possible to shorten the decellularization time has the effect of producing a more efficient decellular support. In addition, the decellular support of the tissue prepared by the method of the present invention can be used by lyophilization and pulverization, long-term preservation, and mixed in various forms can be easily used for cell culture, differentiation, tissue regeneration.

Figure 1 shows a schematic diagram of the manufacturing process of the brain tissue decellular support using the oil and the utilization of the decellular support in accordance with the present invention.
Figure 2 shows a photograph of a brain tissue polymer prepared using oil and acrylamide in the mouse brain tissue in one embodiment of the present invention.
Figure 3 shows the mouse brain tissue has completed the decellularization process in one embodiment of the present invention.
Figure 4 confirms the presence of residual DNA in the mouse brain tissue complete the decellularization process in one embodiment of the present invention.

The present invention relates to a method for producing a decellularized scaffold using soft tissues such as brain tissue, which could not be conventionally produced, and in particular, the present invention relates to a method for preparing a decellularized scaffold for tissue regeneration using oil.

Preferably the method for producing a decellular support for tissue regeneration according to the present invention, (a) preparing a tissue of decellularized target; (b) adding the decellularized tissue to a fixative solution consisting of acrylamide, bis-acrylamide, azo-initiator, and phosphate-buffered saline, and the tissue Penetrating the fixative into the solution; (c) adding the tissue infiltrated with the fixative to oil and polymerizing the polymer at a temperature of 37 ° C. to 45 ° C. for 3 to 4 hours in a vacuum state to prepare a tissue polymerized polymer; And (d) adding the tissue polymer prepared in step (c) to the surfactant solution and performing electrophoresis or physical stirring.

In particular, in the present invention, as mentioned in the prior art, it is to provide a method for producing a successful decellular support of a tissue that was not a general tissue, soft tissue properties and weak physical properties could not produce a decellular support.

Decellularization is involved in the preparation of tissue scaffolds for tissue regeneration, culture or differentiation. Ideal decellularization completely removes cellular components and maintains the physical properties of the tissue itself to minimize immune responses. It is a perfect preservation of extracellular matrix suitable for recellularization by avoiding damage to components such as proteins.

However, conventional preparation of decellularized scaffolds conventionally used uses surfactants, which dissolve cell membranes and remove cellular components other than proteins from tissues, but cause degeneration of remaining proteins in the scaffolds and It has the disadvantage of breaking down the microstructure and removing the growth factor excessively.

On the other hand, tissues with soft physical properties such as brain tissues had technical limitations in the manufacture of decellular scaffolds capable of maintaining the preservation of tissues due to destruction and damage of major tissue structures such as extracellular matrix.

In an effort to solve this problem, the present inventors have found that when the decellularized tissue scaffold is prepared using oil, all of these problems can be solved.

Referring to the manufacturing method of the decellular support for tissue regeneration using the oil provided in the present invention in detail.

First, prepare tissue for decellularization.

The subject for decellularization may use all of various tissues including soft and weak physical tissues. Specifically, the type of tissue for decellularization may be brain tissue or pancreatic tissue, but is not limited thereto. In one embodiment of the mouse-derived brain tissue was used.

The decellularized tissue may be one obtained from a dead body, a living individual (animal), or immediately after sacrifice.

When tissue preparation for decellularization is completed by the above method, the fixative solution is then infiltrated into the decellularized tissue.

A fixative is a solution that stops metabolism in cells or tissues, prevents rotting, and processes for immobilization of the form.

The fixing solution used in the present invention is composed of acrylamide, bis-acrylamide, azo-initiator, and PBS (Phosphate-buffered saline), and preferably, 12 wt% acrylamide, 0.1-0.2 wt% bis-acrylamide and 0.2-0.3 wt% azo-initiator.

As described above, the fixing solution used in the present invention contains acrylamide and bis-acrylamide, so that if the temperature is raised appropriately, the polymerization reaction can be started by covalent bonding to form a polymer. .

Penetrating the fixed solution to the decellularized tissue in the present invention to add the tissue to the fixed solution and stirred for 10 to 14 hours at a low temperature of 4 ~ 6 ℃ to allow the fixed solution to penetrate the tissue well by osmotic pressure.

In addition, the stirring is agitation while maintaining the speed of 40 ~ 70rpm, which is a problem that if the stirring is less than the speed is not well penetrated into the tissue, and if excessively stirred above the speed is damaged tissue It is desirable to stir at the rates described above, as this may cause problems.

Upon completion of the fixative permeation step into the tissue, tissue is then used to prepare tissue polymerisation.

In the preparation of the tissue polymer, the tissue penetrated with the fixative is added to the oil and polymerized at 37 ° C. to 45 ° C. for 3 to 4 hours in a vacuum to prepare a tissue polymer.

For the preparation of the tissue polymer, first, the tissue penetrated into the fixative solution is added to the oil, and the gel polymerization reaction is performed so that the fixer penetrated into the tissue collects more effectively in the tissue by using the property of collecting hydrophilic components in the oil. In addition to the form of the tissue and protein components of the extracellular matrix was to be preserved as it is.

The fixation solution containing acrylamide penetrated into the tissue should be polymerized on the spot without leaving the tissue as much as possible to preserve the composition of the tissue well. The cell support can be said to be a very important factor in the preparation.

However, when the preparation of the polymer is carried out in a hydrophilic environment, the fixative is moved out of the tissue again according to the osmotic phenomenon, and when the tissue is polymerized in the fixed solution of the same composition, not only the tissue but also the entire fixative may be formed into one large gel. Hassle to extract.

However, when the polymer preparation of the tissue penetrating the fixed solution as in the present invention is treated in oil, the above problems can be solved.

The oils that can be used in the present invention can be used as long as they are biohazardous oils, but are not limited thereto, but corn oil, olive oil or mineral oil can be used. In addition, the oil treatment for the tissue penetrated the fixed solution is treated in an amount of 8 ~ 12ml relative to the tissue weight (g) to position the tissue submerged in the oil.

After placing the tissue in the oil in this way in a vacuum to a temperature of 37 ℃ or more, preferably 37 to 45 ℃ polymerization for 3 to 4 hours to prepare a tissue polymer (polymerization).

The principle of formation of the tissue polymer is that the acrylamide monomer contained in the fixative liquid has a linear structure, and the bis-acrylamide monomer is briefly polymerized between the acrylamide linear structures to form a mesh form, and also a thermal initiator. ) Becomes a crosslinker when the temperature is above 37 ° C to form a gel polymer. The principle according to the polymer formation is shown in the following figure.

<Polymer Formation Principle>

When the preparation of the tissue polymer is completed, the tissue polymer is then added to the surfactant solution and decellularized by electrophoresis or physical stirring to prepare a decellular support for tissue regeneration according to the present invention.

Tissue polymers in which the gel is polymerized and the structure is effectively maintained through the method of the present invention can be decellularized by a tissue clearing technique of removing lipids using a surfactant. Although the decellularization rate is proportional to the concentration of the surfactant, the higher the concentration of the surfactant, the more components are lost, and the use of too low a surfactant has the disadvantage of insufficient decellularization efficiency. In consideration of this point, in the present invention, 3-5 wt% of sodium dodecyl sulfate (SDS) solution may be used for decellularization of soft tissues such as brain tissue.

Decellularization of the present invention was performed by applying the method of tissue clearing by electrophoresis or physical stirring, in tissue clearing by physically stirring in the electric field or using a sodium dodecyl sulfate (SDS) as a surfactant from the tissue-hydrogel complex Use a method to remove fat.

In one embodiment of the present invention used a 4% SDS solution, the concentration is higher than the 1% SDS used for general decellularization is effective in decellularization, when combined with electrophoresis in the electric field can be shortened time and high productivity There is an effect that can be derived.

In addition, since the tissue polymerized by the method of the present invention is more effectively fixed through acrylamide gel than the tissue without any treatment, it is possible to decellularize more quickly when the transparent technique is applied, so that it can be removed in a short time within 2 to 3 days. Cell supports can be prepared.

When electrophoresis is performed for the decellularization process, the electrophoresis may be performed at 1.5 kPa for 10 to 14 hours, and when performing the physical agitation for 2 to 3 days at 37 ° C. at 200 to 300 rpm. It can be carried out by stirring with a stirrer.

Furthermore, the present invention provides a tissue decellularization treatment composition for decellularization of tissues including acrylamide, bis-acrylamide, azo-initiator, and PBS (Phosphate-buffered saline). To provide.

In addition, the tissue decellularization composition for decellularization of the tissue may further comprise an oil, the use of such oil allows the composition to effectively penetrate into the tissue, and the composition penetrated into the tissue is maximally tissue The polymerization reaction proceeds in a state where the composition is maintained in an appropriate amount in the tissue so as not to come out. Therefore, the combination of oil and the composition of the present invention can produce a decellular scaffold without damaging the components of the tissue, it is possible to produce a decellular scaffold with only a small amount of gel components.

The composition contains 8 to 12 wt% of acrylamide, 0.1 to 0.2 wt% of bis-acrylamide, and azo-initiator based on the total weight of the composition. It is contained in 0.2 to 0.3 wt%.

In addition, the oil that can be used in the present invention is as described above.

The tissue decellularized treatment composition of the present invention can be applied to decellularized target tissues to which the fixative of the present invention is treated, and can be treated in a form in which the target tissue is precipitated in the composition.

The decellularized tissue scaffold prepared by the method of the present invention is more effective in preserving protein and extracellular matrix in tissues compared to the conventional method because the decellularized tissue scaffold is used to form polymers and perform decellularization using acrylamide gel and oil treatment. In addition, it can not only maintain the appearance form intact, but also effectively reduce the time required for decellularization.

In addition, in the case of the present invention, the use of oil has the advantage that can significantly reduce the amount of hydrogel used compared to the conventional method, and easy to handle as well as decellularized tissue by minimizing the volume by effective polymerization in the oil There is an advantage that can be analyzed further.

The decellularized tissue scaffold prepared by the method of the present invention can be widely used in various fields that require tissue regeneration or tissue differentiation.

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are only for illustrating the present invention in more detail, it will be apparent to those skilled in the art that the scope of the present invention is not limited to these examples.

<Preparation Example>

Fixative

The fixative solution to be used in the following examples is a fixed solution by mixing 40% acrylamide solution, 2% bis-acrylamide solution, 20x PBS, and 10% azo-initiator solution. The fixed solution used was diluted 10% acrylamide, 0.1% bis-acrylamide, 0.25% VA-044 (azo-initiator), 1X PBS.

For reference, the acrylamide monomer contained in the acrylamide fixative used in the present invention has a linear structure, and the bis-acrylamide monomer is briefly polymerized between the acrylamide linear structures to form a mesh. In addition, when the thermal initiator (VA-044) is a temperature of more than 37 ℃ acts as a crosslinker (crosslinker).

 The mesh size of the polymerized polymer varies depending on the acrylamide / bis-acrylamide ratio, which determines the degree of fixation of the tissue. In addition, other components, except for VA-044, can be pre-mixed and stored and used at 4 ° C. for about 6 months, and immediately mixed with VA-044 before use.

<Example 1>

Preparation of decellularized scaffolds using brain tissue

<1-1> Brain tissue preparation for decellularization

① Extraction of brain tissue from animal carcasses or live animals

For the extraction of brain tissue from mouse carcasses or live mice, the whole brain tissue is used, or it is cut to a size of 1 cm ³ (1 cm * 1 cm * 1 cm), and then a closed container of a conical tube The brain tissue is added together with the fixative solution described in Preparation Example, which is mixed in the above, and the mixture is shaken physically with a stirrer for 12 hours at 4 ° C. and 60 rpm to infiltrate the extracted brain tissue well through osmosis. To make it possible. At this time, if the stirring speed is excessive, the tissue may accelerate the degradation (degradation) was stirred carefully.

② Immediately sacrifice animals and extract brain tissue from animals

Infiltration of fixative fluid into tissues has a limitation of low embedding efficiency. Thus, if tissue can be obtained directly at the expense of animals, it is more effective through perfusion. The present inventors anesthetized the mouse and incision of the midline of the rib cage, and after aortic dissection on the right ventricle, 1X PBS was injected into the right ventricle to remove blood. Thereafter, the fixed solution of the preparation was perfused, and the target brain tissue was extracted. Since the fixative fluid can effectively penetrate the target brain tissue through the blood vessels of the mouse, the extracted brain tissue can be directly polymerized by applying heat.

However, the brain tissue extracted after perfusion is added to the sealed container of the conical tube together with the fixative solution, and the fixative solution is extracted through the osmotic phenomenon to the extracted brain tissue while stirring using a stirrer at 4 ° C. and 60 rpm for 10 hours. In addition, it was able to penetrate well.

<1-2> Polymer Preparation Using Oil

The brain tissues of the mouse prepared in the above <1-1> were added to 12-well plates, and 5 ml of corn oil was added to each well, and the brain tissues penetrated with the fixative were placed so as to be immersed in the oil and 30 KPa. The polymer was produced by subjecting to vacuum at 40 ° C. for 4 hours. A photograph of a polymer manufacturing process using oil is shown in FIG. 2. At this time, the brain tissue of the mouse is the average weight of the adult whole brain is about 0.4g, in this experiment used a brain tissue with an average weight of 0.2g cut to the size of 1cm ³ (1cm * 1cm * 1cm).

<1-3> Preparation of decellularized brain tissue scaffold through decellularization

In <1-2>, the gel was polymerized to maintain a well-structured tissue polymer using a surfactant, and a decellularized scaffold was prepared by electrophoresis or physical stirring. Specifically, the present inventors apply a clearing technique to the brain tissue polymer prepared in the above <1-2>, add 4% SDS solution, and then decellularized the brain through electrophoresis for 12 hours (1.5 kPa). Tissue scaffolds were prepared. In addition, as a physical stirring method, 4% SDS solution (50mL) was added to the brain tissue polymer prepared in the above <1-2>, and then physically agitated at a stirring speed of 75 rpm at 37 ° C. for 6 days to clear the decellularized brain. Tissue scaffolds were prepared. A photograph of the cleared decellularized brain tissue support prepared through decellularization is shown in FIG. 3.

<Example 2>

Detect Cells

The present inventors carried out experiments to confirm whether the decellularization was actually good for the decellularized support using the mouse brain tissue prepared by the method of Example 1 above. Decellularization analysis was confirmed by analysis of residual DNA in brain tissues. Since residual DNA in decellularized scaffolds is a direct cause of immunorejection during transplantation, nucleic acid residues were obtained by staining with 4, 6-diamidino-2-phenylindole (DAPI). Check for the presence of water. Specifically, the cleared decellularized brain tissue scaffolds were washed once every 5 hours / day at room temperature using 1 × PBS in a conical tube to remove residual SDS in the scaffolds. The decellularized brain tissue scaffold from which the SDS was removed was then submerged in 10 ml of a solution in which DAPI was mixed in 1 × PBS at a ratio of 1: 5000, and infiltrated by physical stirring at 37 ° C. for 12 hours at 75 rpm. Thereafter, 1X PBS was used to wash once per hour, 5 times per day at room temperature, to remove unbound DAPI, and the presence of nucleic acid in the support was confirmed by adjusting a Z value using a fluorescence microscope.

As a result, it was confirmed that there is little residual DNA in the brain tissue treated by the method of the present invention. Therefore, the present inventors were found to be almost completely decellularized when the method of the present invention is applied (see FIG. 4).

Comparative Example 1

The present inventors prepared decellularized brain tissue scaffolds in the same manner as in Example 1 except that the oil was not treated, and used as a control in Example 3 below.

<Example 3>

Check for decellular support

Brain tissue decellular support of the present invention using the brain tissue decellularized scaffold of the mouse prepared by the method of Comparative Example 1 (n = 6) and the oil prepared by the electrophoretic process for 16 hours in Example 1 (n = Morphology analysis for 6) was performed.

As a result, in the case of the control brain tissue decellular scaffold prepared by the method of Comparative Example 1, tissue damage, gel rupture occurred, soot occurs, such as intact brain tissue decellular scaffold was prepared In addition, the gel swells after decellularization occurs, which makes it difficult to extract proteins present in the tissues, and thus, it is difficult to analyze, whereas brain tissue decellularized by the method of the present invention. The support was found to be clean and intact in shape.

Thus, when the decellularized scaffold was prepared using oil, it was found that the decellularized scaffold, such as brain tissue, could be effectively prepared intactly and quickly and simply without damage to tissues having particularly soft characteristics.

So far I looked at the center of the preferred embodiment for the present invention. Those skilled in the art will appreciate that the present invention can be implemented in a modified form without departing from the essential features of the present invention. Therefore, the disclosed embodiments should be considered in descriptive sense only and not for purposes of limitation. The scope of the present invention is shown in the claims rather than the foregoing description, and all differences within the scope will be construed as being included in the present invention.

Claims (11)

(a) preparing a tissue of a decellularized subject;
(b) The tissue to be decellularized is added to a fixed solution consisting of acrylamide, bis-acrylamide, azo-initiator, and Phosphate-buffered saline (PBS). Penetrating the fixative into the solution;
(c) preparing a tissue polymerized polymer by placing the tissue penetrated into the fixed solution in oil and polymerizing the reaction at 37 ° C. to 45 ° C. for 3 to 4 hours in a vacuum state; And
(d) adding the tissue polymer prepared in step (c) to a surfactant solution and performing electrophoresis or physical stirring,
The fixative contains 8-12 wt% acrylamide, 0.1-0.2 wt% bis-acrylamide and 0.2-0.3 wt% azo-initiator. Characterized by
Method of producing a decellular support for tissue regeneration without tissue damage using oil. The method of claim 1,
Wherein said tissue is brain tissue or pancreatic tissue. delete The method of claim 1,
Penetrating the fixed solution to the tissue in the step (b) is characterized in that the addition of the tissue to the fixed solution and stirred for 10 to 14 hours at a temperature of 4 ~ 6 ℃. The method of claim 1,
The oil in step (c) is characterized in that the corn oil, olive oil or mineral oil. The method of claim 1,
In the step (c), the oil is used in an amount of 8 ~ 12ml per 1g of tissue weight. The method of claim 1,
In the step (d), the surfactant solution is characterized in that 3 ~ 5wt% SDS (sodium dodecyl sulfate) solution. The method of claim 1,
In the step (d) the electrophoresis is to perform electrophoresis for 10-14 hours at 1.5 kPa, the physical stirring is characterized in that the stirring with a stirrer for 2 to 3 days at 200 ~ 300rpm at 37 ℃ . delete delete delete

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