KR20240045745A - Method for Sterilization of Decelluized Tissue-Derived Extracellular Maxtrix - Google Patents

Abstract

The present invention provides a method for effectively sterilizing decellularized tissue-derived extracellular matrix to minimize damage to mechanical strength without affecting the culture activity of a support composition containing it.

Description

Method for sterilization of decelluized tissue-derived extracellular matrix {Method for Sterilization of Decelluized Tissue-Derived Extracellular Maxtrix}

The present invention relates to a method for sterilizing extracellular matrix derived from decellularized tissue.

Organoids are three-dimensional cell structures that are composed of various cells that make up tissues, and have recently attracted attention because they can simulate the in vivo environment. Therefore, it is being actively researched and utilized in various fields ranging from basic biology research to various applied research fields such as new drug development, disease modeling, and regenerative therapy. Currently, animal models are widely used as disease models, but because animals are physiologically different from humans, there are limitations in implementing actual human diseases in animals, and ethical issues are constantly emerging.

The use of organoid models made from patient-derived cells is attracting attention because it enables research on disease mechanisms and even customized patient diagnosis. Currently, various types of organoid models derived from stem cells of various organs in vivo have been constructed. To cultivate these various types of organoids, many researchers around the world are using Matrigel as a culture support. However, Matrigel is an ingredient derived from mouse sarcoma, and concerns have been raised about attempts to transplant organoids cultured from Matrigel into humans due to the risk of infection and immune rejection. Additionally, because it is not a tissue-specific extracellular matrix component, it cannot provide an optimized microenvironment for organoid differentiation.

We present a new platform for organoid culture by utilizing tissue-specific extracellular matrix components obtained by decellularizing various tissues, such as organs such as the lung, liver, stomach, intestines, or tissues such as the heart, as a hydrogel scaffold. I'm doing it. The scaffold obtained by decellularizing the tissue in this way contains various extracellular matrix components and growth factors present in the tissue, and is expected to enhance the development, growth, and differentiation of the tissue organoid. Tissue organoids produced using decellularized scaffolds have a high potential to be applied as disease models for various tissues.

Generally, a scaffold composition containing such a decellularized tissue-derived extracellular matrix includes the steps of decellularizing an isolated tissue to produce a decellularized tissue; It can be manufactured by a method that includes the step of drying the decellularized tissue to produce an extracellular matrix (ECM) derived from the decellularized tissue in a powder state. In addition, the step of gelating the dried decellularized tissue-derived extracellular matrix powder to obtain a ready-to-use decellularized tissue-derived extracellular matrix hydrogel may be further included.

The decellularized tissue-derived extracellular matrix in powder state or the decellularized tissue-derived extracellular matrix in hydrogel state prepared in this way undergoes a sterilization step before being used for organoid culture.

Such sterilization must not only minimally damage the culture activity of the decellularized tissue-derived extracellular matrix, but must also achieve the degree of sterilization required for organoid culture.

In general, a value indicating the degree of sterilization is the sterility assurance level (SAL) according to the ISO11137 standard. This means the probability of one surviving microorganism occurring in the object after sterilization, and the smaller this value, the higher the assurance of sterility.

In order to commercialize the organoid culture support, it may be desirable for the SAL value of the extracellular matrix derived from decellularized tissue to be within the range of 10 ^-2 to 10 ^-3 .

The purpose of the present invention is to provide a sterilization method for extracellular matrix derived from decellularized tissue that can ensure a desired degree of sterilization while minimizing damage to culture performance.

In order to achieve the above object, according to one aspect of the present invention, a method for sterilizing a composite composition containing a decellularized tissue-derived extracellular matrix is provided.

The sterilization method according to one embodiment of the present invention can achieve a desired level of sterilization effect without damaging the culture activity of the support.

Figure 1 is a graph showing the results of mechanical property tests of scaffold compositions containing sterilized decellularized tissue-derived extracellular matrix of Examples 1 to 3 of the present invention.
Figure 2 is a graph showing the results of a gene expression test of a scaffold composition containing sterilized decellularized tissue-derived extracellular matrix of Examples 1 to 3 of the present invention.
Figure 3 is a photograph showing the results of a gene expression test of a scaffold composition containing sterilized decellularized tissue-derived extracellular matrix of Examples 1 to 3 of the present invention.
Figure 4 is a graph showing the results of gene expression tests according to the sterilization method of the scaffold composition containing the sterilized decellularized tissue-derived extracellular matrix of Examples 1 to 3 of the present invention.

Hereinafter, the present invention will be described in detail. However, the present invention may be implemented in various different forms and, therefore, is not limited to the embodiments described herein. When a part is said to "include" a certain component, this means that it does not exclude other components, but may further include other components, unless specifically stated to the contrary.

Unless otherwise defined, it can be performed by conventional techniques commonly used in the fields of molecular biology, microbiology, protein purification, protein engineering, and DNA sequence analysis and recombinant DNA within the capabilities of those skilled in the art. These techniques are known to those skilled in the art and are described in many standardized textbooks and reference books.

Unless otherwise defined herein, all technical and scientific terms used have the same meaning as commonly understood by a person of ordinary skill in the art.

One aspect of the present invention provides a method for sterilizing a composite composition containing decellularized tissue-derived extracellular matrix.

A method of sterilizing decellularized tissue-derived extracellular matrix according to one aspect of the present invention includes the steps of placing the decellularized tissue-derived extracellular matrix into a container; and irradiating the vessel with radiation or treating the vessel with EO gas. Includes.

According to one embodiment of the present invention, the desired degree of sterilization can be obtained without damaging the culture activity of the extracellular matrix.

The extracellular matrix includes collagen, elastin, laminins, glycosaminoglycans, proteoglycans, antimicrobials, chemoattractants, and cytokines. , and growth factors.

The extracellular matrix can be in various forms in mammals and contain about 90% collagen. Extracellular matrix derived from various biological tissues may have different overall structures and compositions due to the unique roles required for each tissue. The biological tissue is not particularly limited, but may be liver, lung, stomach, intestine, heart, etc.

The terms “derive” and “derived” mean ingredients obtained from the source mentioned by any useful method.

According to one embodiment of the present invention, in order to irradiate the decellularized tissue-derived extracellular matrix with radiation or treat it with EO gas, the extracellular matrix is first placed in a suitable container. This container is not particularly limited, and the irradiated extracellular matrix is not particularly limited. When processing EO gas, it is sufficient as long as no physical or chemical changes occur and the radiation or EO gas to be irradiated or treated can penetrate well.

The decellularized tissue-derived extracellular matrix can be placed in a container in powder or solution form.

The decellularized tissue extracellular matrix used in the present invention may be obtained by a known method.

For example, decellularizing the separated tissue to produce a decellularized tissue; and drying the decellularized tissue to produce a decellularized tissue-derived extracellular matrix.

The separated tissue may further include a step of chopping before decellularization. By further including the step of shredding the tissue prior to decellularization, the decellularization process becomes more efficient and complete cell removal is possible. The shredding method may be performed by a known method.

In order to decellularize the separated tissue, the separated tissue may be stirred in a decellularization solution.

The decellularization solution may contain various ingredients for removing cells from tissue, such as hypertonic saline, peracetic acid, Triton X-100, SDS, or May contain other detergent ingredients. According to one embodiment of the present invention, the decellularization solution contains 0.1 to 5% Triton X-100 and 0.01 to 0.5% ammonium hydroxide, more specifically 1% Triton It may include. By using the above-mentioned decellularization solution, DNA in the prepared scaffold can be effectively removed by decellularization under more relaxed conditions compared to the existing process, and at the same time, various proteins in the tissue can be preserved to a greater extent.

The agitation may be performed for 24 to 72 hours, more specifically 36 to 60 hours, most specifically 40 to 56 hours, and as an example, 48 hours. Through this agitation (decellularization) process, tissue cells are 95 to 60 hours old. 99.9%, more specifically 96 to 98%, and most specifically 96.75% may be removed.

The method of drying the decellularized tissue may be performed by a known method and may be natural drying or freeze-drying.

The dried extracellular matrix can be subdivided by a method including tearing, milling, cutting, grinding, and shearing steps. The finely divided extracellular matrix can be processed into powder form by methods such as grinding or milling in a frozen or freeze-dried state.

When the extracellular matrix is placed in a container in a solution state, the cell support powder may be dissolved in, for example, a pepsin solution. At this time, the cell scaffold powder can be added to a 4 mg/ml pepsin solution and then stirred for 48 hours.

The decellularized tissue-derived extracellular matrix is placed in a container and then irradiated with radiation or treated with EO gas.

The radiation may be gamma rays or electron beams. The radiation may be irradiated at an absorbed dose of 2 to 6 kGy (the amount of radiation energy (kJ) delivered per unit mass (kg)) and an absorbed dose of 3 to 5.5 kGy to obtain the desired sterilization effect.

For example, the gamma ray may be irradiated in a wavelength range of about 10 ^-10 to 10 ^-12 for, for example, 2 to 4 hours, and the electron beam may be irradiated with an energy of 8 to 10 MeV in a wavelength range of 2 to 4 GHz. It can be irradiated for from seconds to about 5 seconds.

According to one embodiment of the present invention, the radiation or EO gas may be irradiated or treated two or more times, and there may be a certain period of rest between each time. In this case, the sterilization effect can be improved while the damage to the extracellular matrix can be minimized. The resting period may be, for example, 30 minutes to 24 hours, and the number of irradiations may be 2 to 5 times.

According to one embodiment of the present invention, the decellularized tissue-derived extracellular matrix can be embedded in moisture-proof paper and then placed in a container. When embedded in moisture-proof paper, for example, Tyvek, damage to the culture performance of the extracellular matrix can be effectively prevented while maintaining the sterilization effect.

According to one embodiment of the present invention, the sterility assurance level (SAL) value of the extracellular matrix according to the ISO11137 standard after the irradiation treatment or EO gas treatment may be 10 ^-2 to 10 ^-3 .

According to one embodiment of the present invention, a support composition containing an extracellular matrix sterilized by the above method can be provided.

The support composition can be used for organoid culture in the form of a hydrogel.

For example, the sterilized decellularized tissue-derived extracellular matrix is 0.01 to 10 mg/mL, specifically 0.5 to 9 mg/mL, more specifically 1 mg/mL to 8 mg/mL, most specifically 1, It may be included in the support composition at 3, 5 or 7 mg/mL, with an optimized specific example being 7 mg/mL.

According to one embodiment of the present invention, the composition may have an elastic modulus of 1 to 100 Pa based on 0.1 to 10 Hz, and the composition may form a stable polymer network by having a modulus (elastic modulus) in the above range.

The support composition includes a three-dimensional hydrogel manufactured based on sterilized decellularized tissue-derived extracellular matrix and can be effectively used in organoid culture.

Since the decellularized tissue contains actual tissue-specific extracellular matrix components, it can provide a physical, mechanical, and biochemical environment for the tissue, and is very efficient in promoting differentiation into lung tissue cells and tissue-specific functionality. .

The term “organoid” refers to an ultra-small living organ made by culturing cells derived from tissues or pluripotent stem cells into a 3D form to resemble an artificial organ.

The organoid is a three-dimensional tissue analog containing organ-specific cells that arise from stem cells and self-organize (or self-pattern) in a manner similar to the in vivo state, forming specific tissues by patterning restricted factors (e.g. growth factors). can develop into

The organoids have the original physiological properties of the cells and can have anatomical structures that mimic the original state of the cell mixture (including both defined cell types as well as residual stem cells and proximate physiological niches). . The organoid can have cells and their functions better arranged through a three-dimensional culture method, and have a functional organ-like shape and tissue-specific functions.

The support composition can be prepared by a method comprising the step of gelating an extracellular matrix derived from sterilized decellularized tissue.

Through the gelation, a three-dimensional hydrogel-type scaffold can be produced by cross-linking the decellularized tissue-derived extracellular matrix, and the gelled scaffold can be used in a variety of fields such as experiments and screening, as well as in fields related to organoid culture.

The “hydrogel” is a material in which a liquid using water as a dispersion medium solidifies through a sol-gel phase transition, loses fluidity, and forms a porous structure. Hydrophilic polymers with a three-dimensional network structure and microcrystalline structure expand by containing water. can be formed.

The gelation is performed by solutionizing the sterilized decellularized tissue-derived extracellular matrix with proteolytic enzymes such as pepsin or trypsin in an acidic solution if it is in a powder state, and then using 10X PBS and 1 M NaOH as it is in a solution state. It may be adjusted to neutral pH and electrolyte conditions of 1X PBS buffer and performed at a temperature of 37°C for 30 minutes.

Hereinafter, the present invention will be described in detail with reference to examples. However, the embodiments according to the present invention may be modified into various other forms, and the scope of the present invention should not be construed as being limited to the embodiments described below. The embodiments of this specification are provided to more completely explain the present invention to those skilled in the art.

Example

Preparation Example 1: Preparation of extracellular matrix derived from decellularized liver tissue in powder form

Porcine liver tissue was separated and prepared by mincing. The liver tissue was stirred with 1% Triton Afterwards, the decellularized liver tissue was freeze-dried and pulverized to prepare an extracellular matrix derived from the decellularized liver tissue in powder form.

Preparation Example 2: Preparation of decellularized liver tissue-derived extracellular matrix in solution form

10 mg of decellularized liver tissue-derived extracellular matrix prepared in Preparation Example 1 was dissolved in 4 mg/ml pepsin solution (4 mg of pepsin powder dissolved in 1 ml of 0.02 M HCl) for 48 hours to produce decellularized liver in solution form. Tissue-derived extracellular matrix was prepared.

Examples 1-1 to 1-3, Comparative Examples 1-1 and Comparative Examples 1-2

The powdered decellularized liver tissue-derived extracellular matrix prepared in Preparation Example 1 was placed in different containers and sterilized under the following conditions.

Sterilization Sterility assurance level method century Example 1-1 gamma rays 5kGy 10 ^-2 Example 1-2 electron beam 3.0kGy 10 ^-2 Example 1-3 5.2kGy 10 ^-3 Comparative Example 1-1 EO gas 30℃, 12.5 minutes 10 ^-2 Comparative Example 1-2 30℃, 25 minutes 10 ^-3

Sterilization equipment information, sterilization conditions, and sterility assurance level are as follows.

(1) Gamma irradiation device

Equipment name: JS10000 (Equipment number: IR-203)

Sterilization company name: Soya Greentech Co., Ltd.

Wavelength range: approximately 10 ^-10 to 10 ^-10

Research time: approximately 2 hours and 30 minutes

(2) Electron beam irradiation device

Equipment name: E-Beam Accelerator 1 (Equipment number: MB10-8/6350)

Sterilization company name: Seoul Radiation Service Co., Ltd.

Wavelength range: 2-4Ghz

Electron beam energy: 10 MeV (+10%, -5%)

Scan width (cm)/conveyor speed (m/min): 3kGy (60/3.601), 5.2kGy (60/2.077)

Irradiation time: 3 kGy (about 2 seconds), 5.2 kGy (about 4 seconds)

(3) EO gas

Temperature: 38℃

Relative humidity: 60%

EO gas concentration: 450mg/L

Time: 30 minutes

(4) Sterility assurance level

The sterility assurance level (SAL) was measured using a method according to the ISO11137 standard.

10 mg of the sterilized powdered decellularized liver tissue-derived extracellular matrix was dissolved in 4 mg/ml pepsin solution (4 mg of pepsin powder dissolved in 1 ml of 0.02M HCl) for 48 hours. 10

Reference example 1

10 mg of extracellular matrix derived from decellularized liver tissue in powder form of Preparation Example 1 was dissolved in 4 mg/ml pepsin solution (4 mg of pepsin powder dissolved in 1 ml of 0.02M HCl) for 48 hours. 10

Examples 2-1 to 2-3

10 mg of decellularized liver tissue-derived extracellular matrix in solution form prepared in Preparation Example 2 was dissolved in 4 mg/ml pepsin solution (4 mg of pepsin powder dissolved in 1 ml of 0.02M HCl) for 48 hours.

Each was placed in a different container and sterilized under the following conditions.

Sterilization Sterilization assurance level method century Example 2-1 gamma rays 5kGy 10 ^-2 Example 2-2 electron beam 3.0kGy 10 ^-2 Example 2-3 5.2kGy 10 ^-3

The sterilized decellularized liver tissue-derived extracellular matrix solution was uniformly mixed using 10 A support composition was prepared.

Reference example 2

10 mg of the decellularized liver tissue-derived extracellular matrix in the form of a solution in Preparation Example 2 was mixed uniformly using 10 A support composition in the form of a hydrogel was prepared.

Experimental Example 1: Mechanical strength measurement

The mechanical properties of the hydrogel-type support compositions of Examples 1, 2, and Comparative Example 1 were measured through rheological analysis.

Figure 1 is a graph showing the modulus of the support compositions of Examples 1, 2, and Comparative Example 1.

As shown in Figure 1, both the cases where the extracellular matrix in powder form was sterilized with gamma rays or electron beams and the extracellular matrix in solution state were not sterilized in Reference Example 1 (powder state). It can be seen that the modulus did not drop significantly for each of Example 2 (solution state).

On the other hand, when the powdered extracellular matrix was treated with EO gas (Comparative Examples 1-1 and 1-2), it could be confirmed that the modulus value was significantly lowered compared to Reference Example 1, which was not sterilized.

From this, it can be seen that sterilization treatment by gamma ray or electron beam irradiation does not have a significant effect on the mechanical properties of the extracellular matrix.

Experimental Example 2: Measurement of liver organoid differentiation capacity

The hydrogel-type support composition prepared in the Examples, Comparative Examples, and Reference Examples was applied to mouse liver organoid culture, and the expression of differentiation-related genes in liver organoids cultured for 7 days under each condition was measured by quantitative PCR (qPCR). A comparative analysis was conducted. Matrigel, a commercially available culture support, was used as a control.

Figure 2 is a graph showing the liver organoid gene expression results of the scaffold composition prepared in Example 1 and Comparative Example 1, and Figure 3 is a graph showing the liver organoid gene expression results of the scaffold composition prepared in Example 1 and Comparative Example 1 of the present invention. This is a photo showing.

As shown in Figures 2 and 3, when sterilization was performed, the gene expression performance was equal to or higher than that of Matrigel or Reference Example 1 without sterilization, especially in the case of the gamma ray irradiation of Example 1-1. The expression level of liver differentiation-related marker (Krt19) tended to be highest in the scaffold composition.

Figure 4 is a graph showing the gene expression results in liver organoids of the support composition of Example 2 of the present invention.

As shown in Figure 4, the expression level of the liver differentiation-related marker (Krt19) was slightly decreased compared to Reference Example 2 in which sterilization was not performed, but the decrease in the expression level of the liver differentiation-related marker (Krt19) was most significant in the sample treated with gamma rays. appeared less.

From the above description, those skilled in the art to which the present invention pertains will understand that the present invention can be implemented in other specific forms without changing its technical idea or essential features. In this regard, the embodiments described above should be understood in all respects as illustrative and not restrictive. The scope of the present invention should be construed as including the meaning and scope of the patent claims described below rather than the detailed description above, and all changes or modified forms derived from the equivalent concept thereof are included in the scope of the present invention.

Claims (8)

Method for sterilizing a composite composition containing decellularized tissue-derived extracellular matrix.
In claim 1,
The sterilization method includes placing decellularized tissue-derived extracellular matrix into a container; and
A sterilization method comprising: irradiating the container with radiation or treating the container with EO gas.
In claim 2,
A sterilization method wherein the radiation is gamma rays or electron beams.
In claim 2,
A sterilization method in which the radiation or EO gas is irradiated or treated two or more times, with a certain period of time resting between each time.
In claim 2,
A sterilization method in which the radiation is irradiated at an intensity of 3.0 to 5.5 kGy.
In claim 2
A sterilization method in which the decellularized tissue-derived extracellular matrix is embedded in moisture-proof paper.
In claim 2,
A sterilization method wherein the decellularized tissue-derived extracellular matrix is in powder or solution form.
In claim 2,
A sterilization method in which the sterility assurance level (SAL) value of the extracellular matrix according to the ISO11137 standard is 10 ^-2 to 10 ^-3 after the irradiation treatment or EO gas treatment.