KR101860798B1

KR101860798B1 - An method for decellularizing a brain tissue and an method for culturing a brain tissue

Info

Publication number: KR101860798B1
Application number: KR1020150188998A
Authority: KR
Inventors: 이희경; 조동우
Original assignee: 포항공과대학교 산학협력단; 주식회사 티앤알바이오팹
Priority date: 2015-12-29
Filing date: 2015-12-29
Publication date: 2018-05-25
Also published as: KR20170078460A

Abstract

The present invention relates to a method of decellularizing brain tissue and a method of culturing brain tissue, and more particularly, to a method of decellularizing brain tissue, A step of removing the cells of the first cell, a step of removing the DNA of the tissue treated in the step of removing the first cell using a solution containing the DNA-degrading enzyme, and a step of using a solution containing the second surfactant And a second cell removing step of removing the cells of the tissue treated in the DNA degrading step at a temperature not higher than room temperature, and a method of culturing brain tissue using the same.

Description

[0001] The present invention relates to a method of decellularizing brain tissue and a method of culturing brain tissue,

TECHNICAL FIELD The present invention relates to a decellularization method and a culture method of biological tissue, and more particularly, to a decellularization method and a culture method of brain tissue.

The method of attaching cells derived from body tissues to a culture dish and culturing them in a two-dimensional form of a monolayer is a conventionally widely used in vitro culture method. However, the two - dimensional culture has a disadvantage that it can not reproduce the structural and physico - mechanical properties of the three - dimensional arrangement of the body tissue. Therefore, in order to overcome the difference between the conventional in vitro culture environment and the in-vivo environment, a three-dimensional culture method such as a method of forming a cell sphere and culturing it, a method of encapsulating a cell in a hydrogel such as collagen And these techniques are described in Patent Publication No. 2004-0016984.

The three - dimensional cell culture method showed similar expression to the body tissue as compared with the conventional two - dimensional culture method in terms of cell perception and reaction, cell migration, cell growth and differentiation. In particular, the in vitro culture model of malignant tumor using 3 - D culture method can be used for predicting the response of tumor tissue in the body because it has higher drug resistance than 2 - dimensional culture method.

However, the conventional 3-D tumor cell culture method has the disadvantage that it can not reproduce the characteristics such as the specific structure of the tumor-derived tissue, the mechanical properties, the biochemical constituents, the interaction between the various cells constituting the tumor tissue have.

Patent Publication No. 2004-0016984

DISCLOSURE OF THE INVENTION The present invention has been made to solve the above problems, and it is an object of the present invention to provide a culture method and a decellularization method for culturing tumor tissue which can reproduce the characteristics of original tissue.

In particular, to provide a decellularization method capable of maximizing cell removal and minimizing damage to extracellular matrix in decellularizing brain tissue with high cell density during brain tumor tissue culture.

The present invention provides a method for culturing brain tissue using brain-derived extracellular matrix obtained through decellularization of brain tissue so as to achieve the above objects.

Here, the decellularizing method of brain tissue includes a step of providing a brain tissue to be de-fatigued, a first cell removing step of removing cells of the tissue at a temperature below room temperature using a solution containing the first surfactant , A DNA degradation step of decomposing DNA of the tissue treated in the first cell removal step using a solution containing the DNA degrading enzyme and a step of performing the DNA decomposition step at a temperature below room temperature using a solution containing the second surfactant And removing the cells of the treated tissue from the second cell.

Here, the first cell removing step and the second cell removing step are performed at a temperature of 10 degrees Celsius or less, and more particularly, the first cell removing step and the second cell removing step are performed at a temperature of not less than 2 degrees Celsius Lt; / RTI > or less.

The first surfactant may be an anionic surfactant, and the second surfactant may be a nonionic surfactant.

Specifically, the first cell removing step may use sodium dedecylsulfate as a first surfactant and may be performed at a temperature of 2 to 7 degrees Celsius for 18 hours or more. The second cell removal step may be performed using Triton X-100 (triton x-100) as the second surfactant for a time of 18 hours or more at a temperature of 2 to 7 degrees Celsius.

At this time, the first cell removing step and the second cell removing step may be performed while rotating at a rotating speed of 10 to 20 rpm.

Meanwhile, in the step of providing the brain tissue, the brain tissue extracted from the pig may be separated and frozen for more than 10 hours, and then the frozen brain tissue may be incised and broken down.

The tissue washing step is performed before and after each step, and the tissue washing step can wash the tissue using a solution in which antibiotics are dissolved in a solvent of double distilled water.

Further, after the step of removing the second cells, the method may further include a step of sterilizing the tissue from which cells have been removed using a solution containing acetic acid and ethanol.

According to the present invention, it is possible to obtain an extracellular matrix with minimal cell damage while depleting brain tissue with a large cell retention amount, and to reproduce characteristics similar to the original tissue by culturing brain tissue such as brain tumor Do.

In particular, when the brain tumor tissue according to the present invention is cultured, it is possible to similarly reproduce the main features of brain tumor such as malignant trait expression characteristics and peripheral tissue penetration characteristics, so that it can be effectively applied to the development of new drugs and patient- There are advantages to be able to use.

FIG. 1 is a flowchart showing a procedure of a brain tissue decoltaging method according to an embodiment of the present invention;
FIG. 2 is a graph showing changes in the components of the brain tissue treated by the decellularization method according to FIG. 1,
FIG. 3 is a flow chart showing a procedure for culturing brain tissue using an extracellular matrix,
Figure 4 schematically shows the main steps of Figure 3,
FIG. 5 is a graph showing the degree of malignant expression between two control groups,
Fig. 6 is a photograph showing the penetration pattern of the glioblastoma to be cultured.

Hereinafter, a method of decoltizing brain tissue and a method of culturing brain tissue according to an embodiment of the present invention will be described in detail with reference to the drawings. In the following description, the positional relationship of each component is principally described based on the drawings. The drawings may be simplified for simplicity of the description or exaggerated when necessary. Therefore, the present invention is not limited thereto, and it is needless to say that various devices may be added, changed or omitted.

Meanwhile, in this embodiment, the method of decellularizing brain tissue and culturing brain tumor tissue using the same will be described. However, it should be understood that the present invention is not limited thereto, but can be applied to decellularization of various biological tissues other than brain tissue and culturing of tissues.

In this embodiment, a brain-derived extracellular matrix derived from brain tissue is used for culturing brain tumor tissue. In this case, there is an advantage that the characteristics of the brain tumor tissue can be reproduced more similarly as compared with culturing the tissue using only the hydrogel such as the conventional collagen.

However, since brain tissue has higher cell density and mechanical strength of tissues than other body tissues, extracellular matrix is likely to be damaged when cells are removed using a general decellularization method. Thus, in order to find a decellularization method suitable for brain tissue, experiments were conducted by changing various parameters such as treatment substance, treatment concentration, treatment temperature and treatment time, and found a combination in which the effect was remarkably improved. Hereinafter, Desc / Clms Page number 2 > cell disintegration method capable of removing cells while minimizing damage to the cells.

1 is a flowchart illustrating a procedure of a brain tissue decoltaging method according to an embodiment of the present invention. As shown in FIG. 1, the decolletage method according to the present embodiment includes a tissue preparation step, a primary cell removal step, a DNA degradation step, a secondary cell removal step, and a disinfection step. .

First, the step of preparing the brain tissue (S10) proceeds in such a manner that tissues to be decellularized from the actual brain tissue are obtained, the tissue is frozen at a low temperature, and the tissue is subdivided into small sizes.

Specifically, brain tissue for decellularization is obtained from the brain tissue of the pig. Utilizing wild type pig brain tissue that is easy to supply and receive, tissues are obtained by incising half of the brain tissue from the pig skull. This step can be performed in a low-temperature environment of 10 degrees Celsius or less.

The obtained brain tissue is frozen for at least 12 hours at a temperature of minus 80 degrees Celsius. Then, the frozen brain tissue is subdivided into a size small enough to perform the decellularization process. In this embodiment, the frozen brain tissue is cut at a size of about 1 mm to 10 mm, for example, at room temperature.

When the tissue is prepared through the above steps, a step of washing the tissue is performed (S20). The washing step uses double distilled water as wash water. Here, the secondary distilled water has a lower osmotic pressure than the cytoplasm, which can lead to hemolysis of the cells. Since the brain tissue of the pig is used as a raw material, the washing water may contain an antibiotic for sterilization and disinfection. For example, penicillin / streptomycin, which is an antibiotic in this embodiment, may be contained at a concentration of 1% . Such a washing step can be carried out within about one hour.

When the prepared brain tissue is cleaned, steps such as a primary cell removal step (S30), a DNA degradation step (S50), and a secondary cell removal step (S60) are performed.

Among them, the first and second cell removal steps are performed using a cell removal solution containing a surfactant as a detergent. Since the surfactant has amphiphilic properties, it can dissolve cell membranes and nuclear membranes of tissues and remove cells.

However, since brain tissue has a higher cell density than other tissues, it is difficult to sufficiently remove cells by using a surfactant or the like in a general manner. On the other hand, if the concentration of the surfactant is increased so as to sufficiently remove the cells, the extracellular matrix is damaged and the original characteristics can not be reproduced.

Therefore, in this embodiment, the cell removal step is performed at room temperature, but the cell removal step is performed in a low temperature environment. In addition, a lower concentration of the surfactant solution can be used than in the conventional decolorization process. Thus, the present step can be carried out in such a manner that the temperature and the concentration are combined to maintain the environment in which the functionality of the surfactant in the solution is limited, for a sufficient time. In the case where the surfactant is actively active as in the past, the outer portion of the extracellular matrix is continuously damaged in the inner portion of the tissue mass in a state where the cells are not sufficiently removed, It is possible to minimize the damage of the extracellular matrix of the outer portion while the cells of the inner portion are sufficiently removed.

First, the first cell removing step of FIG. 1 will be described. In the first cell removing step, the brain tissue washed in the washing step (S20) described above is put into the first cell removing solution containing the first surfactant to remove the cell tissue.

As described above, the first cell removing step can be performed in a low-temperature environment below room temperature. As a result of performing at various temperatures, the cell removal step of brain tissue showed good results in a low temperature environment of 10 ° C. or less, and in particular, the cell removal step was performed at a temperature of 2 ° C. to 7 ° C. When the brain tumor tissue was cultured using the obtained extracellular matrix, the reproducibility of the cultured tumor tissue was better.

The first surfactant may be an anionic surfactant. Ionic surfactants have a functional group having a cationic or anionic group in the hydrophilic group, and thus can have a stronger surfactant action than the nonionic surfactant. In this embodiment, sodium dodecylsulfate is used as a first surfactant, and the first cell removing solution used in this step may be a solution containing sodium dodecyl sulfate as a secondary distilled water solvent. At this time, sodium dodecyl sulfate may be a solution containing a low concentration of 1% or less, more specifically, a solution having a concentration of about 0.5% or less. Furthermore, the first cell removal solution can be used further comprising 1% penicillin / streptomycin for sterilization.

In this step, the brain tissue is put into the first cell removing solution, and the process is performed while stirring at 30 rpm using a device such as a stirrer. This process may be carried out for at least 18 hours or more, and more particularly for a period of from 20 hours to 30 hours, so that sufficient cell clearance is achieved in a low temperature / low concentration environment where the functionality of the first surfactant is limited .

When the tissue is treated through the first cell removing step, a step of washing the treated tissue is performed (S40). This step uses a secondary distilled water solution containing penicillin / streptomycin of 1% penicillin and streptomycin. After the treated tissue is put in the solution, washing is carried out while stirring with a stirrer. The temperature environment and the stirring environment in which the present cleaning step proceeds are performed so as to be stirred at a rate of 30 rpm or less at a temperature of 2 to 7 degrees centigrade so as to correspond to the first cell removal step. This step can be carried out for a period of not more than 30 minutes. This washing step can be carried out twice under the same conditions.

When the washing step S40 is completed, a step of decomposing the DNA of the washed tissue is performed (S50). In this step, the solution containing the DNA-degrading enzyme is used to remove the intracellular DNA which can act as an antigen that induces an immune response. Specifically, the DNA degradation solution can be a solution containing 20 U / ml DNase and 1 M sodium chloride (NaCl) in the secondary distilled water. Further, penicillin / streptomycin may be further added to 1% penicillin for sterilization.

In this step, the tissue washed in the washing step (S40) is put into the DNA degradation solution, and the DNA tissue in the tissue is decomposed by stirring with a stirrer. Here, the rotation speed of the stirrer can be performed within 30 rpm. This step proceeds at room temperature in consideration of the reactivity of the DNA degrading enzyme, and proceeds for 10 hours to 15 hours.

When the DNA degradation step is completed, a secondary cell removal step is performed on the treated tissue through the above step (S60). The second cell removal step may use a second cell removal solution containing a second surfactant.

The above-described first cell removing step uses an anionic surfactant, while the present step uses a nonionic surfactant. Thus, damage to various proteins, glycoproteins, glycans and the like of the extracellular matrix can be minimized while the primary cell removal step and the DNA degradation step proceed. In this embodiment, as an example, Triton X-100 (triton X-100) can be used as the second surfactant.

In this case, the second cell removing solution may be a solution containing a low concentration of the second surfactant as in the case of the first cell removing solution, and a second surfactant, Triton X-100, may be added to the second distilled water solvent A solution containing a concentration of 1% or less may be used, more specifically, a solution having a concentration of 0.5% or less. In addition, the second cell removal solution may further comprise 1% penicillin / streptomycin for sterilization.

In this step, the cells are removed by a method in which a tissue subjected to DNA degradation treatment is added to the second cell removal solution and stirred using a stirrer. The temperature environment, the stirring environment, and the time in which the present step proceeds may be designed to correspond to the first cell removal step described above. Therefore, this step is carried out in a low-temperature environment of 10 degrees Celsius or less, particularly in a temperature environment of 2 to 7 degrees Celsius, stirring at a rotation speed of 30 rpm or less, at least 18 hours, more specifically, Or less. As such, the second cell removal step can also minimize damage to the extracellular matrix as it proceeds for a relatively long time in a low / low concentration environment where the functionality of the second surfactant is limited.

When the brain tissue is de-saturatedized by the above-described steps, the treated tissue is washed (S70). This step proceeds in the same manner as the S40 washing step described above, and the tissue from which the cells have been removed is washed while stirring with the washing solution. This step can be carried out twice under the same conditions.

Then, the washed tissue is sterilized and disinfected (S80). This step is performed using a disinfectant solution, and the disinfectant solution may be a solution containing peracetic acid and ethanol, which are effective to destroy the cell membrane of the microorganism. Specifically, the disinfecting solution of this embodiment may be a solution containing 0.1% of peracetic acid in 4% ethanol. Sterilization and disinfection are performed in such a manner that the tissues washed in the previous step are put into the disinfecting solution and then stirred at a rotational speed of 30 rpm or less using a stirrer. This step may be performed in a low temperature environment of 10 degrees Celsius or less and may be performed for a time of 1 hour or more.

The tissue sterilized through the above step may be washed again using a washing solution (S90). The washing step may be carried out by introducing sterilized secondary sterile distilled water (sterile DDW) into the washing solution and agitating through a stirrer.

In the present embodiment, primary washing can be carried out by using a washing liquid containing penicillin / streptomycin at a concentration of 1% in a sterile secondary distilled water solvent with stirring at a rotational speed of 30 rpm or less using a stirrer . The primary cleaning step is performed in a low-temperature environment of 10 degrees Celsius or less, and may be performed for a period of 10 hours to 15 hours or less. The sterilized secondary distilled water not containing any antibiotic may be used as a washing solution and the secondary washing may be performed while stirring at a rotational speed of 30 rpm or less using a stirrer. The secondary washing step is also performed in a low temperature environment of 10 degrees Celsius or less and can be performed for a time of 30 minutes or less. This secondary washing step can be carried out twice in the same environment.

Through the steps described above, the brain tissue is degenerated and the brain-derived cellularization substrate can be obtained. FIG. 2 is a graph showing changes in the component of the brain tissue treated by the decellularization method according to FIG. 1; FIG.

As a result of analyzing the tissues treated by the decellularization method according to the present embodiment, it was found that about 98% of the DNA was removed in the case of DNA as a cellular residual material as compared with the original brain tissue. (GAG, glycosaminoglycan) and hyaluronic acid (HA) remained approximately 140% and 70%, respectively, in the brain tissue Because of the increase in the ratio of GAG per unit mass due to cell depletion).

As described above, in the decellularization method described above, it can be confirmed that the cells are effectively removed from the original brain tissue and the damage of the extracellular matrix is minimized and remained. Therefore, when the brain cells are cultured using the extracellular matrix thus obtained, they are cultured in an environment similar to the original environment, so that tissues having characteristics similar to those of actual brain tissues can be cultured.

Hereinafter, with reference to FIG. 3 and FIG. 4, a method of culturing brain tissue using an extracellular matrix deteriorated in the above-described manner will be described in detail. FIG. 3 is a flowchart showing a procedure for culturing brain tissue using an extracellular matrix, and FIG. 4 is a view schematically showing a main step of FIG.

As shown in FIG. 3, first, a step of preparing an extracellular matrix is performed (S110). The extracellular matrix is an extracellular matrix derived from the brain, and the one obtained by decellularization through the above-described decellularization method can be used. Here, the extracellular matrix can be lyophilized in a state where the cell is decellularized and washed (see Fig. 4 (a)).

When the extracellular matrix is prepared, a step of preparing an extracellular matrix solution is performed (S120). The extracellular matrix solution proceeds by dissolving the extracellular matrix using the digestion of pepsin (see FIG. 4 b).

First, the lyophilized extracellular matrix in 0.01 N HCl with an 80% volume ratio of total volume is quantitatively added to the sterilized container taking into account the desired amount and final concentration. Then, approximately 10% by weight of pepsin is added to the HCl solution containing the extracellular matrix. Then, it is stored at room temperature for 3 days to 5 days using a sterilizing magnetic bar or the like. During this period pepsin acts in an acidic environment to dissolve the added extracellular matrix.

After cooling the HCl solution in which the extracellular matrix is dissolved at a low temperature (for example, 2 to 7 degrees Celsius), phosphate buffered saline (PBS) solution having a volume ratio of 10% To control osmotic pressure with the cultured cells. Then, NaOH solution is added so that the solution in which the extracellular matrix is dissolved can be neutralized to about pH 7.0 to 7.4. Here, a 0.1N NaOH solution having a 10% weight ratio of the previously added 0.01N HCl amount is added, and the pH can be adjusted by adding HCl solution or NaOH, if necessary. Thereafter, distilled water is added to adjust the volume of the extracellular matrix solution.

After the extracellular matrix solution is prepared, cells to be cultured are transplanted into the solution (S130). The transplanted cells may be brain cells. In this embodiment, the malignant brain tumor cells are transplanted so that the malignant brain tumor tissue can be cultured. The transplanted cells are uniformly distributed in the extracellular matrix solution and can maintain the liquid phase in a low temperature environment.

Thereafter, the step of culturing the tissue using the cell-implanted solution is performed (S140). To proceed with tissue culture, the cell-implanted extracellular matrix solution is placed at the culture location. At this time, since the solution to be implanted maintains a liquid state at a low temperature, it can be applied to various three-dimensional culture methods such as injection into a culture mold of various shapes, printing by spraying with a nozzle, and the like. In addition, since the extracellular matrix solution into which the cells have been transplanted is thermosensitive as a hydrogel, the solution is allowed to rise to a temperature close to the body temperature while the solution is positioned at the culture site, and the culture is performed by curing by sol-gel transfer.

As described above, the culture method using the brain-derived dextrinized extracellular matrix hydrogel (BdECM) can uniformly culture the cells by uniformly distributing the extracellular matrix and cells in a low-temperature liquid phase. And can be applied to various culture methods. Further, since it is cured using the sol-gel transfer property, there is an advantage that a curing agent or the like, which may be toxic to cells, is not further required.

Hereinafter, the effect of the brain tissue culture method according to the present invention will be described in detail with reference to FIG. 5 and FIG. One was cultured by transplanting a glioblastoma cell spheroid into an extracellular matrix hydrogel prepared in the above-mentioned manner, and the same cells were transplanted into collagen widely used for three-dimensional culture as a comparative control .

FIG. 5 is a graph showing the degree of malignant expression between two control groups. As shown in FIG. 5 (a), the mitochondrial metabolic activity of intracellular mitochondria was measured. As a result, the culture system using the extracellular matrix hydrogel according to the present invention showed higher activity. In addition, as shown in FIG. 5 (b), in the experiment in which oncogene expression was measured, the culture method using the extracellular matrix hydrogel according to the present invention showed a higher degree of gene expression. In other words, it can be confirmed that the glioblastoma cell is metabolized more actively in the extracellular matrix hydrogel, and the gene expression that can develop into malignant tumor is further increased.

Fig. 6 is a photograph showing the penetration pattern of the glioblastoma to be cultured. As a result of observing the invasion of surrounding tissues, one of the major features of the glioblastoma multiforme, it showed a penetration into a longer and wider area when cultured in extracellular matrix hydrogels as compared with the collagen culture method. Thus, it can be seen that when the glioblastoma is in the extracellular matrix hydrogel, it behaves like a brain tumor originally.

As described above, the present invention can obtain an extracellular matrix that degenerates brain tissue having a large number of cell populations while maintaining intrinsic characteristics by performing a decellularization process using a low concentration cell-removing solution in a low temperature environment for a long period of time . In addition, since the hydrogel is prepared using such an extracellular matrix and the brain tumor cell is transplanted and cultured, a tumor tissue similar to that of a real brain tumor can be produced. As a result, it can be used as a more effective material for the development of new drugs and patient- There is an advantage that can be.

While the present invention has been described with reference to the exemplary embodiments, it is to be understood that the invention is not limited to the disclosed exemplary embodiments. It will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention as defined by the appended claims.

S10: tissue preparing step S30: primary cell removing step
S50: DNA degradation step S60: secondary cell removal step
S110: Extracellular matrix preparation step S120: Extracellular matrix solution preparation step
S130: Cell transplantation step S140: Culture step

Claims

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Obtaining brain-derived extracellular matrix by decellularizing brain tissue;
Dissolving the obtained extracellular matrix using a pepsin-digestion procedure;
And transplanting and culturing tumor cells on a solution in which the extracellular matrix is dissolved,
The step of obtaining the brain derived extracellular matrix comprises:
A first cell removing step of removing the cells of the brain tissue using a second distilled water solution containing sodium dedecylsulfate in an amount of 0.5% or less and stirring at a temperature below room temperature for 18 hours or longer;
A DNA decomposing step of decomposing the DNA of the tissue treated in the first cell removing step using a solution containing the DNA degrading enzyme; And
A second cell for removing cells of the tissue treated in the DNA degradation step by stirring at a temperature below room temperature for 18 hours or longer using a second distilled water solution containing 0.5% or less of Triton X-100 (triton x-100) Comprising:
Wherein the step of transplanting and culturing the tumor cells further comprises a step of solely-gel-transferring the solution in which the tumor cells have been transplanted by raising the temperature.

delete

12. The method of claim 11, wherein the sol-
Wherein the solubilized gel is transferred by elevating the temperature while the solution to which the tumor cells have been transplanted is placed in the culture position.