Decellularized organ tissue-derived scaffold for the culture and transplantation of organoids and method for producing same

Abstract

The present invention relates to a support derived from decellularized organ tissue for organ culture and transplantation of organoids, and a method for producing the same.

Description

[Technical field]

[0001] The present invention relates to a support derived from decellularized organ tissue for the culture and transplantation of organ organoids and a method for producing the same. [Background technology]

[0002] In relation to the decellularized intestinal tissue-derived scaffold for the culture and transplantation of intestinal organoids and its manufacturing method, stem cell-based intestinal organoids are the most effective model capable of embodying the structure and function of human intestinal tissue among the in vitro models constructed so far, and have great potential for use in disease models, new drug development, transplantation therapy, etc., so there is a lot of interest in efficient 3D organoid production, culture, and transplantation technologies.

[0003] Most research on intestinal organoids has used a culture support based on Matrigel, a natural polymeric substance extracted from mouse sarcoma. Matrigel contains rich extracellular matrix components, which allows the growth of various types of tissue organoids, but it has the limitation that it cannot provide a tissue-specific microenvironment during culture.

[0004] In fact, in the body, intestinal stem cells grow and change while constantly interacting with tissue-specific extracellular fluid and tissue-specific extracellular matrix. When producing intestinal organoids using such stem cells, the culture protocol that mimics the tissue-specific extracellular fluid environment through intestinal tissue culture medium is relatively well established, but there is still insufficient research on methods that can provide a tissue-specific extracellular matrix environment, and intestinal organoids are still cultured only on Matrigel. Therefore, intestinal organoids cultured from Matrigel need to further promote the development and maturation of structure and function in the absence of an intestinal tissue-specific extracellular matrix environment.

[0005] In addition, since Matrigel is a substance extracted from mouse sarcoma cancer, there is a possibility of infection and metastasis of animal pathogens and viruses during transplantation treatment of organoids cultured from it, and there is a risk of inducing a serious immune reaction, making it difficult to apply it to clinical research for cell therapy and tissue regeneration, such as organoid transplantation. In addition, Matrigel is an expensive product with a high unit price, so it is difficult to use it when a large amount of culture support is required, such as organoid-based drug screening for new drug development. Therefore, the current situation is that basic research and applied research on intestinal organoids are greatly restricted due to the Matrigel-dependent culture method of intestinal organoids.

[0006] To solve this problem, the present invention developed an intestinal tissue replica artificial scaffold that retains the unique components and characteristics of intestinal tissue-specific extracellular matrix by decellularizing the small intestine of a pig.Through the cultivation of 3D intestinal organoids using a hydrogel scaffold derived from decellularized intestinal tissue, it was confirmed that the proliferation and stable maintenance of intestinal organoids was possible, and the differentiation, development, and function of organoids were promoted.

[0007] The scaffold developed in the present invention uses only extracellular matrix components specific to intestinal tissue, which are pure from pig small intestine, with all cellular components removed, and therefore can solve the problems of the existing organoid culture scaffold, Matrigel, such as lack of tissue specificity, risk of infection and immune reaction, etc. Pig small intestinal tissue is also very advantageous in terms of economy, compared to expensive Matrigel.

[0008] In addition, in relation to a decellularized gastric tissue-derived scaffold for the culture and transplantation of gastric organoids and a manufacturing method thereof, organoids, which have been in the spotlight recently, are tissue analogs that can be used in a variety of fields such as new drug screening, drug toxicity evaluation, disease modeling, and cell therapeutics, and are a technology that is growing rapidly worldwide.

[0009] There are various types of organoids for different organs, such as the brain, intestine, liver, and stomach. Many research teams around the world who are studying these organoids have used Matrigel products as a culture support to culture organoids. However, since Matrigel is an extracellular matrix component extracted from mouse sarcoma cancer tissue, it is difficult to maintain the quality of the product uniformly, and it is expensive. There are also safety issues such as the transfer of animal infectious bacteria and viruses. As a culture system for organoids, Matrigel has many problems to be solved. In particular, as a material derived from cancer tissue, it cannot provide the optimal tissue-specific microenvironment required for the culture of organoids of specific tissues. Although some research has been conducted on the development of polymer-based hydrogels to replace Matrigel, no material that can replace Matrigel has yet been reported.

[0010] Gastric organoids can be generated by extracting stem cells from gastric tissue and culturing them in 3D. Gastric organoids cultured using the Matrigel protocol reported so far have the advantage of being able to be cultured for long periods while maintaining proliferation ability, but there is a problem in that it is difficult to differentiate into cells that perform important functions, such as parietal cells and chief cells, among the various cells that make up gastric tissue. Therefore, there is a need to develop new culture material technologies that can produce gastric organoids that are structurally and functionally more mature and developed than the existing Matrigel-based culture system.

[0011] In addition, for diseases that cause a large amount of tissue damage, such as gastric ulcers and after gastric cancer resection, drug therapy alone has limitations, so the development of cell therapy and tissue engineering techniques that can essentially regenerate tissue is necessary. Gastric organoids contain a variety of cells, including stem cells, that are present in actual gastric tissue, so organoid-based transplantation and therapy are attracting attention. However, there is also an essential demand for the development of transplantation materials that can promote efficient transplantation and engraftment of organoids into the body.

[0012] The present invention proposes a new culture platform in which a hydrogel scaffold based on components of extracellular matrix derived from decellularized gastric tissue is produced from porcine gastric tissue through a decellularization process, and this is applied to the culture of gastric organoids. The developed scaffold derived from decellularized tissue can be mass-produced easily at low cost, and contains a variety of extracellular matrices and growth factors specific to gastric tissue, so that a three-dimensional microenvironment specific to gastric organoids can be provided. It has been verified that the developed scaffold enables the formation and proliferation of gastric organoids at a similar level to existing Matrigel, and that differentiation and function into gastric tissue-specific cells can be further enhanced. Furthermore, the decellularized scaffold improves the transplantation efficiency and engraftment of gastric organoids into damaged gastric tissue, and shows high potential as a material for organoid transplantation.

[0013] Organoids, which have been in the spotlight recently in relation to a scaffold derived from decellularized liver tissue for the culture and transplantation of liver organoids and its manufacturing method, are tissue analogs that can be used in a variety of clinical applications, such as new drug screening, drug toxicity evaluation, disease modeling, cell therapy, tissue engineering, etc., and are a rapidly growing technology worldwide. Organoids are not only composed of various cells that constitute specific organs and tissues in the human body within a three-dimensional structure, but can also embody their interactions, making them applicable to a more accurate in vitro model platform than previously used drug evaluation models such as cell line models and animal models.

[0014] There are various types of organoids according to organs, and many research teams around the world who are studying these have commonly used Matrigel products as a culture support to culture organoids. However, since Matrigel is a component extracted from mouse sarcoma cancer tissue, it is difficult to maintain the quality of the product uniformly, it is expensive, and there are safety issues such as the transfer of animal infectious bacteria and viruses, so Matrigel has many problems to be solved as an organoid culture system. In particular, as a material derived from cancer tissue, it cannot provide the optimal tissue-specific microenvironment required for the culture of organoids of a specific tissue. Although some research has been conducted on the development of polymer-based hydrogels to replace Matrigel, no material of a level that can replace Matrigel has yet been reported.

[0015] Liver organoids are produced by extracting and culturing adult stem cells from liver tissue, or by differentiating pluripotent stem cells such as human induced pluripotent stem cells or embryonic stem cells into hepatocytes and culturing them. Since Matrigel cannot embody the complex liver tissue-specific microenvironment in vivo, there is a need to improve the differentiation efficiency and function of liver organoids. Therefore, there is an urgent need to develop a culture system for producing more mature and functional liver organoids.

[0016] In addition, in the case of intractable liver diseases such as hepatic fibrosis, hepatitis, hepatosclerosis, and hepatic cancer, which cause a large amount of cell loss and liver function decline, drug treatment is difficult, so a therapeutic technique that can essentially regenerate liver tissue is required. Since liver organoids contain various liver tissue constituent cells including stem cells present in actual liver tissue, organoid-based liver tissue engineering and cell therapy techniques have attracted much attention. However, when transplanting large-sized tissue analogs such as organoids, there is also a demand for the development of a support that can help the organoids to be efficiently transplanted and engrafted in the diseased area.

[0017] Thus, in order to solve the current technical problems in the culture and transplantation of hepatic organoids, the present invention presents a new platform in which a decellularized scaffold derived from liver tissue is produced through a decellularization process from liver tissue and used for the culture of hepatic organoids. The developed hydrogel scaffold derived from decellularized liver tissue is rich in a variety of extracellular matrices and growth factors specific to liver tissue, and therefore promotes the differentiation, maturity, and functionality of hepatic organoids compared to when Matrigel was used. Furthermore, the hydrogel derived from decellularized liver tissue shows potential as a transplantation scaffold that enables efficient transplantation of hepatic organoids into living organisms.

[0018] Regarding the scaffold derived from decellularized lung tissue for the culture and transplantation of lung organoids and its manufacturing method, organoids are 3D cellular structures composed of various cells that constitute tissues, and have recently attracted attention because they can mimic the in vivo environment. Therefore, they are being actively studied and used in many areas, from basic biology research to various applied research fields such as new drug development, disease modeling, and regenerative therapy.

[0019] Currently, animal models are widely used for disease models, but because animals differ from humans in physiological aspects, there are limitations to embodying actual human diseases in animals, and ethical issues continue to arise. Organoid models made from patient-derived cells are attracting attention because they enable research into the mechanism of disease and patient-specific diagnosis.

[0020] Currently, various types of organoid models derived from stem cells of various organs in vivo have been constructed. To culture such various types of organoids, many researchers around the world use Matrigel as a culture support. However, Matrigel is a component derived from mouse sarcoma cancer, and concerns have been raised about attempts to transplant organoids cultured from Matrigel into humans due to the risk of infection and immune rejection. In addition, since Matrigel is not a component of tissue-specific extracellular matrix, it cannot provide a microenvironment optimized for organoid differentiation. Therefore, it is necessary to develop a biocompatible and tissue-specific culture support that can replace Matrigel.

[0021] Pulmonary diseases caused by genetic factors, excessive smoking, fine dust particles generated by air pollution, viruses, and bacteria are one of the major causes of death worldwide. However, due to the lack of disease models, it is difficult to study the mechanism of lung disease and it is also very difficult to develop treatments. Therefore, there is an urgent need to develop an in vitro lung disease model that can accurately mimic human lung disease.

[0022] The present invention presents a new platform in which components of lung tissue-specific extracellular matrix obtained by decellularizing lung tissue are utilized as a hydrogel scaffold for culturing lung organoids. The scaffold obtained by decellularizing lung tissue in this way contains various extracellular matrix components and growth factors present in lung tissue, and is expected to promote the development, growth and differentiation of lung organoids. Lung organoids produced using the decellularized scaffold are highly likely to be applicable as a variety of lung disease models. Summary of the Invention [Problem to be solved by the invention]

[0023] The present invention relates to a scaffold derived from decellularized intestinal tissue for the culture and transplantation of intestinal organoids and a manufacturing method thereof. The present invention aims to remove cells through a decellularization process of intestinal tissue, preserve only the intestinal tissue-specific extracellular matrix, and use it as an artificial scaffold for effective 3D culture of intestinal organoids.

[0024] In relation to a scaffold derived from decellularized gastric tissue for the culture and transplantation of gastric organoids and a method for producing the same, the present invention establishes a method for producing a large amount of decellularized scaffolds by performing a simple chemical treatment on gastric tissue, and uses the produced decellularized gastric tissue matrix for the culture of gastric organoids.

[0025] The present invention relates to a support derived from decellularized liver tissue for the culture and transplantation of hepatic organoids and a manufacturing method thereof. The present invention relates to a support derived from decellularized liver tissue for the culture and transplantation of hepatic organoids, and to a method for producing the same, in which decellularized liver tissue is decellularized to remove all cellular components and preserve liver-specific extracellular matrix components, and a three-dimensional hydrogel based on the decellularized liver tissue is applied to the culture of hepatic organoids.

[0026] The present invention relates to a scaffold derived from decellularized lung tissue for the culture and transplantation of pulmonary organoids and a method for producing the same. The present invention relates to a scaffold derived from decellularized lung tissue for the culture and transplantation of pulmonary organoids, and a method for producing the same. The present invention relates to a scaffold derived from decellularized lung tissue obtained by chemically treating porcine lung tissue, and a method for producing the scaffold based on the scaffold, and a method for culturing the scaffold using the scaffold.

[0027] However, the technical problems that the present invention aims to achieve are not limited to those mentioned above, and other problems not mentioned will be clearly understood by those skilled in the art from the following description. [Means for solving the problem]

[0028] The present invention will now be described with reference to the accompanying drawings. However, the present invention may be embodied in various different forms and is not limited to the embodiments described herein. When a part is described as "comprising" a certain element, this means that the part may further include the other element, without excluding the other element, unless otherwise specified.

[0029] Unless otherwise specified, the procedures may 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, which are within the capabilities of those skilled in the art and are known to those skilled in the art and are described in many standard textbooks and reference books.

[0030] Unless otherwise defined herein, all technical and scientific terms used have the same meaning as commonly understood by one of ordinary skill in the art.

[0031] Various scientific dictionaries, including the terms contained herein, are well known and available in the art. It is understood that any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present application, and several methods and materials are described. The present invention is not limited to specific methodologies, protocols, and reagents, as they can be used in a variety of contexts by those of skill in the art. The present invention is described in more detail below.

[0032] The present invention provides a support composition comprising an extracellular matrix derived from decellularized organ tissue.

[0033] In one embodiment of the present invention, the organ may be a support composition for any one of the intestine, stomach, liver, and lung.

[0034] In one specific example of the present invention, the extracellular matrix derived from the decellularized organ tissue may be contained in a concentration of 0.01 to 10 mg / mL.

[0035] The present invention also provides a method for producing a support composition, comprising the steps of: (a) decellularizing isolated organ tissue to produce decellularized organ tissue; (b) drying the decellularized organ tissue to produce an extracellular matrix derived from the decellularized organ tissue; and (c) gelating the extracellular matrix derived from the dried decellularized organ tissue.

[0036] In one embodiment of the present invention, the organ may be any one of an intestine, a stomach, a liver, and a lung.

[0037] As a specific example of the present invention, in step (a), the organ tissue may be agitated with the decellularization solution.

[0038] As a specific example of the present invention, the decellularization solution may contain 0.1 to 5% Triton X-100 and 0.01 to 0.5% ammonium hydroxide.

[0039] As a specific example of the present invention, the decellularization in step (a) may result in removal of 95 to 99.9% of organ tissue cells.

[0040] As a specific example of the present invention, after the step (b), the method may further comprise a step of adjusting the extracellular matrix derived from the decellularized organ tissue so that it is contained in a range of 0.01 to 10 mg / mL.

[0041] The present invention provides a method for organ culturing organoids on the support composition or a support composition produced by the production method.

[0042] In one embodiment of the present invention, the organ may be any one of an intestine, a stomach, a liver, and a lung.

[0043] The present invention will be described in more detail below with respect to each organ.

[0044] In relation to a support derived from decellularized intestinal tissue for the culture and transplantation of intestinal organoids and a method for producing the same, one aspect of the present invention provides a support composition comprising an extracellular matrix (Intestinal Extracellular Matrix; IEM) derived from decellularized intestinal tissue.

[0045] The term "extracellular matrix" refers to a natural support for cell growth produced through decellularization of tissues found in mammals and multicellular organisms. The extracellular matrix can be further processed through dialysis or crosslinking.

[0046] The extracellular matrix can be a mixture of structural and non-structural biomolecules, including but not limited to collagens, elastins, laminins, glycosaminoglycans, proteoglycans, antimicrobials, chemoattractants, cytokines, and growth factors.

[0047] The extracellular matrix may contain about 90% collagen in various forms in mammals. Extracellular matrices derived from various biological tissues may have different overall structures and structures due to the specific roles required for each tissue, and in one embodiment of the present invention, the intestinal tissue may be the entire mucosal layer of the pig intestine, more specifically, the entire mucosal layer of the pig small intestine and / or large intestine. This is an improvement over the conventional technology that only used the submucosa of the pig small intestine, and since the raw materials are not limited, the convenience of production is increased, and the produced support composition can have a richer amount of extracellular matrix proteins than the conventional process.

[0048] The terms "derive" and "derived" refer to ingredients that are obtained from the stated source in any useful manner.

[0049] In addition, as a specific example of the present invention, the extracellular matrix derived from the decellularized intestinal tissue may be contained at a concentration of 0.01 to 10 mg / mL, specifically 0.5 to 8 mg / mL, more specifically 1 mg / mL to 5 mg / mL, most specifically 1, 2, 3, 4 or 5 mg / mL, and as an optimized specific example, 2 mg / mL. If it is contained at a concentration outside the above range, the intended effect of the present invention may not be obtained, or it may be incompatible for production or use.

[0050] As a specific example of the present invention, the composition may have a 0.1-1 Hz reference elastic modulus of 1-80 Pa, and when the composition has an elastic modulus in this range, a stable polymer network can be formed.

[0051] The support composition comprises a three-dimensional hydrogel produced based on a decellularized intestinal tissue matrix composition, and can be effectively used in the culture of intestinal organoids.

[0052] The decellularized intestinal tissue contains components of the actual tissue-specific extracellular matrix, and thus can provide the physical, mechanical and biochemical environment of the tissue, which is highly efficient in promoting differentiation into intestinal tissue cells and tissue-specific functionality.

[0053] The term "organoid" refers to a very small biological organ produced by culturing differentiated cells from adult stem cells or omnipotent stem cells in a tissue in a 3D form and fabricating the organ-like form of an artificial organ.

[0054] The organoids are three-dimensional tissue analogs containing organ-specific cells that arise from stem cells and self-organize (or self-pattern) in a manner similar to the in vivo state, and can develop into specific tissues through restricted growth factor patterning.

[0055] The organoids can have the original physiological properties of cells and an anatomical structure that mimics the original state of a cell mixture (including not only a limited cell type, but also residual stem cells and adjacent physiological niches). The organoids can have a functional organ-like morphology and tissue-specific functions, with cells and cell functions better arranged through a 3D culture method.

[0056] Another aspect of the present invention provides a method for producing a support composition, comprising the steps of: (a) decellularizing isolated intestinal tissue to produce decellularized intestinal tissue; (b) drying the decellularized intestinal tissue to produce an extracellular matrix (Intestinal Extracellular Matrix; IEM) derived from the decellularized intestinal tissue; and (c) gelating the dried decellularized intestinal tissue-derived extracellular matrix.

[0057] The step (a) is a step of decellularizing the isolated intestinal tissue to produce decellularized intestinal tissue.

[0058] In one embodiment of the present invention, in step (a), the intestinal tissue may be the entire mucosal layer of the porcine intestine, more specifically, the entire mucosal layer of the porcine small intestine and / or large intestine. As described above, this is an improvement over the conventional technique that only used the submucosa of the porcine small intestine, and since the raw materials can be used without limitations, the convenience of production is increased, and the produced support composition can have a richer amount of extracellular matrix proteins than the conventional process.

[0059] In one embodiment of the present invention, in step (a), the intestinal tissue may be agitated with the decellularization solution.

[0060] The decellularization solution may contain various components for removing cells from intestinal tissue, for example, hypertonic saline, peracetic acid, Triton X-100, SDS, or other detergent components, and in one embodiment of the present invention, the decellularization solution may contain 0.1 to 5% Triton X-100 and 0.01 to 0.5% ammonium hydroxide, more specifically, 1% Triton X-100 and 0.1% ammonium hydroxide. By using the above-mentioned decellularization solution, decellularization can be carried out under milder conditions than existing processes, and DNA in the prepared support can be effectively removed while various proteins in the intestinal tissue can be more preserved.

[0061] The stirring may be carried out for 24 to 72 hours, more specifically 36 to 60 hours, most specifically 44 to 52 hours, and for example 48 hours, and through this stirring (decellularization) process, 95 to 99.9%, more specifically 96 to 98%, and most specifically 97.68% of the intestinal tissue cells may be removed. If decellularization is carried out for a time outside the above range, there is a problem that the quality of the produced support composition may decrease or the economic efficiency of the process may decrease.

[0062] The step (b) is a step of drying the decellularized intestinal tissue to produce an extracellular matrix (Intestinal Extracellular Matrix; IEM) derived from the decellularized intestinal tissue.

[0063] The method for drying the decellularized intestinal tissue may be performed by a known method, and the tissue may be naturally dried or freeze-dried. For sterilization, the tissue may be exposed to ethylene oxide gas or supercritical carbon dioxide after drying, or by electron beam or gamma radiation.

[0064] In one embodiment of the present invention, after step (b), the method may further include a step of adjusting the concentration of the extracellular matrix derived from decellularized intestinal tissue to 0.01 to 10 mg / mL, specifically 0.5 to 8 mg / mL, more specifically 1 mg / mL to 5 mg / mL, most specifically 1, 2, 3, 4 or 5 mg / mL, and in an optimized embodiment, 2 mg / mL. If the concentration is outside the above range, the intended effect of the present invention may not be obtained, or the method may be incompatible with production or utilization.

[0065] The dried extracellular matrix may be comminuted by methods including steps of tearing, milling, cutting, grinding, and shearing. The comminuted extracellular matrix may be processed into a powder form in the frozen or freeze-dried state by methods such as grinding or milling.

[0066] The step (c) is a step of gelling the extracellular matrix derived from the dried decellularized intestinal tissue.

[0067] Through the gelation, the extracellular matrix derived from the decellularized intestinal tissue can be crosslinked to produce a three-dimensional hydrogel-type scaffold, and the gelled scaffold can be used in a variety of ways in fields related to organoid culture as well as experiments and screening.

[0068] The "hydrogel" is a material in which a liquid with water as a dispersion medium becomes solid and loses fluidity through a sol-gel phase transition, forming a porous structure, and can be formed when a hydrophilic polymer with a three-dimensional network structure and an amorphous structure absorbs water and expands.

[0069] The gelation may be carried out by dissolving the extracellular matrix derived from decellularized intestinal tissue in an acidic solution with a proteolytic enzyme such as pepsin or trypsin, mixing the mixture with water:IEM solution:PBS buffer:NaOH in a ratio of 69:20:10:1, and then mixing uniformly at 37°C for 30 minutes.

[0070] Another aspect of the present invention provides a method for culturing intestinal organoids from the support composition or a support composition produced by the production method.

[0071] Existing matrigel-based culture systems are extracts derived from animal cancer tissues, and have large differences in arrangement, making it impossible to mimic the actual intestinal environment, and therefore the efficiency of differentiation and development into intestinal organoids is insufficient. In contrast, the support composition can create an environment similar to intestinal tissue, making it suitable for culturing intestinal organoids.

[0072] The culturing refers to the process of maintaining and growing cells under suitable conditions, which may refer, for example, to the temperature at which the cells are maintained, the solubility of nutrients, the content of atmospheric CO2, and the cell density.

[0073] Appropriate culture conditions for maintaining, growing, expanding and differentiating different types of cells are known and documented in the art. The conditions suitable for the formation of said organoids may be conditions that facilitate or allow the differentiation of cells and the formation of multicellular structures.

[0074] In relation to a support derived from decellularized gastric tissue for the culture and transplantation of gastric organoids and a method for producing the same, one aspect of the present invention provides a support composition comprising an extracellular matrix (SEM) derived from decellularized gastric tissue.

[0075] The term "extracellular matrix" refers to a natural support for cell growth produced through decellularization of tissues found in mammals and multicellular organisms. The extracellular matrix can be further processed through dialysis or crosslinking.

[0076] The extracellular matrix can be a mixture of structural and non-structural biomolecules, including but not limited to collagens, elastins, laminins, glycosaminoglycans, proteoglycans, antimicrobials, chemoattractants, cytokines, and growth factors.

[0077] The extracellular matrix may contain about 90% collagen in various forms in mammals. Extracellular matrices derived from various biological tissues may differ in overall structure and composition due to the specific roles required for each tissue.

[0078] The terms "derive" and "derived" refer to ingredients that are obtained from the stated source in any useful manner.

[0079] In one specific example of the present invention, the extracellular matrix derived from the decellularized gastric tissue may be at a concentration of 0.01 to 10 mg / mL, specifically 0.5 to 8 mg / mL, more specifically 1 mg / mL to 5 mg / mL, most specifically 1, 3, 5 or 7 mg / mL, and in an optimized specific example, 5 mg / mL. If the concentration is outside the above range, the intended effect of the present invention may not be obtained, or the production or utilization may be incompatible.

[0080] As a specific example of the present invention, the composition may have a 0.1-10 Hz reference elastic modulus of 1-100 Pa. When the composition has an elastic modulus in the above range, a stable polymer network can be formed.

[0081] The support composition comprises a three-dimensional hydrogel produced based on a decellularized stomach tissue matrix composition, and can be effectively used in the culture of gastric organoids.

[0082] The decellularized gastric tissue contains components of the actual tissue-specific extracellular matrix, and thus can provide the physical, mechanical and biochemical environment of the tissue, which is highly efficient in promoting differentiation into gastric tissue cells and tissue-specific functionality.

[0083] The term "organoid" refers to a micro-organ produced by culturing cells derived from tissues or full-potential stem cells in a 3D form and fabricating them in the form of an artificial organ.

[0084] The organoids are three-dimensional tissue analogs containing organ-specific cells that arise from stem cells and self-organize (or self-pattern) in a manner similar to the in vivo state, and can develop into specific tissues through restricted growth factor patterning.

[0085] The organoids have the inherent physiological properties of cells and can have an anatomical structure that mimics the original state of a cell mixture (including not only a limited cell type, but also residual stem cells and adjacent physiological niches). The organoids can have a functional organ-like morphology and tissue-specific functions, with cells and cell functions better arranged through a 3D culture method.

[0086] Another aspect of the present invention provides a method for producing a support composition, comprising the steps of: (a) decellularizing isolated gastric tissue to produce decellularized gastric tissue; (b) drying the decellularized gastric tissue to produce decellularized gastric tissue (Stomach Extracellular Matrix; SEM); and (c) gelating the extracellular matrix derived from the dried decellularized gastric tissue.

[0087] The step (a) is a step of decellularizing the isolated gastric tissue to produce decellularized gastric tissue.

[0088] In one embodiment of the present invention, the gastric tissue separated in step (a) may further be minced prior to decellularization. The present invention includes a step of mincing the gastric tissue prior to decellularization, which allows for more efficient and complete removal of cells in the decellularization process. The method for mincing the separated gastric tissue may be a known method.

[0089] In one specific example of the present invention, in step (a), the gastric tissue may be agitated with a decellularization solution.

[0090] The decellularization solution may contain various components for removing cells from gastric tissue, for example, hypertonic saline, peracetic acid, Triton X-100, SDS or other detergent components, and in one embodiment of the present invention, the decellularization solution may contain 0.1 to 5% Triton X-100 and 0.01 to 0.5% ammonium hydroxide, more specifically, 1% Triton X-100 and 0.1% ammonium hydroxide. By using the above decellularization solution, decellularization can be carried out under milder conditions than in the existing process, and at the same time, various proteins in the gastric tissue can be more preserved while effectively removing DNA in the prepared support.

[0091] The stirring may be carried out for 24 to 72 hours, more specifically 36 to 60 hours, most specifically 40 to 56 hours, and for example 48 hours, and 95 to 99.9%, more specifically 96 to 98%, of the gastric tissue cells may be removed through this stirring (decellularization) process. If decellularization is carried out for a time outside the above range, there is a problem that the quality of the produced support composition may decrease or the economic efficiency of the process may decrease.

[0092] The step (b) is a step of drying the decellularized stomach tissue to produce a decellularized stomach tissue (Stomach Extracellular Matrix; SEM).

[0093] The decellularized gastric tissue can be dried by a known method, and can be naturally dried or freeze-dried. For sterilization, the tissue can be exposed to ethylene oxide gas or supercritical carbon dioxide after drying, or by electron beam or gamma radiation.

[0094] In one embodiment of the present invention, after step (b), the method may further include a step of adjusting the concentration of the extracellular matrix derived from decellularized gastric tissue to be within 0.01 to 10 mg / mL, specifically 0.5 to 8 mg / mL, more specifically 1 mg / mL to 5 mg / mL, most specifically 1, 3, 5 or 7 mg / mL, and an optimized embodiment is 5 mg / mL. If the concentration is outside the above range, the intended effect of the present invention may not be obtained, or the method may be incompatible with production or utilization.

[0095] The dried extracellular matrix may be comminuted by methods including steps of tearing, milling, cutting, grinding, and shearing. The comminuted extracellular matrix may be processed into a powder form in a frozen or freeze-dried state by methods such as grinding or milling.

[0096] The step (c) is a step of gelating the extracellular matrix derived from the dried decellularized gastric tissue.

[0097] Through the gelation, the extracellular matrix derived from the decellularized gastric tissue can be crosslinked to prepare a three-dimensional hydrogel-type scaffold, and the gelled scaffold can be used in a variety of ways in fields related to organoid culture as well as in experiments and screening.

[0098] The "hydrogel" is a material in which a liquid with water as a dispersion medium becomes solid and loses fluidity through a sol-gel phase transition, forming a porous structure, and can be formed when a hydrophilic polymer with a three-dimensional network structure and an amorphous structure absorbs water and expands.

[0099] The gelation may be carried out by dissolving the extracellular matrix from decellularized intestinal tissue in an acidic solution with a proteolytic enzyme such as pepsin or trypsin, adjusting the neutral pH and electrolyte conditions of 1X PBS buffer using 10X PBS and 1M NaOH, at a temperature of 37°C for 30 minutes.

[0100] In one embodiment of the present invention, the method for producing the scaffold composition may not include a perfusion method, which can reduce waste of decellularized solution.

[0101] Another aspect of the present invention provides a method for culturing gastric organoids on the support composition or on a support composition produced by the production method.

[0102] Existing matrigel-based culture systems are extracts derived from animal cancer tissues, and have large differences in configuration, making it impossible to mimic the actual stomach environment and resulting in insufficient efficiency in differentiation and development into gastric organoids. In contrast, the support composition is capable of creating an environment similar to stomach tissue, making it suitable for culturing gastric organoids.

[0103] The culturing refers to the process of maintaining and growing cells under suitable conditions, which may refer, for example, to the temperature at which the cells are maintained, the solubility of nutrients, the content of atmospheric CO2, and the cell density.

[0104] Appropriate culture conditions for maintaining, growing, expanding and differentiating different types of cells are known and documented in the art. The conditions suitable for the formation of said organoids may be conditions that facilitate or allow the differentiation of cells and the formation of multicellular structures.

[0105] In relation to a support derived from decellularized liver tissue for culturing and transplanting hepatic organoids and a method for producing the same, one aspect of the present invention provides a support composition comprising a decellularized liver tissue-derived extracellular matrix (LEM).

[0106] The term "extracellular matrix" refers to a natural support for cell growth produced through decellularization of tissues found in mammals and multicellular organisms. The extracellular matrix can be further processed through dialysis or crosslinking.

[0107] The extracellular matrix can be a mixture of structural and non-structural biomolecules, including but not limited to collagens, elastins, laminins, glycosaminoglycans, proteoglycans, antimicrobials, chemoattractants, cytokines, and growth factors.

[0108] The extracellular matrix may contain about 90% collagen in various forms in mammals. Extracellular matrices derived from various biological tissues may differ in overall structure and composition due to the specific roles required for each tissue.

[0109] The terms "derive" and "derived" refer to ingredients that are obtained from the stated source in any useful manner.

[0110] In addition, as a specific example of the present invention, the extracellular matrix derived from the decellularized liver tissue may be contained at a concentration of 0.01 to 10 mg / mL, specifically 0.5 to 9 mg / mL, more specifically 1 mg / mL to 8 mg / mL, most specifically 2, 4, 6 or 8 mg / mL, and as an optimized specific example, 4 or 6 mg / mL. If it is contained at a concentration outside the above range, the intended effect of the present invention may not be obtained, or it may be incompatible for production or utilization.

[0111] As a specific example of the present invention, the composition may have a 0.1-10 Hz reference elastic modulus of 20-100 Pa. When the composition has an elastic modulus in this range, a stable polymer network can be formed.

[0112] The support composition comprises a three-dimensional hydrogel produced based on a decellularized liver tissue matrix composition, and can be effectively used in culturing liver organoids.

[0113] The decellularized liver tissue contains components of the actual tissue-specific extracellular matrix, and thus can provide the physical, mechanical and biochemical environment of the tissue, which is highly efficient in promoting differentiation into liver tissue cells and tissue-specific functionality.

[0114] The term "organoid" refers to a micro-organ produced by culturing cells derived from tissues or full-potential stem cells in a 3D form and fabricating them in the form of an artificial organ.

[0115] The organoids have the inherent physiological properties of cells and can have an anatomical structure that mimics the original state of a cell mixture (including not only a limited cell type, but also residual stem cells and adjacent physiological niches). The organoids can have a functional organ-like morphology and tissue-specific functions, with cells and cell functions better arranged through a 3D culture method.

[0116] Another aspect of the present invention provides a method for producing a support composition, comprising the steps of: (a) decellularizing isolated liver tissue to produce decellularized liver tissue; (b) drying the decellularized liver tissue to produce a decellularized liver tissue-derived extracellular matrix (Liver Extracellular Matrix; LEM); and (c) gelating the dried decellularized liver tissue-derived extracellular matrix.

[0117] The step (a) is a step of decellularizing the isolated liver tissue to produce decellularized liver tissue.

[0118] In one embodiment of the present invention, the step (a) may further include a step of chopping the isolated liver tissue before decellularization. The present invention includes a step of chopping the liver tissue before decellularization, which allows for more efficient and complete removal of cells in the decellularization process. The method for chopping the isolated liver tissue may be performed using known methods (instruments) and sizes.

[0119] In one specific example of the present invention, in the step (a), the liver tissue may be agitated with the decellularization solution.

[0120] The decellularization solution may contain various components for removing cells from liver tissue, for example, hypertonic saline, peracetic acid, Triton-X, SDS or other detergent components. In one embodiment of the present invention, the decellularization solution may contain 0.1-5% Triton X-100 and 0.01-0.5% ammonium hydroxide, more specifically, 1% Triton X-100 and 0.1% ammonium hydroxide. By using the above-mentioned decellularization solution, decellularization can be carried out under milder conditions than in the existing process, and at the same time, various proteins in the liver tissue can be more preserved while effectively removing DNA in the prepared support.

[0121] The stirring may be carried out for 24 to 72 hours, more specifically 36 to 60 hours, most specifically 40 to 56 hours, and for example 48 hours, and through this stirring (decellularization) process, 95 to 99.9%, more specifically 96 to 98%, and most specifically 97.18% of the liver tissue cells may be removed. If decellularization is carried out for a time or to a level of liver tissue cell removal outside the above range, there is a problem that the quality of the produced scaffold composition may decrease or the economic efficiency of the process may decrease.

[0122] The step (b) is a step of drying the decellularized liver tissue to produce a decellularized liver tissue-derived extracellular matrix (LEM).

[0123] The method for drying the decellularized liver tissue may be performed by a known method, and the tissue may be naturally dried or freeze-dried. For sterilization, the tissue may be exposed to ethylene oxide gas or supercritical carbon dioxide after drying, or by electron beam or gamma radiation.

[0124] In one embodiment of the present invention, after step (b), the extracellular matrix derived from decellularized liver tissue may be contained at 0.01 to 10 mg / mL, specifically 0.5 to 9 mg / mL, more specifically 1 mg / mL to 8 mg / mL, most specifically 2, 4, 6 or 8 mg / mL, and in an optimized embodiment, 4 or 6 mg / mL. If it is contained at a concentration outside the above range, the intended effect of the present invention may not be obtained, or it may be incompatible for production or utilization.

[0125] The dried extracellular matrix may be comminuted by methods including steps of tearing, milling, cutting, grinding, and shearing. The comminuted extracellular matrix may be processed into a powder form in a frozen or freeze-dried state by methods such as grinding or milling.

[0126] The step (c) is a step of gelating the extracellular matrix derived from the dried decellularized liver tissue.

[0127] Through the gelation, the extracellular matrix derived from the decellularized liver tissue can be crosslinked to prepare a 3D hydrogel-type scaffold, and the gelled scaffold can be used in a variety of ways in fields related to organoid culture as well as experiments and screening.

[0128] The "hydrogel" is a material in which a liquid with water as a dispersion medium becomes solid and loses fluidity through a sol-gel phase transition, forming a porous structure, and can be formed when a hydrophilic polymer with a three-dimensional network structure and an amorphous structure absorbs water and expands.

[0129] The gelation may be carried out by dissolving the extracellular matrix derived from decellularized liver tissue in an acidic solution with a proteolytic enzyme such as pepsin or trypsin, adjusting the pH to neutral and the electrolyte conditions of 1X PBS buffer using 10X PBS and 1M NaOH, and at a temperature of 37°C for 30 minutes.

[0130] Another aspect of the present invention provides a method for culturing hepatic organoids from the support composition or a support composition produced by the production method.

[0131] Existing Matrigel-based culture systems are extracts derived from animal cancer tissues, and have large differences in configuration, making it impossible to mimic the actual liver environment and therefore inefficient in differentiating and developing into hepatic organoids. In contrast, the support composition can create an environment similar to liver tissue, making it suitable for culturing hepatic organoids.

[0132] The culturing refers to the process of maintaining and growing cells under suitable conditions, which may refer, for example, to the temperature at which the cells are maintained, the solubility of nutrients, the content of atmospheric CO2, and the cell density.

[0133] Appropriate culture conditions for maintaining, growing, expanding and differentiating different types of cells are known and documented in the art. Conditions suitable for the formation of said organoids may be conditions that facilitate or permit the differentiation of cells and the formation of multicellular structures.

[0134] In relation to a support derived from decellularized lung tissue for the culture and transplantation of lung organoids and a method for producing the same, one aspect of the present invention provides a support composition comprising a lung tissue-derived extracellular matrix (LuEM).

[0135] The term "extracellular matrix" refers to a natural support for cell growth produced through decellularization of tissues found in mammals and multicellular organisms. The extracellular matrix can be further processed through dialysis or crosslinking.

[0136] The extracellular matrix can be a mixture of structural and non-structural biomolecules, including but not limited to collagens, elastins, laminins, glycosaminoglycans, proteoglycans, antimicrobials, chemoattractants, cytokines, and growth factors.

[0137] The extracellular matrix may contain about 90% collagen in various forms in mammals. Extracellular matrices derived from various biological tissues may differ in overall structure and composition due to the specific roles required for each tissue.

[0138] The terms "derive" and "derived" refer to ingredients that are obtained from the stated source in any useful manner.

[0139] In one embodiment of the present invention, the extracellular matrix derived from the decellularized lung tissue may be at a concentration of 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, 3, 5 or 7 mg / mL, and an optimized embodiment is 7 mg / mL. If the concentration is outside the above range, the intended effect of the present invention may not be obtained, or the production or utilization may be inappropriate.

[0140] As a specific example of the present invention, the composition may have a 0.1-10 Hz reference elastic modulus of 1-100 Pa. When the composition has an elastic modulus in the above range, a stable polymer network can be formed.

[0141] The support composition comprises a three-dimensional hydrogel produced based on a decellularized lung tissue matrix composition, and can be effectively used for culturing lung organoids.

[0142] The decellularized lung tissue contains components of the actual tissue-specific extracellular matrix, and thus can provide the physical, mechanical and biochemical environment of the tissue, which is highly efficient in promoting differentiation into lung tissue cells and tissue-specific functionality.

[0143] The term "organoid" refers to a micro-organ produced by culturing cells derived from tissues or full-potential stem cells in a 3D form and fabricating them in the form of an artificial organ.

[0144] The organoids are three-dimensional tissue analogs containing organ-specific cells that arise from stem cells and self-organize (or self-pattern) in a manner similar to the in vivo state, and can develop into specific tissues through restricted growth factor patterning.

[0145] The organoids have the inherent physiological properties of cells and can have an anatomical structure that mimics the original state of a cell mixture (including not only a limited cell type, but also residual stem cells and adjacent physiological niches). The organoids can have a functional organ-like morphology and tissue-specific functions, with cells and cell functions better arranged through a 3D culture method.

[0146] Another aspect of the present invention provides a method for producing a support composition, comprising the steps of: (a) decellularizing isolated lung tissue to produce decellularized lung tissue; (b) drying the decellularized lung tissue to produce an extracellular matrix of decellularized lung tissue (Lung Extracellular Matrix, LuEM); and (c) gelating the extracellular matrix derived from the dried decellularized lung tissue.

[0147] The step (a) is a step of decellularizing the isolated lung tissue to produce decellularized lung tissue.

[0148] In one embodiment of the present invention, in the step (a), the separated lung tissue may further be minced before decellularization. The present invention includes a step of mincing the lung tissue before decellularization, which allows for more efficient and complete removal of cells in the decellularization process. The method for mincing the separated lung tissue may be performed using known methods (instruments) and sizes.

[0149] In one embodiment of the present invention, in step (a), the lung tissue may be agitated with the decellularization solution.

[0150] The decellularization solution may contain various components for removing cells from lung tissue, for example, hypertonic saline, peracetic acid, Triton X-100, SDS or other detergent components, and in one embodiment of the present invention, the decellularization solution may contain 0.1 to 5% Triton X-100 and 0.01 to 0.5% ammonium hydroxide, more specifically, 1% Triton X-100 and 0.1% ammonium hydroxide. By using the above-mentioned decellularization solution, decellularization can be carried out under milder conditions than in the existing process, and at the same time, various proteins in the lung tissue can be more preserved while effectively removing DNA in the prepared support.

[0151] The stirring may be carried out for 24 to 72 hours, more specifically 36 to 60 hours, most specifically 40 to 56 hours, and for example 48 hours, and through this stirring (decellularization) process, 95 to 99.9%, more specifically 96 to 98%, and most specifically 96.75% of the lung tissue cells may be removed. If decellularization is carried out for a time or to a level of removal of lung tissue cells outside the above range, there is a problem that the quality of the produced scaffold composition may be reduced or the economic efficiency of the process may be reduced.

[0152] The step (b) is a step of drying the decellularized lung tissue to produce a lung tissue extracellular matrix (Lung Extracellular Matrix, LuEM).

[0153] The decellularized lung tissue can be dried by a known method, and can be naturally dried or freeze-dried. For sterilization, the tissue can be exposed to ethylene oxide gas or supercritical carbon dioxide after drying, or by electron beam or gamma radiation.

[0154] In one embodiment of the present invention, after step (b), the concentration of the extracellular matrix derived from decellularized lung tissue may be 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, 3, 5 or 7 mg / mL, and in an optimized embodiment, 7 mg / mL. If the concentration is outside the above range, the intended effect of the present invention may not be obtained, or the production or utilization may be incompatible.

[0155] The dried extracellular matrix may be comminuted by methods including steps of tearing, milling, cutting, grinding, and shearing. The comminuted extracellular matrix may be processed into a powder form in a frozen or freeze-dried state by methods such as grinding or milling.

[0156] The step (c) is a step of gelating the extracellular matrix derived from the dried decellularized lung tissue.

[0157] Through the gelation, the extracellular matrix derived from the decellularized lung tissue can be crosslinked to produce a three-dimensional hydrogel-type scaffold, and the gelled scaffold can be used in a variety of ways in fields related to organoid culture as well as experiments and screening.

[0158] The "hydrogel" is a material in which a liquid with water as a dispersion medium becomes solid and loses fluidity through a sol-gel phase transition, forming a porous structure, and can be formed when a hydrophilic polymer with a three-dimensional network structure and an amorphous structure absorbs water and expands.

[0159] The gelation may be carried out by dissolving the extracellular matrix from decellularized intestinal tissue in an acidic solution with a proteolytic enzyme such as pepsin or trypsin, adjusting the neutral pH and electrolyte conditions of 1X PBS buffer using 10X PBS and 1M NaOH, at a temperature of 37°C for 30 minutes.

[0160] Another aspect of the present invention provides a method for culturing pulmonary organoids on the support composition or on a support composition produced by the production method.

[0161] Existing Matrigel-based culture systems are extracts derived from animal cancer tissues, and have large differences in configuration, making it impossible to mimic the actual lung environment, and therefore the efficiency of differentiation and development into pulmonary organoids is insufficient. In contrast, the support composition can create an environment similar to lung tissue, making it suitable for culturing pulmonary organoids.

[0162] The culturing refers to the process of maintaining and growing cells under suitable conditions, which may refer, for example, to the temperature at which the cells are maintained, the solubility of nutrients, the content of atmospheric CO2, and the cell density.

[0163] Appropriate culture conditions for maintaining, growing, expanding and differentiating different types of cells are known and documented in the art. Conditions suitable for the formation of said organoids may be conditions that facilitate or permit the differentiation of cells and the formation of multicellular structures. Effect of the Invention

[0164] In relation to the scaffold derived from decellularized intestinal tissue for the culture and transplantation of intestinal organoids and its manufacturing method, the artificial scaffold derived from decellularized intestinal tissue developed in the present invention has been developed as a new culture scaffold for intestinal organoids that overcomes the limitations of Matrigel, a representative existing scaffold for organoid culture, and is expected to be utilized as a core technology for a variety of preclinical and clinical research, such as a large-scale new drug screening platform based on intestinal organoids and cell therapeutics for tissue regeneration, creating high added value from industrial and economic perspectives and promoting the development of a new medical industry.

[0165] The artificial matrix scaffold derived from decellularized intestinal tissue is expected to be widely used in various fields, such as disease modeling research to clarify the mechanism of various intractable intestinal diseases (inflammatory bowel disease, Crohn's disease, colon cancer, etc.) by ex vivo expression and construction of transplantation treatment platforms. As the prevalence of such intractable intestinal diseases has increased significantly recently and much research is required, it is possible to generate revenue as a research reagent. In addition, by combining intestinal organoids with in situ microbial co-culture in the microenvironment of intestinal tissue-specific extracellular matrix, it can be used for research on the microbiome, which has been attracting much attention recently but has been difficult to study substantively, and is expected to have further application potential.

[0166] The artificial scaffold developed in this invention can be applied to intestinal organoids derived from human induced pluripotent stem cells and embryonic stem cells, and 3D spheroid cultures derived from colorectal cancer, which contributes to the construction of disease models specific to intractable diseases and cancer patients and can be used as a precision medicine platform technology. Considering the size of the precision medicine market, which has been rapidly expanding recently, it is expected to create enormous added value.

[0167] Overall, as mentioned above, for the application of intestinal organoids, a culture scaffold called Matrigel is essentially required. Compared to Matrigel, the artificial scaffold developed in the present invention shows functionality equal to or greater than Matrigel as a culture system, is safer, and has a very advantageous advantage in terms of cost. Unlike the existing commonly used Matrigel culture scaffold, it can be commercialized to a GMP standard that allows transplantation into humans. Therefore, it is expected that the replacement effect of Matrigel will generate huge economic profits.

[0168] In relation to the scaffold derived from decellularized gastric tissue for the culture and transplantation of gastric organoids and its manufacturing method, the artificial scaffold derived from decellularized gastric tissue developed in the present invention has been developed as a new culture scaffold for gastric organoids that overcomes the limitations of Matrigel, a representative existing scaffold for organoid culture. It is expected to be utilized as a core technology for a variety of preclinical and clinical research, such as a large-scale new drug screening platform based on gastric organoids and cell therapeutics for tissue regeneration, and to create high added value from industrial and economic perspectives, thereby promoting the development of a new medical industry.

[0169] The artificial matrix scaffold derived from decellularized gastric tissue is expected to be widely used in various fields, such as disease modeling research to clarify the mechanism of various intractable gastric diseases (gastric ulcer, gastric cancer, etc.) by realizing them in vitro and establishing a transplantation treatment platform. As the prevalence of such intractable gastric diseases has increased significantly recently and many studies are required, it is possible to generate revenue as a research reagent. In addition, in conjunction with co-culture of gastric organoids with infectious bacteria (e.g. Helicobacter pylori) in the microenvironment of gastric tissue-specific extracellular matrix, it can be used in basic research that has been attracting much attention recently but has been difficult to conduct practically, so it is expected to have further application potential.

[0170] The artificial support developed in the present invention can be applied to the culture of not only gastric organoids derived from tissue stem cells but also organoids derived from gastric cancer, contributing to the construction of disease models specific to intractable diseases and cancer patients, and can also be used as a precision medicine platform technology. Considering the size of the precision medicine market, which has been rapidly expanding recently, it is expected to create enormous added value.

[0171] Overall, as mentioned above, for the application of gastric organoids, a culture scaffold called Matrigel is essentially required. Compared to Matrigel, the artificial scaffold developed in the present invention shows functionality equal to or greater than Matrigel as a culture system, is safer, and has a very advantageous advantage in terms of cost. Therefore, it is predicted that the replacement effect of Matrigel alone will generate huge economic profits.

[0172] In relation to the scaffold derived from decellularized liver tissue for the culture and transplantation of hepatic organoids and its manufacturing method, the artificial scaffold derived from decellularized liver tissue developed in the present invention has been developed as a new culture scaffold for hepatic organoids that overcomes the limitations of Matrigel, a representative existing scaffold for organoid culture, and is expected to be utilized as a core technology for a variety of preclinical and clinical research, such as a large-scale new drug screening platform based on hepatic organoids and cell therapeutics for tissue regeneration, creating high added value from industrial and economic perspectives and promoting the development of a new medical industry.

[0173] By using the scaffold derived from the decellularized liver tissue developed in the present invention, it is possible to produce more advanced liver organoids than the existing culture methods, and it can be used as an economical and more accurate platform that surpasses the existing in vitro models for drug testing. Since hepatotoxicity is an important indicator that must be analyzed in new drug development and drug evaluation, it is expected that the advanced in vitro model platform based on liver organoids will greatly increase the success rate of new drug development and greatly reduce costs and time required, thereby greatly contributing to the development of the medical industry.

[0174] The artificial matrix scaffold derived from decellularized liver tissue is expected to be widely used in various fields, such as disease modeling research to clarify the mechanisms of various intractable liver diseases (liver sclerosis, liver fibrosis, non-alcoholic hepatitis, etc.) by ex vivo expression and construction of transplant treatment platforms. As the prevalence of such intractable liver diseases has increased significantly recently and much research is required, it is possible to generate revenue as a research reagent.

[0175] The artificial support developed in this invention can be applied to the culture of not only liver organoids derived from tissue stem cells but also liver cancer organoids, contributing to the construction of disease models specific to intractable diseases and cancer patients, and can also be used as a precision medicine platform technology. Considering the size of the precision medicine market, which has been rapidly expanding recently, it is expected to create enormous added value.

[0176] Overall, as mentioned above, for the application of liver organoids, a culture scaffold called Matrigel is essentially required. Compared to Matrigel, the artificial scaffold developed in the present invention shows functionality equal to or greater than Matrigel as a culture system, is safer, and has a very advantageous advantage in terms of cost. Therefore, it is predicted that the creation of huge economic profits will be achieved just by replacing Matrigel.

[0177] In relation to the scaffold derived from decellularized lung tissue for the culture and transplantation of pulmonary organoids and its manufacturing method, the artificial scaffold derived from decellularized lung tissue developed in the present invention has been developed as a new culture scaffold for pulmonary organoids that overcomes the limitations of Matrigel, a representative existing scaffold for organoid culture. It is expected to be utilized as a core technology for various preclinical and clinical research, such as a large-scale new drug screening platform based on pulmonary organoids and cell therapeutics for tissue regeneration, and to create high added value from industrial and economic perspectives and promote the development of a new medical industry.

[0178] The lung organoids established in the present invention can be used to create various lung disease models that are currently difficult to realize because they can structurally and functionally copy lung tissue very precisely. Currently, fine dust caused by air pollution induced in the process of industrialization and urbanization has become a serious social problem, and the harmful effects on the human body have not yet been clearly investigated, and there is no suitable model for studying it. In addition, it is necessary to create various intractable lung disease models such as cystic fibrosis, idiopathic pulmonary fibrosis, and chronic obstructive pulmonary disease. The culture technique of mature lung organoids established through the present invention is expected to make it easier to create such lung disease models and be used in research on the mechanism of lung disease, thereby making a great contribution to medical research.

[0179] The decellularized scaffold developed in the present invention can be applied to the culture of not only lung organoids derived from tissue stem cells but also organoids derived from lung cancer, contributing to the construction of disease models specific to intractable diseases and cancer patients, and can also be utilized as a precision medicine platform technology. Considering the size of the precision medicine market, which has been rapidly expanding recently, it is expected to create enormous added value.

[0180] For the culture of pulmonary organoids, Matrigel is indispensably required as a culture support. Compared to Matrigel, which has various problems, the decellularized support developed in the present invention shows functionality equal to or greater than Matrigel as a culture system, is safer, and has very advantageous advantages in terms of cost. Therefore, it is predicted that the replacement effect of Matrigel alone will generate huge economic profits. [Brief description of the drawings]

[0181] [Figure 1] FIG. 1 shows a schematic diagram of the fabrication of an artificial scaffold derived from decellularized intestinal tissue. [Diagram 2] FIG. 14. Fabrication and microstructural analysis of decellularized intestinal tissue-derived hydrogel scaffolds. [Diagram 3] FIG. 1 shows an analysis of the components of an artificial support derived from decellularized intestinal tissue. [Figure 4] FIG. 1 shows the results of a comparison between the decellularization protocol of the present invention and existing protocols, comparing matrix components and physical properties. [Diagram 5] FIG. 1 shows the results of a comparison between the decellularization protocol of the present invention and existing protocols, comparing matrix components and physical properties. [Figure 6] FIG. 1 is a diagram showing the results of a comparison between the decellularization protocol of the present invention and existing protocols, comparing the aspects of organoid culture. [Figure 7] FIG. 1 is a comparative analysis of the protein structure of the scaffold composition of the present invention and that of the existing culture scaffold (Matrigel). [Figure 8] FIG. 1 is a comparative analysis of the protein structure of the scaffold composition of the present invention and that of the existing culture scaffold (Matrigel). [Figure 9] FIG. 1 is a comparative analysis of the protein structure of the scaffold composition of the present invention and that of the existing culture scaffold (Matrigel). [Figure 10] FIG. 1 shows an analysis of the physical properties of a hydrogel scaffold derived from decellularized intestinal tissue. [Figure 11] FIG. 1 shows the optimal concentration of decellularized intestinal tissue-derived hydrogel scaffolds for the culture of intestinal organoids. [Figure 12] FIG. 1 shows the growth pattern of intestinal organoids cultured on a hydrogel scaffold derived from decellularized intestinal tissue. [Figure 13] FIG. 1 shows the analysis of the expression of stem cell and differentiation markers in intestinal organoids cultured from hydrogel scaffolds derived from decellularized intestinal tissue (cell immunostaining analysis). [Figure 14] FIG. 1 shows the analysis of the expression of stem cell and differentiation markers of intestinal organoids cultured from hydrogel supports derived from decellularized intestinal tissue (analysis of gene expression through quantitative PCR). [Figure 15] FIG. 1 shows the functionality of intestinal organoids cultured from decellularized intestinal tissue-derived hydrogel scaffolds. [Figure 16]This figure shows the analysis of gene expression profiles of intestinal organoids cultured from hydrogel supports derived from decellularized intestinal tissue (RNA-sequencing analysis). [Figure 17] This figure shows the analysis of gene expression profiles of intestinal organoids cultured from hydrogel supports derived from decellularized intestinal tissue (RNA-sequencing analysis). [Figure 18] These results are the results of analyzing the survival and development of intestinal organoids as a tissue-specific effect of extracellular matrix support derived from decellularized intestinal tissue. [Figure 19] These are the results of a comparative analysis of tissue-specific extracellular matrix components of scaffolds derived from decellularized tissue. [Figure 20] This is the result of a comparative analysis of the culture appearance and extracellular matrix of organoids depending on the tissue age of the scaffold derived from decellularized intestinal tissue. [Figure 21] FIG. 14: The ability of decellularized intestinal tissue-derived hydrogel scaffolds to support organ culture of intestinal organoids was examined. [Figure 22] This figure examines the possibility of long-term frozen storage of a biomimetic artificial scaffold derived from decellularized intestinal tissue. [Figure 23] This figure examines the possibility of long-term refrigerated storage of a biomimetic artificial scaffold derived from decellularized intestinal tissue. [Figure 24] FIG. 1 shows the application of decellularized intestinal tissue-derived hydrogel as a support for the transplantation of intestinal organoids in vivo. [Diagram 25] FIG. 1 shows the transplantation and engraftment induction of intestinal organoids into a living body using a hydrogel derived from decellularized intestinal tissue. [Figure 26] FIG. 1 shows the transplantation and engraftment induction of intestinal organoids into a living body using a hydrogel derived from decellularized intestinal tissue. [Figure 27] This figure confirms the feasibility of culturing intestinal organoids derived from human induced pluripotent stem cells and embryonic stem cells on a hydrogel scaffold derived from decellularized intestinal tissue. [Figure 28]FIG. 1 shows the formation and culture of colorectal cancer-derived 3D organoids on decellularized intestinal tissue-derived hydrogel scaffolds. [Figure 29] These results confirm the feasibility of cryopreserving organoids using a hydrogel support derived from decellularized intestinal tissue. [Diagram 30] These results confirmed the feasibility of mass culturing intestinal organoids using a hydrogel scaffold derived from decellularized intestinal tissue and a microfluidic chip. [Diagram 31] FIG. 1 shows the fabrication of a support derived from decellularized stomach tissue (Stomach Extracellular Matrix; SEM) for the culture of gastric organoids. [Diagram 32] FIG. 1 shows an analysis of the characteristics of the fabricated decellularized gastric tissue-derived scaffold (SEM). [Diagram 33] FIG. 1 shows an analysis of the physical properties of a scaffold derived from decellularized gastric tissue (SEM) depending on its concentration. [Diagram 34] This is the result of a comparative analysis of gastric tissue decellularization methods. [Diagram 35] This is the result of a comparative analysis of gastric tissue decellularization methods. [Diagram 36] This is the result of comparing the culture conditions of gastric organoids using different methods of decellularization of gastric tissue. [Figure 37] FIG. 1 shows the protein content of scaffolds (SEM) derived from decellularized gastric tissue. [Figure 38] This shows the results of the type and quantitative analysis of matrisome proteins in scaffolds (SEM) derived from decellularized gastric tissue. [Figure 39] This shows the results of the type and quantitative analysis of matrisome proteins in scaffolds (SEM) derived from decellularized gastric tissue. [Diagram 40] This is the result of an analysis of non-matrisome proteins in scaffolds (SEM) derived from decellularized gastric tissue. [Diagram 41] FIG. 1 shows the optimum concentration of hydrogel scaffold derived from decellularized stomach tissue for culturing gastric organoids. [Diagram 42]FIG. 13 shows a comparison of the culture state of gastric organoids depending on the concentration of hydrogel scaffold derived from decellularized gastric tissue. [Diagram 43] FIG. 1 shows a comparison of the growth of gastric organoids formed on a hydrogel scaffold derived from decellularized gastric tissue and on an existing culture scaffold (Matrigel). [Diagram 44] FIG. 1 shows a comparison of gastric organoids formed on a hydrogel scaffold derived from decellularized gastric tissue and on an existing culture scaffold (Matrigel). [Diagram 45] FIG. 13 shows a comparative analysis of the acid secretion function of gastric organoids cultured from a hydrogel scaffold derived from decellularized gastric tissue and from Matrigel. [Diagram 46] FIG. 1 shows the effect of promoting differentiation of gastric organoids by mixing a hydrogel support derived from decellularized gastric tissue with Matrigel. [Figure 47] FIG. 1 shows the effect of enhancing the function of gastric organoids by mixing a hydrogel support derived from decellularized gastric tissue with Matrigel. [Figure 48] FIG. 13 confirms the tissue-specific effect of a support derived from decellularized gastric tissue for the culture of gastric organoids. [Figure 49] These results confirm the differences in the components of the extracellular matrix between skin tissue and stomach tissue. [Figure 50] This shows the results of an analysis of differences in the components of the extracellular matrix of scaffolds derived from decellularized gastric tissue according to tissue age. [Figure 51] FIG. 1 shows optimization of culture medium conditions for organ culture of gastric organoids on hydrogel scaffolds derived from decellularized gastric tissue. [Figure 52] FIG. 1 shows organ culture of gastric organoids using a hydrogel scaffold derived from decellularized gastric tissue. [Diagram 53] FIG. 1 shows an analysis of transcriptome expression in gastric organoids cultured using hydrogel scaffolds derived from decellularized gastric tissue. [Figure 54] FIG. 1 shows an analysis of transcriptome expression in gastric organoids cultured using hydrogel scaffolds derived from decellularized gastric tissue. [Figure 55] FIG. 1 shows the transplantation of gastric organoids into a living body using a hydrogel scaffold derived from decellularized gastric tissue. [Figure 56] This figure examines the possibility of long-term storage of hydrogel derived from decellularized gastric tissue. [Figure 57] This figure examines the possibility of long-term storage of hydrogel derived from decellularized gastric tissue. [Figure 58] FIG. 1 shows the culture of gastric cancer organoids using a hydrogel scaffold derived from decellularized gastric tissue. [Figure 59] The results confirmed the feasibility of mass culturing gastric organoids using a microfluidic device and a hydrogel derived from decellularized gastric tissue. [Figure 60] FIG. 1 shows the preparation of a support derived from decellularized liver tissue (Liver Extracellular Matrix; LEM) for the culture of hepatic organoids. [Figure 61] FIG. 13: Analysis of decellularized liver tissue-derived supports (LEM) for the culture of hepatic organoids. [Figure 62] FIG. 1 shows an analysis of the physical properties of a scaffold derived from decellularized liver tissue (LEM) depending on its concentration. [Figure 63] FIG. 1 shows the protein content of the scaffold (LEM) derived from decellularized liver tissue. [Figure 64] FIG. 1 shows the protein content of the scaffold (LEM) derived from decellularized liver tissue. [Figure 65] FIG. 1 shows the culture and concentration selection of hepatic organoids (cholangiocyte-derived liver organoids; CLO) depending on the concentration of hydrogel support derived from decellularized liver tissue. [Figure 66] FIG. 13 shows an analysis of the difference in differentiation potential of hepatic organoids (bile duct cell-derived hepatic organoids, CLO) depending on the LEM concentration of the hydrogel scaffold derived from decellularized liver tissue. [Figure 67]FIG. 1 shows the analysis of hepatic organoids (cholangiocyte-derived hepatic organoids, CLO) cultured from hydrogel scaffolds derived from decellularized liver tissue. [Figure 68] FIG. 1 shows an analysis of the functionality of hepatic organoids (cholangiocyte-derived hepatic organoids, CLO) cultured from hydrogel scaffolds derived from decellularized liver tissue. [Figure 69] FIG. 1 shows organ culture of hepatic organoids (bile duct cell-derived hepatic organoids, CLO) using a hydrogel support derived from decellularized liver tissue. [Figure 70] FIG. 1 shows the culture and analysis of human hepatic organoids (human cholangiocyte-derived hepatic organoids - hCLO) using a hydrogel support derived from decellularized liver tissue. [Figure 71] FIG. 1 shows the culture and analysis of human hepatic organoids (human cholangiocyte-derived hepatic organoids - hCLO) using a hydrogel support derived from decellularized liver tissue. [Figure 72] FIG. 1 shows the culture and analysis of human hepatic organoids (human cholangiocyte-derived hepatic organoids - hCLO) using a hydrogel support derived from decellularized liver tissue. [Figure 73] FIG. 1 shows the culture of hepatic organoids (hepatocyte-derived liver organoids; HLO) using different concentrations of hydrogel scaffolds derived from decellularized liver tissue, and the selection of the optimal concentration. [Figure 74] FIG. 1 shows a comparison of the growth of hepatic organoids (HLO) formed on a hydrogel scaffold (LEM) derived from decellularized liver tissue and on a conventional culture scaffold (MAT). [Figure 75] FIG. 1 shows the analysis of hepatic organoids (HLO) derived from hepatocytes cultured from decellularized liver tissue-derived hydrogel scaffolds (LEM). [Figure 76] FIG. 1 shows the culture of hepatic organoids derived from human-induced pluripotent stem cells (hiPSCs) using a hydrogel scaffold derived from decellularized liver tissue. [Figure 77]FIG. 13. Analysis of human induced pluripotent stem cell-derived hepatic organoids (hiPSC-LO) cultured from decellularized liver tissue-derived hydrogel supports. [Figure 78] FIG. 1 shows a liver fibrosis model created using hepatic organoids (CLOs) cultured from decellularized liver tissue-derived hydrogel scaffolds (LEM). [Figure 79] FIG. 1 shows the construction of a liver fibrosis model using hepatic organoids (hCLO) cultured from a hydrogel scaffold derived from decellularized liver tissue. [Figure 80] FIG. 1 shows the transplantation of hepatic organoids into a living body using a hydrogel scaffold derived from decellularized liver tissue. [Figure 81] FIG. 1 is a diagram verifying the possibility of long-term storage of hydrogel derived from decellularized liver tissue. [Figure 82] FIG. 1 is a diagram verifying the possibility of long-term storage of hydrogel derived from decellularized liver tissue. [Figure 83] These are the results of culturing liver cancer organoids using a hydrogel scaffold derived from decellularized liver tissue. [Figure 84] 1 shows the results of verifying the superiority of the scaffold derived from decellularized liver tissue of the present invention through a comparison of liver tissue decellularization protocols. [Figure 85] 1 shows the results of verifying the superiority of the scaffold derived from decellularized liver tissue of the present invention through a comparison of liver tissue decellularization protocols. [Figure 86] 1 shows the results of verifying the superiority of the scaffold derived from the decellularized liver tissue of the present invention through a comparison of liver tissue decellularization protocols (organoid culture experiment). [Figure 87] Results of an interspecies comparison analysis of decellularized liver tissue-derived hydrogel scaffolds (pig vs. human). [Figure 88] Comparison of human hepatic organoids (human cholangiocyte-derived hepatic organoids - hCLO) cultured on hydrogel supports derived from human and porcine decellularized liver tissue. [Figure 89]FIG. 1 shows the fabrication and analysis of a decellularized lung tissue-derived extracellular matrix support (Lung Extracellular Matrix, LuEM) for the culture of lung organoids. [Figure 90] This figure shows an analysis of the physical properties of the extracellular matrix support (Lung Extracellular Matrix, LuEM) derived from decellularized lung tissue for the culture of lung organoids. [Figure 91] This figure shows the protein content of Lung Extracellular Matrix (LuEM), a decellularized lung tissue-derived extracellular matrix support for the culture of lung organoids. [Figure 92] These are the results of a quantitative analysis of the types of matrisome proteins in the extracellular matrix support (Lung Extracellular Matrix, LuEM) derived from decellularized lung tissue. [Figure 93] These are the results of an analysis of non-matrisome proteins in the extracellular matrix support (Lung Extracellular Matrix, LuEM) derived from decellularized lung tissue. [Figure 94] 1 shows the results of a comparative analysis of decellularized lung tissues obtained using different decellularization methods. [Figure 95] 1 shows the results of a comparative analysis of decellularized lung tissues obtained using different decellularization methods. [Figure 96] This figure shows an analysis of mouse lung organoid formation at different concentrations of extracellular matrix support (Lung Extracellular Matrix, LuEM) derived from decellularized lung tissue. [Figure 97] This figure shows an analysis of the degree of differentiation of mouse lung organoids depending on the concentration of extracellular matrix support (Lung Extracellular Matrix, LuEM) derived from decellularized lung tissue. [Figure 98] This figure shows a comparative analysis of the expression of specific marker proteins in mouse lung organoids cultured from a support of extracellular matrix derived from decellularized lung tissue (Lung Extracellular Matrix, LuEM) and Matrigel. [Figure 99]These results are the results of an analysis of the functionality of mouse lung organoids cultured from a support of extracellular matrix (Lung Extracellular Matrix, LuEM) derived from decellularized lung tissue and Matrigel. [Figure 100] FIG. 1 shows the analysis of mouse lung organoids cultured on a support made of extracellular matrix (Lung Extracellular Matrix, LuEM) derived from decellularized lung tissue. [Figure 101] FIG. 1 shows the analysis of mouse lung organoids cultured on a support made of extracellular matrix (Lung Extracellular Matrix, LuEM) derived from decellularized lung tissue. [Figure 102] These results confirm the tissue-specific effects of an extracellular matrix support derived from decellularized lung tissue (Lung Extracellular Matrix, LuEM). [Figure 103] These results show the culture of human lung organoids using a support made of extracellular matrix (Lung Extracellular Matrix, LuEM) derived from decellularized lung tissue. [Figure 104] FIG. 1 shows the feasibility of creating a pulmonary fibrosis model using mouse lung organoids cultured from a decellularized lung tissue-derived extracellular matrix support (Lung Extracellular Matrix, LuEM). [Figure 105] FIG. 1 shows the creation of a human pulmonary fibrosis model using human lung organoids cultured from a decellularized lung tissue-derived extracellular matrix support (Lung Extracellular Matrix, LuEM). [Fig. 106] These are the results of an analysis of the functionality of a human pulmonary fibrosis model constructed from an extracellular matrix scaffold derived from decellularized lung tissue (Lung Extracellular Matrix, LuEM). [Figure 107] FIG. 1 shows the creation of a fine dust disease model using mouse lung organoids cultured from a support of extracellular matrix (Lung Extracellular Matrix, LuEM) derived from decellularized lung tissue. [Figure 108]These are the results of an investigation into the possibility of long-term storage of an extracellular matrix support (Lung Extracellular Matrix, LuEM) derived from decellularized lung tissue. [Fig. 109] This figure confirms the possibility of forming lung organoids under support-free culture conditions using components of the extracellular matrix (Lung Extracellular Matrix, LuEM) derived from decellularized lung tissue. [Figure 110] This shows the results of an analysis of gene and protein expression in mouse lung organoids formed without a culture support. [Figure 111] These are the results of an analysis of protein expression in human lung organoids formed without a culture support. [Figure 112] These are the results of an analysis of protein expression in human lung organoids formed without a culture support. [Figure 113] FIG. 1 shows an experiment on the treatment of fine dust of mouse lung organoids formed without a culture support. [Fig. 114] FIG. 1 shows the results of promoting alveolar differentiation of mouse lung organoids using a support of extracellular matrix (Lung Extracellular Matrix, LuEM) derived from decellularized lung tissue. [Fig. 115] This is the formation of a monolayer of human lung organoids cultured on a support of extracellular matrix (Lung Extracellular Matrix, LuEM) derived from decellularized lung tissue, and the results of analyzing this. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0182] The present invention provides a support composition comprising an extracellular matrix derived from decellularized organ tissue.

[0183] In one embodiment of the present invention, the organ may be a support composition for any one of the intestine, stomach, liver, and lung.

[0184] In one specific example of the present invention, the extracellular matrix derived from the decellularized organ tissue may be contained in a concentration of 0.01 to 10 mg / mL.

[0185] The present invention also provides a method for producing a support composition, comprising the steps of: (a) decellularizing isolated organ tissue to produce decellularized organ tissue; (b) drying the decellularized organ tissue to produce an extracellular matrix derived from the decellularized organ tissue; and (c) gelating the extracellular matrix derived from the dried decellularized organ tissue.

[0186] In one embodiment of the present invention, the organ may be any one of an intestine, a stomach, a liver, and a lung.

[0187] As a specific example of the present invention, in step (a), the organ tissue may be agitated with the decellularization solution.

[0188] As a specific example of the present invention, the decellularization solution may contain 0.1 to 5% Triton X-100 and 0.01 to 0.5% ammonium hydroxide.

[0189] As a specific example of the present invention, the decellularization in step (a) may result in removal of 95 to 99.9% of organ tissue cells.

[0190] As a specific example of the present invention, after the step (b), the method may further comprise a step of adjusting the extracellular matrix derived from the decellularized organ tissue so that it is contained in a range of 0.01 to 10 mg / mL.

[0191] The present invention provides a method for organ culturing organoids on the support composition or a support composition produced by the production method.

[0192] In one embodiment of the present invention, the organ may be any one of an intestine, a stomach, a liver, and a lung.

[0193] The present invention relates to a scaffold derived from decellularized intestinal tissue for the culture and transplantation of intestinal organoids and a manufacturing method thereof. The present invention is a technology that removes cells through a decellularization process of intestinal tissue, preserves only the intestinal tissue-specific extracellular matrix, and uses this as an artificial scaffold for effective 3D culture of intestinal organoids. The hydrogel based on the decellularized intestinal tissue matrix shows potential as an alternative to Matrigel.

[0194] The artificial support of the present invention is composed of components of the extracellular matrix of pure water from which cellular antigens have been removed, and therefore does not cause inflammatory reactions and immune rejection of tissues during transplantation, has excellent biocompatibility, and is inexpensive. Therefore, the safety and economics of organoid culture and transplantation therapy can be greatly improved. In addition, compared with Matrigel, which is an existing, commonly used culture support for organoids, it provides a tissue-specific microenvironment for the growth of organoids, and therefore can greatly enhance the differentiation, development, and function of organoids. In other words, it is possible to provide a culture system suitable for the advancement of organoids.

[0195] It was confirmed that artificial scaffolds derived from decellularized intestinal tissue can be produced and applied at various concentrations, and that intestinal organoids can be formed at any concentration, and the various cells that make up intestinal tissue can be developed.

[0196] In addition, intestinal organoids cultured on the hydrogel scaffold derived from decellularized intestinal tissue showed similar characteristics to those cultured on Matrigel in terms of formation efficiency, growth rate, size, cell development and functionality, confirming that the scaffold developed in the present invention has the ability to replace Matrigel.

[0197] In the culture of intestinal organoids, it was confirmed that the intestinal tissue-derived scaffold had the most excellent efficacy compared to artificial matrix scaffolds made by decellularizing other types of tissues (heart, skin, lymph, stomach, muscle). This result suggests the importance of developing tissue-specific matrices for organoid culture through decellularization technology. It was found that scaffolds containing components of intestinal tissue-specific extracellular matrix provide a microenvironment optimized for the culture of intestinal organoids.

[0198] The decellularized intestinal tissue scaffold of the present invention allows long-term subculture of intestinal organoids. The characteristics and differentiation degree of intestinal stem cells are maintained at a constant level similar to that of Matrigel even during the organ culture period, and the developed scaffold is confirmed to be an effective culture system that can maintain stable organ culture of intestinal organoids. In addition, it has been confirmed that the hydrogel solution derived from decellularized intestinal tissue can be used for culturing intestinal organoids without problems even when used after long-term freezing (3 months or more) and refrigeration (1 month or more) storage, and it can be developed as a product that can be stored for a long period of time during use and is highly convenient for users.

[0199] The hydrogel scaffold derived from decellularized intestinal tissue of the present invention can be applied not only as a culture system but also as a transplantation material for the delivery of organoids to the body. In fact, it was confirmed through animal experiments that it is effective in transplanting and inducing engraftment of organoids into the damaged site of the intestine. It was confirmed that it can be successfully applied to intestinal organoids derived from various stem cells (e.g., human induced pluripotent stem cells, embryonic stem cells) as well as adult stem cells derived from intestinal tissue, and 3D spheroid culture derived from colon cancer, providing an in vitro model system capable of realizing various disease models.

[0200] In the following, preferred examples are presented to aid in understanding the present invention. However, the following examples are merely provided to facilitate understanding of the present invention, and the contents of the present invention are not limited to the following examples.

[0201] Example 1: Preparation of a scaffold composition comprising extracellular matrix derived from decellularized intestinal tissue

[0202] A scaffold composition comprising extracellular matrix derived from decellularized intestinal tissue was prepared as follows (see FIG. 1).

[0203] Example 1-1. Preparation of decellularized intestinal tissue-derived extracellular matrix (IEM)

[0204] The entire mucosal layer of the small intestine of a pig was prepared, and the intestinal tissue was stirred with 1% Triton X-100 and 0.1% ammonium hydroxide for 48 hours to remove only the cells of the intestinal tissue and produce decellularized intestinal tissue.

[0205] The decellularized intestinal tissue was then freeze-dried and pulverized to produce an extracellular matrix (IEM) derived from the decellularized intestinal tissue.

[0206] Example 1-2. Preparation of support composition

[0207] 10 mg of the decellularized intestinal tissue-derived extracellular matrix was dissolved in 4 mg / ml pepsin solution (4 mg of pepsin powder derived from porcine gastric mucosa dissolved in 1 ml of 0.02 M HCl) for 48 hours. Water: IEM solution: PBS buffer: NaOH were mixed in a ratio of 69:20:10:1, homogenously mixed, and gelled at 37°C for 30 minutes to prepare a hydrogel-type support composition.

[0208] Experimental Example 1: Analysis of extracellular matrix derived from decellularized intestinal tissue

[0209] The extracellular matrix derived from the decellularized intestinal tissue of Example 1-1 was analyzed as follows.

[0210] Experimental Example 1-1. Confirmation of the decellularization level of extracellular matrix derived from decellularized intestinal tissue

[0211] The decellularization level of the extracellular matrix derived from the decellularized intestinal tissue prepared in Example 1-1 and the presence or absence of hydrogel formation were confirmed.

[0212] As a result, as shown in Figure 2, the decellularized intestinal tissue of Example 1-1 was visible to the naked eye as the cells were removed by the decellularization process and the tissue turned white, but the general intestinal tissue structure remained intact (Figure 2(a)). When the hydrogel prepared using this was placed in a glass container and turned over after the gelation process, the solution did not spill out, confirming that the hydrogel had solidified through a crosslinking reaction caused by temperature (Figure 2(b)). In addition, when the microstructure inside the 3D IEM hydrogel prepared was analyzed using a scanning electron microscope (SEM), it was confirmed that it was composed of a nanofiber structure similar to collagen (Figure 2(c)).

[0213] Experimental Example 1-2. Analysis of components of extracellular matrix of decellularized intestinal tissue

[0214] The components of the extracellular matrix derived from the decellularized intestinal tissue prepared in Example 1-1 were analyzed and the preservation level was confirmed.

[0215] Specifically, DNA was extracted from intestinal tissue before and after decellularization and compared, and the glycosaminoglycan (GAG) content was compared through analysis of 1,9-dimethylmethylene blue dye solution.

[0216] For histological analysis, the tissues before and after decellularization were fixed in 4% paraformaldehyde solution for 1 day, embedded in paraffin, sectioned, and then stained with hematoxylin and eosin (H&E).

[0217] To analyze the protein content of the scaffold derived from decellularized intestinal tissue, the scaffold was dissolved in a dissolution buffer [4% sodium dodecyl sulfate (SDS), 0.1M Tris-HCl (pH 7.6) and 1X protease inhibitor], and the protein components were extracted and decomposed to the peptide level. The types and relative values ​​of peptides were then detected using a mass spectrometer and MaxQuant software. Detailed classification of matrisomes, etc. was then carried out using an existing library.

[0218] As a result, as shown in Figure 3, the amount of remaining DNA in the intestinal tissue before decellularization (before) and after decellularization (after) was compared, and it was confirmed that 97.68% of cells were efficiently removed through the decellularization process (Figure 3(A)). The amount of remaining GAG (glycosaminoglycan) in the intestinal tissue before decellularization (before) and after decellularization (after) was compared, and it was confirmed that the components of the extracellular matrix were maintained even after decellularization (Figure 3(B)). The results of comparing the hematoxylin and eosin staining of the intestinal tissue before decellularization and after decellularization confirmed that most of the cell nuclei were removed from the tissue after the decellularization process, while the components of the extracellular matrix were preserved (Figure 3(C)). The proteomic analysis of the decellularized intestinal tissue by batch using mass spectrometry confirmed that various types of intestinal tissue-specific extracellular matrix components commonly remained (Figure 3(D)).

[0219] Experimental Example 1-3. Comparison of the extracellular matrix derived from decellularized intestinal tissue of the present invention with the prior art (1)

[0220] The extracellular matrix derived from decellularized intestinal tissue of the present invention was compared with that of the prior art. Specifically, the decellularization process developed in the present invention was designated protocol 1, and the decellularization process of the prior art was designated protocol 2, and the same pig small intestine was used for the decellularization process.

[0221] Specifically, DNA was extracted from intestinal tissue before and after decellularization and compared, and the glycosaminoglycan (GAG) content was compared through analysis of 1,9-dimethylmethylene blue dye solution.

[0222] For histological analysis, the tissues before and after decellularization were fixed in 4% paraformaldehyde solution for 1 day, embedded in paraffin, sectioned, and then stained with hematoxylin and eosin (H&E).

[0223] To analyze the protein content of the scaffold derived from decellularized intestinal tissue, the scaffold was dissolved in a dissolution buffer [4% sodium dodecyl sulfate (SDS), 0.1M Tris-HCl (pH 7.6) and 1X protease inhibitor], and the protein components were extracted and decomposed to the peptide level. The types and relative values ​​of peptides were then detected using a mass spectrometer and MaxQuant software. Detailed classification of matrisomes, etc. was then carried out using an existing library.

[0224] As a result, when the amount of remaining DNA was compared between intestinal tissue that had not been decellularized (Native), intestinal tissue that had been decellularized using the protocol of the present invention (protocol 1), and intestinal tissue that had been decellularized using a previously reported protocol (protocol 2), it was confirmed that both processes were able to efficiently remove cells within the tissue (Figure 4(a)).

[0225] Furthermore, when comparing the remaining amount of GAG (glycosaminoglycan), it was confirmed that when the process of the present invention (protocol 1) was used, the GAG ​​content was preserved at the native tissue level, whereas when the existing process (protocol 2) was used, only about 16% of the GAG ​​content in the native tissue was preserved (Figure 4(b)).

[0226] Furthermore, when comparing the results of hematoxylin and eosin staining (H&E staining), it was confirmed that after the decellularization process, most of the cell nuclei were removed from the tissue, while the components of the extracellular matrix were preserved (Figure 4(c)).

[0227] Furthermore, when comparing the types of proteins detected in each extracellular matrix, it was confirmed that a greater variety of proteins remained in the intestinal tissue treated with the protocol of the present invention (Figure 4(d)).

[0228] On the other hand, when a 5 mg / ml hydrogel scaffold was prepared using the decellularized intestinal tissue prepared by each protocol and the mechanical properties (modulus) were compared, it was confirmed that the properties of the hydrogel scaffold derived from the decellularized intestinal tissue prepared by the protocol of the present invention were superior even at the same concentration (Figure 4(e)).

[0229] In contrast to the previously reported protocol (protocol 2), which uses an ionic sodium deoxycholate-based decellularization solution, the protocol of the present invention uses milder conditions based on a non-ionic solution of 1% Triton X-100 and 0.1% ammonium hydroxide, which allows more proteins in the intestinal tissue to be preserved, and the physical properties of the hydrogel scaffold fabricated based on this were also confirmed to be superior.

[0230] Experimental Example 1-4. Comparison of the extracellular matrix derived from decellularized intestinal tissue of the present invention with the prior art (2)

[0231] The extracellular matrix derived from decellularized intestinal tissue of the present invention was compared with that of the prior art. Specifically, the decellularization process developed in the present invention was designated protocol 1, and a general decellularization process not optimized for intestinal tissue was designated protocol 3. The same pig small intestine was used for the decellularization process.

[0232] Specifically, DNA was extracted from intestinal tissue before and after decellularization and compared, and the glycosaminoglycan (GAG) content was compared through analysis of 1,9-dimethylmethylene blue dye solution.

[0233] For histological analysis, the tissues before and after decellularization were fixed in 4% paraformaldehyde solution for 1 day, embedded in paraffin, sectioned, and then stained with hematoxylin and eosin (H&E).

[0234] To analyze the protein content of the scaffold derived from decellularized intestinal tissue, the scaffold was dissolved in a dissolution buffer [4% sodium dodecyl sulfate (SDS), 0.1M Tris-HCl (pH 7.6) and 1X protease inhibitor], and the protein components were extracted and decomposed to the peptide level. The types and relative values ​​of peptides were then detected using a mass spectrometer and MaxQuant software. Detailed classification of matrisomes, etc. was then carried out using an existing library.

[0235] Comparing the amount of remaining DNA in intestinal tissue that had not been decellularized (Native), intestinal tissue that had been decellularized using the protocol of the present invention (protocol 1), and intestinal tissue that had been decellularized using a general protocol (protocol 3), it was confirmed that both protocols were able to efficiently remove cells from within the tissue (Figure 5(a)).

[0236] Furthermore, a comparison of the remaining amount of GAG (glycosaminoglycan) confirmed that when the process of the present invention (protocol 1) was used, the GAG ​​content was preserved at the native tissue level, whereas when the general protocol (protocol 2) was applied, only about 33% of the GAG ​​content in the native tissue was preserved (Figure 5(b)).

[0237] In addition, a comparison of the results of hematoxylin and eosin staining (H&E staining) confirmed that after the decellularization process, most of the cell nuclei were removed from the tissue, while the components of the extracellular matrix were preserved (Figure 5(c)).

[0238] By comparing the types of proteins detected, it was confirmed that a greater number of proteins were preserved in the intestinal tissue treated with the protocol of the present invention (Figure 5(d)).

[0239] A 5 mg / ml hydrogel scaffold was prepared using the decellularized intestinal tissue prepared by each protocol, and the mechanical properties (modulus) were compared. As a result, it was confirmed that the modulus of the hydrogel scaffold derived from the decellularized intestinal tissue prepared by the protocol of the present invention was superior even at the same concentration (Figure 5(e)).

[0240] These results confirmed that while the general decellularization process (protocol 3) used in the comparative protocol uses Triton X-100 and ionic sodium deoxycholate for decellularization, the process of the present invention (protocol 1) uses only a mixed solution of 1% Triton X-100 and 0.1% ammonium hydroxide, which allows more proteins in the intestinal tissue to be preserved, and the physical properties of the hydrogel scaffold fabricated based on this are also superior.

[0241] Experimental Example 1-5. Comparison of the extracellular matrix derived from decellularized intestinal tissue of the present invention with the prior art (3)

[0242] The extracellular matrix derived from the decellularized intestinal tissue of the present invention was compared with the conventional technology through the aspect of intestinal organoid culture. Specifically, the decellularization process developed in the present invention was designated as protocol 1, and a general decellularization process not optimized for intestinal tissue was designated as protocol 3. The same pig small intestine was decellularized, hydrogel was produced, and an experiment was conducted to use the hydrogel as a culture support for intestinal organoids.

[0243] Specifically, intestinal crypts containing intestinal stem cells were extracted and 3D cultured for six days in hydrogels derived from decellularized intestinal tissue prepared using protocol 1 and protocol 3. The appearance of the organoids formed on the sixth day was confirmed, and their formation efficiency was quantified.

[0244] As a result, as can be seen from Figures 6(a) and 6(b), when comparing the formation patterns and formation efficiency of intestinal organoids cultured from a hydrogel derived from decellularized intestinal tissue prepared according to the protocol of the present invention (protocol 1) and a hydrogel derived from intestinal tissue prepared by decellularization according to a general protocol (protocol 3), it was confirmed that in the scaffold derived from decellularized intestinal tissue prepared according to the general protocol (protocol 3), intestinal organoids grew in an irregular pattern and the formation efficiency was low, whereas in the scaffold derived from decellularized intestinal tissue prepared according to the protocol of the present invention (protocol 1), intestinal organoids were formed in a normal pattern and the formation efficiency was also high.

[0245] These results demonstrate that, whereas the extracellular matrix hydrogel prepared by a general decellularization process has weak physical properties and contains many lost extracellular matrix proteins specific to intestinal tissue, the extracellular matrix hydrogel prepared by the protocol of the present invention has better physical properties and contains an abundance of extracellular matrix proteins specific to intestinal tissue, making the decellularization process of the present invention even more suitable for producing a culture scaffold for intestinal organoids.

[0246] Experimental Example 1-6. Comparative analysis of the extracellular matrix derived from decellularized intestinal tissue of the present invention and the protein bodies of the prior art

[0247] The extracellular matrix derived from decellularized intestinal tissue of the present invention was compared with the proteinaceous body of the prior art (Matrigel).

[0248] Specifically, to analyze the protein structure of the scaffold derived from decellularized intestinal tissue, the scaffold was dissolved in a dissolution buffer [4% sodium dodecyl sulfate (SDS), 0.1M Tris-HCl (pH 7.6) and 1X protease inhibitor], and the protein components were extracted and decomposed to the peptide level. Then, the types and relative values ​​of peptides were detected using a mass spectrometer and MaxQuant software, and detailed classification such as matrisomes was carried out using an existing library.

[0249] As a result, as can be seen in Figure 7, the content ratios (pie charts) of matrisome proteins [extracellular matrix proteins (ECM proteins) + proteins associated with extracellular matrix components (ECM-associated proteins)] in the extracellular matrix support (IEM) derived from decellularized intestinal tissue and the 10 matrisome proteins with the highest content were confirmed, as well as the content ratios (pie charts) of matrisome proteins in Matrigel, an existing culture support for organoids, and the 10 matrisome proteins with the highest content (Figures 7(a) and 7(b)).

[0250] We were able to confirm the types of proteins that were specifically and abundantly expressed in actual intestinal tissue present in the extracellular matrix support (IEM) derived from decellularized intestinal tissue, and the types of proteins that were specifically and abundantly expressed in actual intestinal tissue present in Matrigel (Figures 7(c) and 7(d)).

[0251] Through this, it was confirmed that the IEM scaffold derived from decellularized intestinal tissue has a high content of collagen type 6 and matrisome proteins such as decorin (DCN), dermatopontin (DPT), and biglycan (BGN) that are present in real intestinal tissue. It was also confirmed that real intestinal tissue contains five types of matrisome proteins and 22 types of non-matrisome proteins, and that unlike real intestinal tissue, Matrigel is mostly composed of glycoproteins, and that the proteins specific to real intestinal tissue are only four types of non-matrisome proteins.

[0252] Based on these results, it is possible to predict that the IEM scaffold derived from decellularized intestinal tissue can provide an extracellular matrix microenvironment that is more suitable for the formation and differentiation of organoids in situ than the existing Matrigel.

[0253] As can be seen in Figure 8, when the types of matrisome proteins contained in the IEM scaffold derived from decellularized intestinal tissue and Matrigel were compared, it was confirmed that the IEM scaffold contained a greater variety of matrisome proteins and matrisome-associated proteins than Matrigel (Figure 8(a)).

[0254] It was also confirmed that the IEM scaffold derived from decellularized intestinal tissue contained the most abundant collagen at approximately 45%, followed by proteoglycan at 35% and glycoprotein at approximately 16%. On the other hand, in the case of Matrigel, an existing culture scaffold, it was confirmed that glycoproteins accounted for the majority at approximately 96%, with the remaining proteins being very small at around 1% (Figure 8(b)). In other words, it was confirmed that the IEM scaffold has a matrisome protein composition ratio that is more similar to that of intestinal tissue compared to Matrigel.

[0255] Meanwhile, as can be seen from FIG. 9, approximately 59% of the IEM scaffold derived from the decellularized intestinal tissue was composed of non-matrisomal proteins, rather than matrisomal proteins. The gene functions of the proteins were analyzed through a gene ontology of biological processes to determine what functions the non-matrisomal proteins, rather than matrisomal proteins, were responsible for. It was found that the non-matrisomal proteins contained in the IEM scaffold were mainly responsible for functions related to cell organization, skeletal development, and structure formation (FIG. 9(a)).

[0256] On the other hand, in the case of Matrigel, approximately 12% is composed of matrisome proteins, and the rest is mostly non-matrisome proteins, which are responsible for functions related to translation and RNA and peptide metabolism (Figure 9(b)).

[0257] Based on these results, the IEM scaffold derived from decellularized intestinal tissue prepared according to the present invention not only contains matrisome proteins similar to those in actual intestinal tissue, but also contains non-matrisome proteins that are mainly responsible for the generation of cellular components and the formation of structures, and therefore it is predicted to be more effective in the formation and development of intestinal organoids than Matrigel.

[0258] Experimental Example 2: Analysis of scaffold compositions containing extracellular matrix derived from decellularized intestinal tissue

[0259] The support composition prepared in Example 1-2 was analyzed as follows.

[0260] Experimental Example 2-1. Confirmation of the physical properties of the support composition

[0261] The physical properties of the support compositions (hydrogels) prepared in Examples 1-2 were compared and confirmed.

[0262] Specifically, the elastic modulus (G') and viscous modulus (G'') at frequencies in the range of 0.1-1 Hz were measured using a rotational rheometer, and the physical properties of hydrogel scaffolds based on components of extracellular matrix derived from decellularized intestinal tissue at concentrations of 1-5 mg / ml (IEM 1-5 mg / ml) were analyzed.

[0263] As a result, it was confirmed that all concentrations of IEM hydrogels, including the lowest concentration of 1 mg / ml, formed an internally stable polymer network (G' > G'') after 30 minutes of crosslinking reaction (Figure 10(a)). It was also found that the mechanical properties (modulus) of the IEM hydrogel increased with increasing IEM concentration (Figure 10(b)).

[0264] Experimental Example 2-2. Confirmation of optimal conditions for support composition

[0265] The optimal conditions for the support composition (hydrogel) prepared in Example 1-2 were confirmed.

[0266] Specifically, the upper part of the mouse small intestine was taken, the internal food and villi were removed, and the tissue was physically washed multiple times. Then, the crypt part where the intestinal stem cells exist was isolated efficiently through EDTA (ethylenediaminetetraacetic acid) treatment and multiple washing processes. The crypt tissue was cultured on the hydrogel scaffold of the present invention and Matrigel according to the concentration, and the developmental patterns of 3D intestinal organoids were compared and confirmed.

[0267] As a result, as can be seen from Figure 11, the above analysis confirmed that intestinal organoids could be cultured using IEM hydrogel scaffolds derived from decellularized intestinal tissue at all concentrations, but that the stem cell ability and differentiation ability of intestinal organoids into intestinal-specific cells was best with IEM hydrogel at a concentration of 2 mg / ml.

[0268] Experimental Example 2-3. Confirmation of the growth pattern of intestinal organoids cultured from the support composition

[0269] The growth pattern of intestinal organoids cultured from the support composition (hydrogel) produced in Example 1-2 was confirmed.

[0270] Specifically, the growth patterns of intestinal organoids developed from intestinal crypts 3D cultured on the support composition (hydrogel) and Matrigel of the present invention were tracked over time (from day 1 to day 6 of culture).

[0271] As a result, as can be seen in Figure 12, it was confirmed that intestinal organoids grown on the IEM hydrogel scaffold derived from decellularized intestinal tissue grew at a similar growth rate and size to those grown on Matrigel. It was found that the culture ability of intestinal organoids on the developed IEM hydrogel scaffold was similar to that of the conventional Matrigel scaffold.

[0272] Experimental Example 2-4. Analysis of the expression of stem cell and differentiation markers in intestinal organoids cultured from the support composition (cell immunostaining analysis)

[0273] The expression of stem cell and differentiation markers of the intestinal organoids cultured from the support composition (hydrogel) prepared in Example 1-2 was analyzed by cell immunostaining.

[0274] Specifically, the cultured organoids were fixed with 4% paraformaldehyde for 1 hour, treated with 0.5% Triton X-100 for 1 hour, and then blocked with 5% BSA solution (to suppress non-specific protein reactions).Tissue-specific antibodies were then used to perform cell immunostaining.

[0275] As a result, as can be seen in Figure 13, the results of immunostaining of cells for the analysis of the expression of stemness markers (LGR5, SOX9) of intestinal organoids cultured from IEM hydrogel and Matrigel derived from decellularized intestinal tissue (Figure 13(A)) and the results of immunostaining of cells for the analysis of the expression of differentiation markers (MUC2, LYZ1, CHGA, VILLIN, ZO1) of intestinal organoids cultured from IEM hydrogel and Matrigel derived from decellularized intestinal tissue (Figure 13(B)) were obtained.

[0276] Through the above analysis, it was found that there was no difference in the level of expression of specific markers between intestinal organoids cultured from IEM hydrogel and Matrigel, derived from decellularized intestinal tissue, confirming that the induction of proliferation and differentiation of intestinal organoids by IEM hydrogel is similar to that of Matrigel.

[0277] * LGR5, SOX9: intestinal stem cell markers / ECAD: intestinal epithelial markers

[0278] *MUC2: goblet cell marker / LYZ1: Paneth cell marker / CHGA: enteroendocrine cell marker / VILLIN, ZO1: enterocyte marker

[0279] Experimental Example 2-5. Analysis of expression of stem cell and differentiation markers in intestinal organoids cultured from support composition (quantitative PCR analysis)

[0280] The expression of stem cell and differentiation markers of intestinal organoids cultured from the support composition (hydrogel) prepared in Example 1-2 was analyzed by quantitative PCR.

[0281] Specifically, we extracted mRNA samples from intestinal organoids using an RNA extraction kit, synthesized cDNA samples from the extracted mRNA using a cDNA synthesis kit, and then performed quantitative PCR analysis using tissue-specific gene primers.

[0282] As a result, as shown in Figure 14, through qPCR analysis data on the expression of stemness markers in intestinal organoids cultured from IEM hydrogel and Matrigel derived from decellularized intestinal tissue, it was confirmed that the expression of stemness markers was somewhat higher in intestinal organoids cultured from the IEM hydrogel scaffold derived from decellularized intestinal tissue than in intestinal organoids cultured from Matrigel (Figure 14(A)). This indicates that the proliferation ability of intestinal organoids cultured from IEM hydrogel is superior.

[0283] In addition, qPCR analysis data on the expression of differentiation markers in intestinal organoids cultured from IEM hydrogel and Matrigel derived from decellularized intestinal tissue confirmed that the expression levels of differentiation markers were similar between intestinal organoids cultured from IEM hydrogel scaffold derived from decellularized intestinal tissue and intestinal organoids cultured from Matrigel (Figure 14(B)). These results confirmed that the IEM hydrogel scaffold has the ability to induce organoid differentiation to the extent that it can replace the commonly used Matrigel scaffold.

[0284] Experimental Example 2-6. Verification of the functionality of intestinal organoids cultured from the support composition

[0285] The functionality of the intestinal organoids cultured from the support composition (hydrogel) produced in Example 1-2 was verified.

[0286] Specifically, 5 μM forskolin was added to the culture medium of intestinal organoids, and then the change in organoid size over time was quantified.

[0287] As a result, as shown in Figure 15, when treated with forskolin (a chemical that stimulates the opening of ion channels to promote water excretion), an antagonist of CFTR (cystic fibrosis transmembrane conductance regulator) present in the epithelial cells of intestinal tissue, the lumens of the IEM hydrogel derived from decellularized intestinal tissue and the intestinal organoids cultured from Matrigel swelled to a similar extent (Figure 15 (A)). After treating the IEM hydrogel derived from decellularized intestinal tissue and the intestinal organoids cultured from Matrigel, respectively, with forskolin, it was confirmed through the results of a quantitative analysis of the change in area over time that the degree of swelling due to forskolin treatment was similar for the intestinal organoids cultured from the IEM hydrogel derived from decellularized intestinal tissue and the intestinal organoids cultured from Matrigel. This confirmed that the intestinal organoids grown on the developed IEM hydrogel scaffold have intestinal tissue-specific functionality that regulates the secretion of electrolytes and water in the human body (Figure 15 (B)).

[0288] Experimental Example 2-7. Analysis of gene expression profile of intestinal organoids cultured from support composition (1) (RNA-sequencing analysis)

[0289] Gene expression profile analysis was performed on the intestinal organoids cultured from the support composition (hydrogel) prepared in Example 1-2.

[0290] Specifically, we classified the genetic information of organoids grown in hydrogel and Matrigel derived from decellularized intestinal tissue, and classified the differentially expressed genes (DEGs) at the statistical level of 2-fold, p-value<0.05, and FDR<0.1. As a result, 270 genes were obtained and illustrated by heatmap. We then confirmed that the morphology of the organoids generated when cultured from each hydrogel was almost similar, but a very large number of genes were differentially expressed (Figure 16(A)).

[0291] In addition, in the intestinal organoids cultured from the IEM hydrogel derived from decellularized intestinal tissue, the expression of 213 genes out of 270 DEGs was increased compared to that of the intestinal organoids grown in Matrigel, with the largest increase observed in genes related to the extracellular matrix. The expression of 57 genes out of 270 DEGs was decreased, with the largest decrease observed in genes related to oxidation / reduction reactions and metabolism (Figure 16(B)).

[0292] Meanwhile, when genes with a difference of 4 times or more among DEGs were selected compared to intestinal organoids cultured from Matrigel, it was confirmed that the expression of genes belonging to categories related to extracellular matrix, wound healing response, cell proliferation, and cytokine activity was significantly increased in intestinal organoids cultured from IEM hydrogel derived from decellularized intestinal tissue (Figure 16(C)). This showed that IEM hydrogel derived from decellularized intestinal tissue significantly activated genes related to the synthesis of extracellular matrix and cell proliferation in intestinal organoids.

[0293] Experimental Example 2-8. Analysis of gene expression profile of intestinal organoids cultured from support composition (2) (RNA-sequencing analysis)

[0294] Gene expression profile analysis was performed on the intestinal organoids cultured from the support composition (hydrogel) prepared in Example 1-2.

[0295] Specifically, when we looked at genes that fall into the category of genes related to the extracellular matrix environment and cell-matrix interactions, we found that the number of genes whose expression levels were increased in organoids in the decellularized intestinal tissue-derived hydrogel group was greater than that in the Matrigel group, and the degree of increase was also greater (Figure 17(A)).

[0296] It was confirmed that the expression of extracellular matrix protein-related genes (Col4a2, Nid1, Lama3), cytoskeleton-related genes (Flna, Gsn, Tuba1), and intestinal epithelial gene involved in thiamine absorption (Tm4sf4) in the hydrogel group derived from decellularized intestinal tissue was similar to that of actual tissue. On the other hand, in the case of organoids cultured from Matrigel, it was confirmed that the degree of expression of the genes was significantly different from that of actual tissue. In addition, it was confirmed that the expression of genes involved in wound regeneration, inflammation, and immune response (Procr, Mcpt2, Icam1, Cxcl10, Cxcl16, Timp3) was similar between organoids cultured from hydrogel derived from decellularized intestinal tissue and actual tissue (Figure 17(B)).

[0297] These genes are involved in maintaining the homeostasis of the intestinal wall under physiological conditions, which confirms that the hydrogel scaffold of the present invention effectively preserves the inherent ability of intestinal organoids to respond to injury and maintain tissue homeostasis.

[0298] Experimental Example 2-9. Confirmation of tissue-specific effects of intestinal organoids cultured from support composition

[0299] The tissue-specific effects of intestinal organoids cultured from the support composition (hydrogel) prepared in Examples 1-2, specifically the survival and development of intestinal organoids, were analyzed.

[0300] Specifically, intestinal crypts containing intestinal stem cells were encapsulated in hydrogels and Matrigel derived from six types of decellularized tissues (intestine, stomach, skin, lymph, heart, and muscle) and cultured in 3D. After six days of culture, the formation of organoids generated from each hydrogel was confirmed. The efficiency of organoid formation in hydrogels and Matrigel derived from three types of decellularized tissues (intestine, stomach, and skin) was measured, and mRNA was extracted from each organoid and the expression of stem cell and differentiation markers was analyzed using quantitative PCR.

[0301] As a result, as can be seen from Figure 18, when comparing the formation patterns (concentration 2mg / ml) of intestinal organoids cultured from hydrogels and Matrigel derived from six types of decellularized tissues (intestine, stomach, skin, lymph, heart, muscle), it was observed that intestinal organoids were not properly formed on decellularized scaffolds derived from other organs (skin, lymph, heart, muscle). In addition, quantitative PCR (qPCR) analysis was performed on the formation efficiency of intestinal organoids and the stemness marker (Lgr5) and differentiation marker (Muc2) of intestinal organoids, and it was confirmed that the expression of stem cell markers and differentiation markers was the lowest in intestinal organoids cultured from hydrogel derived from decellularized skin tissue.

[0302] These results confirmed that the formation and development of intestinal organoids was superior on decellularized scaffolds derived from intestinal tissue compared to decellularized scaffolds derived from other tissues, verifying that the microenvironment provided by the intestinal tissue-specific extracellular matrix has an important effect on the culture of intestinal organoids.

[0303] * SEM: stomach extracellular matrix, SkEM: skin extracellular matrix, LyEM: lympHnode extracellular matrix, HEM: heart extracellular matrix, MEM: muscle extracellular matrix

[0304] Experimental Example 2-10. Comparative analysis of tissue-specific extracellular matrix components of scaffolds derived from decellularized intestinal tissue (1)

[0305] The tissue-specific extracellular matrix components of the scaffold compositions prepared in Examples 1-2 were comparatively analyzed.

[0306] Specifically, a comparison of the extracellular matrix protein content in decellularized skin tissue-derived scaffolds (SkEM) and decellularized intestinal tissue-derived scaffolds (IEM) was performed.

[0307] As a result, as can be seen from FIG. 19, it was confirmed that extracellular matrix proteins such as laminin and fibronectin are very important in the formation and development of intestinal organoids, and that the content of these extracellular matrix proteins was higher in the scaffold derived from decellularized intestinal tissue than in the scaffold derived from decellularized skin tissue.

[0308] Additionally, while the scaffold derived from decellularized skin tissue contained a large amount of collagen types 1 and 3, the scaffold derived from decellularized intestinal tissue contained a large amount of collagen types 6 and 14. Collagen type 6 is known to be an important factor in regulating the interaction between intestinal epithelial cells and fibronectin.

[0309] Through this, it was confirmed that by using a scaffold derived from decellularized intestinal tissue for the culture of intestinal organoids, it is possible to better embody the microenvironment of extracellular matrix specific to intestinal tissue compared to decellularized scaffolds derived from other tissues, and therefore it can be used as a matrix more suitable for the culture of intestinal organoids.

[0310] Experimental Example 2-11. Comparative analysis of tissue-specific extracellular matrix components of scaffolds derived from decellularized intestinal tissue (2)

[0311] The expression of stem cell and differentiation markers of intestinal organoids cultured from the support compositions (hydrogels) prepared in Examples 1-2 was comparatively analyzed.

[0312] Specifically, small intestines were extracted from 6-month-old pigs weighing 100-120 kg (old) and 2-month-old pigs weighing 20 kg (young), and a decellularization process was carried out on each. Quantitative PCR (qPCR) analysis was carried out on the expression of stemness markers (Lgr5, Axin2, Olfm4) and differentiation markers (Lyz1, Muc2, Chga, Vil1) of intestinal organoids cultured from the scaffolds derived from the decellularized intestinal tissue.

[0313] As a result, it was confirmed that intestinal organoid stem cell genes were highly expressed in IEM scaffolds made from tissue extracted from young individuals (Figure 20(A)).

[0314] In addition, the quantitative analysis of extracellular matrix proteins in the IEM scaffolds derived from decellularized tissue prepared from the small intestine of 6-month-old pigs (old) weighing 100-120 kg and 2-month-old pigs (young) weighing 20 kg was performed.

[0315] As a result, the fibronectin content of the IEM scaffold made from tissues from young individuals was higher, and fibronectin is known to be very important for the early development of intestinal organoids (Figure 20(B)).

[0316] These results confirmed that even in the same tissue, the content of extracellular matrix proteins in the tissue varies depending on age, and that decellularized scaffolds prepared using tissues from individuals of different ages also have differences in the content of extracellular matrix proteins. Therefore, it can be seen that preparing a decellularized scaffold using tissues from younger individuals and applying it to the culture of organoids is more advantageous for the growth of organoids.

[0317] Experimental Example 3: Verification of the utility of intestinal organoids cultured from the support composition

[0318] Experimental Example 3-1. Verification of the ability to maintain organ culture of intestinal organoids cultured from the support composition

[0319] The ability of intestinal organoids cultured from the support composition (hydrogel) produced in Example 1-2 to maintain organ culture was examined.

[0320] As a result, we obtained long-term subculture data for intestinal organoids cultured from IEM hydrogel and Matrigel derived from decellularized intestinal tissue (Figure 21(A)), and qPCR analysis results for the expression of stemness markers (Lgr5, Axin2, Olfm4) in P0 (day 6), P2 (day 15), and P6 (day 33) intestinal organoids cultured from IEM hydrogel and Matrigel derived from decellularized intestinal tissue (P=passage number, D=culture day) (Figure 21(B)).

[0321] These results confirmed that intestinal organoids cultured on the IEM hydrogel scaffold derived from decellularized intestinal tissue did not show any significant changes in cell growth or formation during the organ culture process, and the expression of stemness markers was maintained at a constant level, similar to that of intestinal organoids cultured on Matrigel. Therefore, it was verified that the developed IEM hydrogel artificial scaffold is suitable for stable organ culture of intestinal organoids.

[0322] Experimental Example 3-2. Verification of the possibility of freezing and storing the support composition for culturing intestinal organoids

[0323] The possibility of freezing and storing the support composition produced in Example 1-2 was examined.

[0324] Specifically, to confirm the feasibility of long-term frozen storage of the IEM hydrogel pre-gel solution derived from decellularized intestinal tissue, an experiment was conducted in which the solution was thawed after long-term frozen storage and used to culture intestinal organoids (Figure 22(A)). The morphology of intestinal organoids cultured from IEM hydrogel prepared immediately before culture and from IEM hydrogel derived from decellularized intestinal tissue that was thawed again after 1, 2, and 3 months of frozen storage (-80°C) was compared with that of intestinal organoids cultured from Matrigel (Figure 22(B)).

[0325] As a result, as shown in Figure 22, it was confirmed that intestinal organoids were well formed even in IEM hydrogels that had been frozen for a long period of time, with no difference from Matrigel (Figure 22(B)). When the expression of stemness (stemness-Lgr5) and differentiation (differentiation-Lyz1, Muc2) markers of intestinal organoids cultured from IEM hydrogels prepared immediately before culture and from IEM hydrogels derived from decellularized intestinal tissues that had been frozen (-80℃) and then melted after 1 month, 2 months, and 3 months was analyzed by quantitative PCR (qPCR), it was confirmed that there was no significant difference in the expression of each marker (Figure 22(C)).

[0326] These results confirmed that the IEM hydrogel scaffold derived from decellularized intestinal tissue can be stored frozen for a long period of time after preparation, and can be thawed and used again when necessary without any abnormalities in terms of performance for culturing intestinal organoids. This is a very advantageous advantage for commercialization of the developed hydrogel scaffold.

[0327] Experimental Example 3-3. Verification of the possibility of refrigerated storage of support composition for culturing intestinal organoids

[0328] The possibility of storing the support composition produced in Example 1-2 in a refrigerator was examined.

[0329] Specifically, to confirm the feasibility of long-term refrigerated storage of the IEM hydrogel scaffold derived from decellularized intestinal tissue, a refrigerated IEM hydrogel pre-gel solution was applied to the culture of intestinal organoids (Figure 23(A)), and the morphology of intestinal organoids cultured from IEM hydrogel prepared immediately before culture and from IEM hydrogel derived from decellularized intestinal tissue that had been refrigerated for a certain period of time was compared with that of intestinal organoids cultured from Matrigel (Figure 23(B)).

[0330] As a result, as shown in Figure 23, it was confirmed that intestinal organoids were formed well even in the IEM hydrogel stored in a refrigerator, just like in the case of Matrigel (Figure 23(B)).

[0331] In addition, when the expression of stemness (stemness-Lgr5, Axin2) and differentiation (differentiation-Lyz1, Muc2, Chga) markers of intestinal organoids cultured from IEM hydrogels prepared immediately before culture and IEM hydrogels derived from decellularized intestinal tissue 1 and 4 weeks after refrigerated storage was analyzed by quantitative PCR (qPCR), it was confirmed that there were no significant differences in the expression of each marker (Figure 23(C)).

[0332] These results confirmed that the IEM hydrogel scaffold derived from decellularized intestinal tissue can be stored in a refrigerator for a long period of time after preparation, and that the hydrogel stored in a refrigerator has no abnormalities in terms of performance for culturing intestinal organoids, which is a very advantageous advantage for commercialization of the developed hydrogel scaffold.

[0333] Experimental Example 3-4. Verification of applicability of the scaffold composition as a scaffold for transplantation of intestinal organoids into the body

[0334] The applicability of the support composition (hydrogel) produced in Example 1-2 as a support for transplantation of intestinal organoids into the body was examined.

[0335] Specifically, intestinal organoids labeled with the fluorescent substance DiI dye were transplanted into the damaged site of the intestine of a mouse using IEM hydrogel derived from decellularized intestinal tissue. A part of the mouse's small intestine was fixed with a surgical clamp, and 40 μl of 1% acetic acid was injected into the site to induce tissue damage for 2 minutes. Then, the organoids were mixed with a hydrogel solution for transplantation and injected into the damaged site (Figure 24(A)).

[0336] In addition, to adjust the viscosity of the IEM hydrogel derived from decellularized intestinal tissue so that it is suitable for transplantation in vivo, conditions in which the IEM hydrogel and culture medium were mixed in various ratios were applied to the culture of intestinal organoids (Figure 24(B)).

[0337] As a result, it was confirmed that intestinal organoids survived well under all IEM hydrogel conditions for transplantation, as can be seen in Figure 24. In existing literature, when transplanting organoids, Matrigel and culture medium were mixed at a volume ratio of 1:20, so the IEM hydrogel conditions for transplantation were also similarly applied with a mixture ratio (v / v) of 1:20 with culture medium.

[0338] Experimental Example 3-5. Verification of in vivo transplantation and engraftment induction of intestinal organoids cultured from the support composition

[0339] The possibility of transplanting and inducing engraftment of the intestinal organoids cultured from the support composition (hydrogel) produced in Example 1-2 into a living body was examined.

[0340] Specifically, intestinal organoids labeled with DiI fluorescence were transplanted into the damaged intestinal tissue using an IEM hydrogel solution for transplantation (IEM:medium = 1:20 ratio), and the next day, DiI fluorescence was observed from the transplanted tissue site, confirming that the intestinal organoids had been successfully engrafted (Figure 25 (A)).

[0341] In addition, when the intestinal tissue from the site where the intestinal organoids were transplanted was collected and the expression of fluorescence was confirmed, it was confirmed that DiI (transplanted intestinal organoid) fluorescence was present in the intestinal epithelial tissue. It was confirmed that the IEM hydrogel scaffold derived from decellularized intestinal tissue can be used not only for in vitro culture of intestinal organoids, but also as a material for in vivo transfer and transplantation (Figure 25 (B)).

[0342] On the other hand, as shown in Figure 26(A), an experiment was conducted in which intestinal organoids derived from EGFP-expressing mice were transplanted into the site of intestinal damage using an IEM hydrogel scaffold derived from decellularized intestinal tissue.

[0343] Specifically, a portion of the mouse small intestine was fixed with a surgical clamp, and 40 μl of 1% acetic acid was injected into the site to induce tissue damage for 2 minutes. Then, organoids were mixed with a hydrogel solution for transplantation and injected into the damaged site, and the results were confirmed one day later.

[0344] Before transplantation, it was confirmed that green fluorescence (EGFP) was well expressed throughout the intestinal organoids produced by extracting intestinal stem cells from EGFP-expressing mice (Figure 26(B)). When the intestinal organoids were transplanted into the intestinal tissue using an IEM hydrogel solution for transplantation (IEM:medium = 1:20 ratio), and the transplanted area was removed to check for fluorescence expression, it was confirmed that EGFP (transplanted EGFP-expressing intestinal organoid) fluorescence was detected within the intestinal epithelial tissue (Figure 26(C)).

[0345] These results confirmed that the IEM hydrogel scaffold derived from decellularized intestinal tissue can be used not only for in vitro culture of intestinal organoids but also as a material for in vivo delivery and transplantation.

[0346] Experimental Example 3-6. Confirmation of the possibility of culturing intestinal organoids derived from human induced pluripotent stem cells and embryonic stem cells using the scaffold composition

[0347] It was confirmed that the support composition (hydrogel) of Example 1-2 can be used to culture intestinal organoids derived from human induced pluripotent stem cells and embryonic stem cells.

[0348] Specifically, human induced pluripotent stem cells and embryonic stem cells were seeded as single cells onto a culture dish, and when the cells reached over 90% full, Activin A and FBS were added to initiate intestinal differentiation. After that, FGF-4 and CHIR99021 were treated, and once mid-hindgut spheroids were formed, they were transferred to IEM hydrogel or Matrigel derived from decellularized intestinal tissue, and cultured in 3D using intestinal organoid culture medium.

[0349] As a result, as can be seen in Figure 27, it was confirmed that intestinal organoids derived from human induced pluripotent stem cells (hiPSCs) and human embryonic stem cells (hESCs) could be successfully cultured using IEM hydrogel and Matrigel derived from decellularized intestinal tissue (image of organoid morphology on the 7th day of culture) (Figure 27(A)).

[0350] When cell survival was analyzed for intestinal organoids in which differentiation was induced from human embryonic stem cells (hESCs) using IEM hydrogel and Matrigel derived from decellularized intestinal tissue (Live / Dead analysis), it was confirmed that the cells in the intestinal organoids were well-lived when cultured for 6 days (Figure 27(B)).

[0351] It was confirmed that the IEM hydrogel scaffold derived from decellularized intestinal tissue is a highly effective culture scaffold for the formation, maintenance, and growth of intestinal organoids derived from mouse intestinal tissue adult stem cells, as well as organoids induced to differentiate from human induced pluripotent stem cells and human embryonic stem cells, which are stem cell types with totipotency. Therefore, it is highly versatile as a scaffold capable of culturing intestinal organoids derived from various tissues and cells, regardless of the type of stem cells, which provides an advantageous advantage for commercialization. It was confirmed that the applicability of IEM hydrogel is very high as a component technology of a culture system for constructing disease models based on intestinal organoids derived from patient stem cells.

[0352] Experimental Example 3-7. Confirmation of the possibility of forming and culturing 3D organoids derived from colon cancer using scaffold composition

[0353] It was confirmed that the support composition (hydrogel) of Example 1-2 enables the formation and cultivation of three-dimensional organoids derived from colon cancer.

[0354] Specifically, as shown in Figure 28, the formation patterns of 3D cancer organoids (8 days after culture) created from colon cancer-derived cell lines (DLD-1, HT29) in IEM hydrogel derived from decellularized intestinal tissue and Matrigel were compared. As a result, it was confirmed that colon cancer organoids were well formed in IEM hydrogel without any significant difference from Matrigel (Figure 28(A)).

[0355] Analysis of immunostaining for Ki67, a cell proliferation marker, of 3D cancer organoids (8 days after culture) created from colon cancer-derived cell lines (DLD-1, HT29) in decellularized intestinal tissue-derived IEM hydrogel and Matrigel confirmed that there was no significant difference in the proliferation ability of cells in colon cancer organoids cultured in IEM hydrogel and Matrigel (Figure 28(B)).

[0356] These results confirmed that the IEM hydrogel scaffold derived from decellularized intestinal tissue is a highly effective culture scaffold for the formation and growth of not only stem cell-derived intestinal organoids, but also cancer organoids created from colon cancer cell lines. The applicability of IEM hydrogel as a core technology for a culture system for constructing in vitro models of colon cancer patients is extremely high.

[0357] Experimental Example 3-8. Confirmation of the possibility of cryopreservation of organoids using the support composition

[0358] In general, the cryopreservation of intestinal organoids involves removing the hydrogel (support) on which the organoids were cultured, and freezing only the organoids using a freezing culture medium. In this case, after thawing, the organoids must be encapsulated in hydrogel again and cultured, which is inconvenient, and during this process, the function and activity of the cells in the organoids may be damaged.

[0359] The possibility of cryopreserving organoids using the support composition (hydrogel) of Example 1-2 was confirmed.

[0360] Specifically, intestinal organoids cultured for six days in IEM hydrogel and Matrigel derived from decellularized intestinal tissue were frozen and stored without removing the respective culture supports, and after thawing, they were cultured without any additional support encapsulation process.

[0361] Live / Dead staining of thawed organoids was analyzed after they were frozen and stored encapsulated in IEM hydrogel or Matrigel derived from decellularized intestinal tissue. It was confirmed that the majority of cells in the organoids encapsulated in IEM hydrogel and frozen and then thawed were alive (green fluorescence), while the organoids encapsulated in Matrigel and frozen and then thawed contained a large number of dead cells (red fluorescence) (Figure 29(A)).

[0362] In addition, after Live / Dead staining, the images taken were quantitatively analyzed (left side: area comparison of Live+ cell regions (green fluorescence) within the organoid, right side: area comparison of Dead+ cell regions (red fluorescence) within the organoid) (Figure 29(B)).

[0363] In addition, images of the organoid morphology before and after freezing and storage were obtained when the organoids were encapsulated in IEM hydrogel and Matrigel derived from decellularized intestinal tissue. The results showed that the intestinal organoids that were encapsulated in Matrigel, frozen and then thawed had a lot of destroyed structure and many areas where the cells had died, whereas the intestinal organoids that were encapsulated in IEM hydrogel derived from decellularized intestinal tissue, frozen and then thawed had their structure and pattern well maintained at a level almost similar to that before freezing and storage (Figure 29(c)).

[0364] Expression of apoptosis genes (Caspase-3) in organoids encapsulated in IEM hydrogel and Matrigel derived from decellularized intestinal tissue before and after freezing and storage was analyzed by qPCR. The results showed that the expression of apoptosis genes was increased after thawing in organoids encapsulated in Matrigel and frozen, but there was no difference between before and after freezing in the case of organoids encapsulated in IEM hydrogel derived from decellularized intestinal tissue and frozen (Figure 29(D)).

[0365] Based on these results, if the IEM hydrogel scaffold derived from decellularized intestinal tissue is used for cryopreservation of organoids, the frozen intestinal organoids can be immediately cultured without complicated processes after thawing, and it protects the organoids from damage during the freezing and thawing process, which is extremely advantageous for organoid storage and post-thaw culture.

[0366] Experimental Example 3-9. Confirmation of the feasibility of mass culturing of intestinal organoids using support composition and microfluidic chip

[0367] The possibility of mass culturing of intestinal organoids using the support composition (hydrogel) of Example 1-2 and the microfluidic chip was confirmed.

[0368] Specifically, the microfluidic chip is composed of multiple organoid chambers, allowing simultaneous mass culture of organoids, and even with a simple rocker, a fine flow of culture fluid can be provided to the organoids, enabling effective dynamic culture (Figure 30(A)). Intestinal organoids were cultured using the support of the present invention in this device.

[0369] When intestinal organoids were cultured using an IEM hydrogel support derived from decellularized intestinal tissue in a microfluidic chip capable of mass culture of organoids (six days after culture) (Figure 30(B)), it was confirmed that many intestinal organoids could be formed and cultured in each chamber.

[0370] These results suggest that intestinal organoids 3D cultured within the IEM hydrogel scaffold derived from decellularized intestinal tissue can be grafted with microfluidic chip technology to combine the tissue-specific extracellular matrix microenvironment with precise dynamic culture of microfluidic flow, enabling efficient mass production of organoids.

[0371] In relation to a scaffold derived from decellularized gastric tissue for the culture and transplantation of gastric organoids and a method for producing the same, the present invention establishes a method for producing a large amount of decellularized scaffolds by performing a simple chemical treatment on gastric tissue, and uses the produced decellularized gastric tissue matrix for the culture of gastric organoids.

[0372] It was confirmed that the decellularized scaffold developed in the present invention has all cellular components removed and contains the various extracellular matrices and growth factors contained in gastric tissue, and can provide an optimal three-dimensional microenvironment for the culture of gastric organoids.

[0373] It was confirmed that gastric organoids could be generated and grow within the developed hydrogel scaffold derived from decellularized gastric tissue. In order to efficiently use the scaffold for culturing gastric organoids, the decellularized scaffold was tested at various concentrations and the concentration optimized for culturing gastric organoids was selected.

[0374] It was confirmed that gastric organoids could be cultured in a similar manner on the developed decellularized scaffold and on the control Matrigel, and differentiation into various gastric tissue cells also occurred. This confirmed the possibility that the decellularized scaffold can be used as an alternative to Matrigel. In addition, conditions were found that can further promote differentiation of organoids by appropriate combination with Matrigel when necessary. This indicates the possibility that gastric organoids cultured from the scaffold developed in the present invention can better mimic the structure and function of actual gastric tissue than existing organoids.

[0375] The hydrogel scaffold derived from decellularized gastric tissue developed in the present invention was found to promote not only the differentiation of organoids but also the acid secretion function. Through this, we have established a culture technique that can generate gastric organoids with enhanced differentiation and function, overcoming the limitations of gastric organoids cultured from existing matrigel. It was confirmed that the stemness of organoids during organ culture was maintained at a similar level compared to matrigel conditions.

[0376] In order to confirm the tissue-specific effect of the decellularized scaffold developed in the present invention, gastric organoids were cultured on the decellularized scaffolds derived from stomach, intestine, skin, lymph, heart, and muscle, respectively, and compared. It was observed that the formation efficiency and expression of stem cell-related genes of gastric organoids cultured on the scaffold derived from decellularized gastric tissue were the best compared to the decellularized scaffolds derived from other tissues such as skin, lymph, heart, and muscle, and it was confirmed that the developed scaffold can have a tissue-specific effect.

[0377] In the present invention, a gastric ulcer model was created in a mouse, and when gastric organoids were transplanted using a decellularized scaffold, it was confirmed that the organoids were able to take root in the gastric tissue and be transplanted, and therefore the high applicability of the decellularized scaffold as a transplant material was also confirmed.

[0378] In the present invention, it was verified that when the decellularized scaffold was stored in a refrigerated or frozen state for a long period of time and then used to culture gastric organoids, gastric organoids could be formed at a similar level compared to a newly produced scaffold. This indicates the possibility and safety of long-term storage of the developed scaffold, and suggests that there is a high possibility of commercializing and industrializing the decellularized scaffold in the future.

[0379] In the present invention, it was confirmed that not only stem cell-derived organoids but also gastric cancer organoids can be successfully cultured using the decellularized scaffold. Therefore, it can be applied as a customized in vitro cancer model and has a high degree of utility as an advanced culture element technology that can be applied to anti-cancer drug screening and new drug development.

[0380] In the following, preferred examples are presented to aid in understanding the present invention. However, the following examples are merely provided to facilitate understanding of the present invention, and the contents of the present invention are not limited to the following examples.

[0381] Example 2: Preparation of a support composition comprising extracellular matrix derived from decellularized gastric tissue

[0382] A support composition comprising extracellular matrix derived from decellularized gastric tissue was prepared as follows (see FIG. 31(A)).

[0383] Example 2-1. Preparation of decellularized stomach tissue-derived extracellular matrix (SEM)

[0384] Pig stomach tissue was prepared by isolating and mincing, and the stomach tissue was stirred with 1% Triton X-100 and 0.1% ammonium hydroxide to remove only the stomach tissue cells, producing decellularized stomach tissue.

[0385] The decellularized gastric tissue was then freeze-dried and pulverized to produce decellularized gastric tissue-derived extracellular matrix (SEM).

[0386] Example 2-2. Preparation of support composition

[0387] 10 mg of the extracellular matrix derived from the decellularized stomach tissue was dissolved in a 4 mg / ml pepsin solution (4 mg of pepsin powder derived from porcine gastric mucosa was dissolved in 1 ml of 0.02 M HCl) for 48 hours. The mixture was uniformly mixed with 10X PBS and 1 M NaOH at a neutral pH and in the electrolyte state of 1X PBS buffer, and gelled at 37° C. for 30 minutes to prepare a hydrogel-type support composition (FIG. 31(B)).

[0388] Experimental Example 4: Analysis of extracellular matrix derived from decellularized gastric tissue

[0389] The extracellular matrix derived from the decellularized gastric tissue of Example 2-1 was analyzed as follows.

[0390] Experimental Example 4-1. Analysis of the characteristics of extracellular matrix derived from decellularized gastric tissue

[0391] The characteristics of the extracellular matrix derived from the decellularized gastric tissue of Example 2-1 were analyzed.

[0392] To confirm whether cells had been sufficiently removed from the fabricated extracellular matrix (SEM) scaffold derived from decellularized gastric tissue, the amount of DNA was compared before and after the decellularization process. To confirm whether extracellular matrix components remained, the amount of glycosaminoglycans (GAG), a representative component of extracellular matrix, was quantitatively compared. As a result, it was confirmed that all cells had been removed after the decellularization process, and that GAG was maintained at a level similar to that of actual gastric tissue (Figure 32(A)).

[0393] Furthermore, to confirm whether all cells had been removed after the decellularization process and whether the matrix structure of the gastric tissue had been well maintained, analysis was performed through H&E tissue staining. As a result, no cell nuclei were observed, it was confirmed that the majority of the cells had been removed (Figure 32(B)).

[0394] Meanwhile, to confirm that the overall matrix structure of the stomach tissue remained intact, the hydrogel was formed on the scaffold derived from the decellularized stomach tissue, and the internal structure of the hydrogel was confirmed through scanning electron microscopy (SEM). As a result, it was observed that the SEM hydrogel had a nanofiber structure similar to that of collagen hydrogel, confirming that it had an internal structure suitable for the attachment and growth of stomach organoids (Figure 32(C)).

[0395] Experimental Example 4-2. Analysis of concentration-specific characteristics of extracellular matrix derived from decellularized gastric tissue

[0396] The concentration-specific characteristics of the extracellular matrix derived from the decellularized gastric tissue of Example 2-1 were analyzed.

[0397] Hydrogels were formed using scaffold compositions with different concentrations of extracellular matrix derived from decellularized gastric tissue, and the changes in physical properties due to the four SEM concentrations were measured through rheological analysis.

[0398] As a result, as can be seen in Figure 33, the storage modulus (G') value was consistently higher than the loss modulus (G'') value under all concentration conditions, confirming that a stable polymer network was formed through cross-linking within the hydrogel. It was also confirmed that the mechanical properties (modulus) increased as the SEM concentration increased.

[0399] Experimental Example 4-3. Comparative analysis of decellularization conditions

[0400] We conducted an experiment to compare the decellularization method established in this study (Protocol 1) with a decellularization method that has been reported in existing literature (Protocol 2).

[0401] Existing literature has reported treating the tissue with 4% sodium deoxycholate (SDC) for 4 hours, followed by Dnase I to remove DNA, whereas in this study, we used only a solution of non-ionic 1% Triton X-100 and 0.1% ammonium hydroxide, which is an even milder condition, to minimize damage to proteins in the tissue.

[0402] As a result, when comparing the amount of remaining DNA after the decellularization process, it was confirmed that DNA was successfully removed from both protocols. However, when comparing the remaining extracellular matrix components through GAG quantification, it was confirmed that the GAG ​​components were well preserved in the tissue treated with protocol 1, whereas the remaining GAG components were significantly reduced in the tissue treated with protocol 2 (Figure 34(a)).

[0403] In addition, after preparing a hydrogel using the prepared decellularized matrix, the mechanical properties (modulus) were measured. It was confirmed that the mechanical properties of the hydrogel prepared by Protocol 1 were significantly high (Figure 34(b)).

[0404] Comparison of H&E histological analysis confirmed that both methods effectively removed cells, but protocol 1 preserved extracellular matrix components better (Figure 34(c)).

[0405] Proteomics analysis of the matrices produced by the decellularization methods was performed, and it was confirmed that Protocol 1 was effective in preserving a greater number of gastric tissue-specific extracellular matrix proteins than Protocol 2 (Figure 34(d)).

[0406] Through this, it can be predicted that the decellularization protocol established in this study can better preserve the components of the extracellular matrix of gastric tissue than existing methods, and therefore will be more advantageous for the cultivation of gastric organoids.

[0407] Meanwhile, we conducted an experiment to compare the decellularization method established in this study (Protocol 1) with a commonly used existing decellularization method (Protocol 3).

[0408] Specifically, protocol 3 is a protocol in which the decellularization protocol 1 established in this study is treated with an additional 24 hours of 3% sodium dodecyl sulfate (SDS), which is often used as a decellularization reagent.

[0409] As a result, when comparing the amount of DNA remaining after the decellularization process, it was confirmed that DNA was successfully removed in both protocols, but when comparing the remaining extracellular matrix components through GAG quantification, it was confirmed that the remaining GAG components were significantly reduced in the tissue treated with protocol 3 (Figure 35(a)).

[0410] In addition, after preparing hydrogels using the prepared decellularized matrices, the mechanical properties (modulus) were measured, and it was confirmed that there was no significant difference in the properties of the hydrogels prepared using each protocol (Figure 35(b)).

[0411] Comparison of H&E histological analysis confirmed that both methods effectively removed cells, but protocol 1 preserved extracellular matrix components better (Figure 35(c)).

[0412] Proteomics analysis of the matrices produced by each decellularization method was performed, and the results confirmed that Protocol 1 was effective in preserving a greater number of gastric tissue-specific extracellular matrix proteins than Protocol 3 (Figure 35(d)).

[0413] Through this, it can be predicted that the decellularization protocol established in this study can better preserve the components of the extracellular matrix of gastric tissue than methods using SDS reagents, and therefore may be more advantageous for the culture of gastric organoids.

[0414] Experimental Example 4-4. Confirmation of differences in the appearance of gastric organoid culture depending on decellularization conditions

[0415] In the decellularization process for producing a culture matrix for gastric organoids, an experiment was conducted to compare the decellularization method established in this study (Protocol 1) with the commonly used existing decellularization method (Protocol 3).

[0416] Specifically, gastric organoids were cultured in the decellularized matrix hydrogels prepared by each protocol, and the formation efficiency and gene expression were analyzed.

[0417] As a result, through organoid morphology analysis on the 5th day of culture, it was confirmed that the gastric organoids cultured from the decellularized matrix prepared by protocol 1 were larger and maintained a spherical structure better than those prepared by protocol 3. In addition, the formation efficiency was quantified, and it was confirmed that protocol 1 was significantly higher (Figure 36(a)).

[0418] On the fifth day of culture, quantitative qPCR experiments were performed to compare the expression of the Lgr5 gene, which is important for stemness, and it was confirmed that the gastric organoids formed using the matrix derived from the decellularized gastric tissue prepared in Protocol 1 showed significantly higher expression (Figure 36(b)).

[0419] Through this, we verified that the decellularization protocol established in this study is more suitable for culturing gastric organoids than existing methods.

[0420] Experimental Example 4-5. Analysis of protein bodies of extracellular matrix (SEM) derived from decellularized gastric tissue

[0421] The fabricated scaffold derived from decellularized gastric tissue (SEM) contains various gastric tissue-specific proteins present in actual gastric tissue. Therefore, to understand the detailed components, proteomics analysis was performed on the SEM scaffold, and it was confirmed that various extracellular matrix-related proteins (glycoproteins, proteoglycans, collagens, secreted factors, etc.) are contained in the decellularized matrix.

[0422] As a result, as can be seen in Figure 37, the scaffold (SEM) derived from the decellularized gastric tissue produced contained various gastric tissue-specific proteins present in actual gastric tissue. When SEM samples derived from four other tissue configurations were analyzed, the components of these extracellular matrices were observed to be generally similar. Therefore, it can be inferred that a uniform level of gastric organoids can be generated through culture using the SEM hydrogel scaffold via the decellularization process.

[0423] Experimental Example 4-6. Kinds and quantitative analysis of proteins in the extracellular matrix derived from decellularized gastric tissue

[0424] The types and quantities of proteins in the extracellular matrix of the present invention were analyzed.

[0425] Specifically, decellularized gastric tissue was dissolved in lysis buffer [4% sodium dodecyl sulfate (SDS), 0.1M Tris-HCl (pH 7.6), and 1X protease inhibitor], and protein components were extracted and decomposed to peptide level. Then, the types and relative values ​​of peptides were detected using a mass spectrometer and MaxQuant software. Then, detailed classification such as matrisome was carried out using existing libraries.

[0426] As a result, the matrisome proteins contained in Matrigel and SEM were detected through protein analysis and classified by type. It was confirmed that the number of matrisome proteins detected in SEM was greater than that in Matrigel (Figure 38(a)).

[0427] In addition, a relative quantitative analysis of the matrisome proteins that make up Matrigel and SEM was conducted and the results were shown in numerical values. The results showed that Matrigel is composed of more than 96% glycoproteins, and SEM showed that each matrisome protein was evenly distributed with collagens at 40%, proteoglycans at 35%, and glycoproteins at 15% (Figure 38(b)).

[0428] Therefore, since the SEM scaffold contains a wider variety of extracellular matrix components than the existing Matrigel, it is expected that it will provide a more suitable microenvironment for the culture of gastric organoids.

[0429] As a result of performing a quantitative analysis on the matrisome protein data, it was confirmed that in the case of Matrigel, more than 95% of the proteins were glycoproteins, and when examining the 10 most abundant matrisome proteins, it was confirmed that laminin, a representative glycoprotein, accounted for a very high proportion. Furthermore, no proteins known to be specifically expressed in large amounts in actual stomach tissue were detected (Figure 39(a)).

[0430] Similarly, when quantitative analysis of matrisome proteins contained in SEM was performed, it was confirmed that SEM contained a large amount of matrisome proteins in the order of collagens, proteoglycans, and glycoproteins, and that they were uniformly distributed. In addition, when the 10 most abundant matrisome proteins were observed, it was confirmed that collagen was present in a very large amount, unlike matrigel. In addition, five non-matrisome proteins that are known to be specifically expressed in actual stomach tissue were detected in SEM. It is expected that such stomach tissue-specific components will also have a positive effect on the growth and development of gastric organoids (Figure 39(b)).

[0431] Therefore, it is expected that the SEM scaffold with this protein structure can be used as a hydrogel more suitable for culturing gastric organoids than existing Matrigel.

[0432] Experimental Example 4-7. Analysis of non-matrisome proteins in decellularized stomach tissue

[0433] The non-matrisome proteins contained in the decellularized gastric tissue of the present invention were analyzed in comparison with Matrigel.

[0434] Specifically, the decellularized gastric tissue was dissolved in lysis buffer [4% sodium dodecyl sulfate (SDS), 0.1M Tris-HCl (pH 7.6), and 1X protease inhibitor], and the protein components were extracted and decomposed to the peptide level. The types and relative values ​​of peptides were then detected using a mass spectrometer and MaxQuant software. Detailed classification, such as matrisomes, was then carried out using an existing library.

[0435] As a result, as can be seen in Figure 40, it was confirmed that most of the proteins that make up Matrigel are matrisome proteins, and among these, non-matrisome proteins were analyzed using the GOBP (gene ontology biological process) method to analyze and compare what biological processes are involved in the proteins contained in abundance. It was confirmed that Matrigel contains many translation or metabolism-related proteins (Figure 40(a)).

[0436] SEM confirmed that the sample contained relatively more non-matrisome proteins. By analyzing the roles of these proteins using the GOBP method, it was confirmed that the sample contained a particularly large amount of proteins involved in the formation of cells and tissues, such as organelle organization and cytoskeleton organization (Figure 40(b)).

[0437] Therefore, it is expected that SEM scaffolds containing such non-matrisome proteins can be used as hydrogels more suitable for culturing gastric organoids than existing Matrigel.

[0438] Experimental Example 5: Analysis of scaffold compositions containing extracellular matrix derived from decellularized gastric tissue

[0439] Experimental Example 5-1. Selection of the optimal concentration of decellularized gastric tissue-derived extracellular matrix in the scaffold composition

[0440] In relation to the support composition (hydrogel) prepared in Example 2-2, the optimal concentration of the extracellular matrix derived from decellularized gastric tissue was selected.

[0441] Specifically, to select the optimal hydrogel concentration when applying a scaffold derived from decellularized gastric tissue to organoid culture, hydrogels were prepared with different SEM concentrations (1, 3, 5, and 7 mg / mL) and gastric organoids were cultured in them. Gastric gland tissue, the most functional unit, was extracted from mouse stomach tissue and cultured in 3D in each hydrogel to induce the formation of gastric organoids. On the fifth day of culture, the morphology and formation efficiency of gastric organoids formed under each SEM concentration condition were compared with gastric organoids formed in Matrigel.

[0442] As a result, as can be seen from Figure 41, it was confirmed that the gastric organoids cultured from SEM hydrogels under all concentration conditions formed spherical shapes, similar to the organoids cultured from Matrigel used as a control group. At a concentration of 1 mg / ml, relatively small organoids were formed (Figure 41 (A)). Comparing the formation efficiency of gastric organoids for each SEM concentration, it was confirmed that in the case of culture using SEM hydrogel, the formation efficiency was lower than that of Matrigel at most concentrations, but the formation efficiency was highest in SEM hydrogel with a concentration of 5 mg / ml compared to the other concentrations (Figure 41 (B)).

[0443] Experimental Example 5-2. Comparison of the culture state of gastric organoids cultured from support compositions with different concentrations of extracellular matrix derived from decellularized gastric tissue

[0444] In relation to the support composition (hydrogel) prepared in Example 2-2, the culture state of gastric organoids was compared according to the concentration of extracellular matrix derived from decellularized gastric tissue.

[0445] Specifically, to select the optimal SEM hydrogel concentration when applying the scaffold derived from decellularized gastric tissue to the culture of gastric organoids, gastric organoids were cultured from hydrogels prepared with different SEM concentrations (1, 3, 5, and 7 mg / mL). Matrigel was used as a control.

[0446] As a result, the mRNA expression levels of gastric organoids cultured for 5 days in SEM hydrogels prepared at each concentration were compared through quantitative PCR (q-PCR) analysis. It was confirmed that Lgr5, a gene related to stemness, was significantly decreased at concentrations of 1mg / ml and 7mg / ml. In addition, Pgc (chief cell), Atp4a, and Atp4b (parietal cell), which are genes expressed in specific cell types in gastric tissue, showed a tendency to increase in expression as the concentration increased. Overall, it is considered that the 5mg / ml SEM concentration is a suitable concentration for maintaining stemness (stemness-Lgr5 expression) and promoting differentiation into various gastric tissue-specific cell types compared to the control Matrigel group (Figure 42 (A)).

[0447] Furthermore, on the same day, 5 days, the expression levels of gastric tissue cell markers Muc5ac (gastric pit cell) and HK (parietal cell) were compared through immunostaining, and it was confirmed that the expression levels of gastric tissue-specific marker proteins increased overall as the SEM hydrogel concentration increased (Figure 42 (B)). Through screening of the SEM hydrogel concentration, follow-up experiments were carried out at 5mg / ml SEM, which is expected to promote both the proliferation and differentiation of gastric organoids.

[0448] Experimental Example 5-3. Comparison of the culture state of gastric organoids cultured from the support composition of the extracellular matrix derived from decellularized gastric tissue and the existing culture support

[0449] The culture aspects of gastric organoids cultured from the support composition of the present invention and an existing culture support (Matrigel) were compared.

[0450] Specifically, we compared gastric organoids grown on Matrigel, the most widely used medium for culturing organoids, and on SEM hydrogel (5 mg / ml) supports derived from decellularized gastric tissue.

[0451] As a result, as shown in Figure 43, it was confirmed that the gastric organoids formed on each support grew large and could be cultured well up to 5 to 6 days just before subculture.

[0452] Experimental Example 5-4. Comparison of the support composition of the extracellular matrix derived from decellularized gastric tissue with gastric organoids cultured from an existing culture support

[0453] We performed multiple comparative analyses between gastric organoids cultured on Matrigel, the most widely used medium for culturing organoids, and on SEM hydrogel (5 mg / ml) scaffolds derived from decellularized gastric tissue.

[0454] Specifically, gene expression and protein expression of gastric organoids cultured from each culture support on day 5 of culture were compared.

[0455] When comparing the expression of markers at the mRNA level in gastric organoids cultured from decellularized SEM hydrogel scaffolds and Matrigel, it was confirmed that the expression of stem cell markers Lgr5 and Axin2 was at similar levels.In addition, it was confirmed that the expression levels of genes Muc6 (neck cell), Gif (parietal cell), Pgc, and Pga5 (chief cell), which are expressed in the various cell types mentioned above, were also similar (Figure 44(A)).

[0456] In addition, by comparing protein expression through immunostaining, it was confirmed that both groups of organoids expressed similar levels of stem cell markers and proliferation-related markers LGR5, SOX9, KI67, and differentiation-related markers MUC5AC (gastric pit cell), CHGA (endocrine cell), and HK (parietal cell).ECAD and ZO1, which are related to cell-cell interaction or tight junctions, were also well expressed in each organoid, and morphologically, it was confirmed that the organoids were composed of gastric tissue-constituting cells and that the shape of the epithelium was well maintained (Figure 44 (B)).

[0457] Therefore, it was confirmed that the SEM hydrogel derived from decellularized gastric tissue is a support capable of culturing gastric organoids at a similar level compared to the existing culture support, Matrigel.

[0458] Experimental Example 5-5. Comparative analysis of acid secretion of gastric organoids cultured from a support composition of extracellular matrix derived from decellularized gastric tissue and an existing culture support

[0459] The functionality of gastric organoids cultured for 5 days from the hydrogel scaffold of the present invention was analyzed in comparison with gastric organoids cultured for the same period in Matrigel.

[0460] Specifically, the acid secretion function, one of the important functions mentioned above, was confirmed by Acridine Orange staining experiments.

[0461] As a result, as can be seen from Figure 45, the more acidic the solution is, the more green fluorescence (F 500-550 ) becomes weaker, and the red fluorescence (F 600-650) increased. After staining with Acridine Orange, the fluorescence expression was compared for each group. SEM confirmed that the acid secretion of gastric organoids cultured from hydrogel was at a similar level to that of gastric organoids cultured from Matrigel. Therefore, it is considered that gastric organoids cultured on a scaffold derived from decellularized gastric tissue can functionally replace the organoids cultured from existing Matrigel.

[0462] Experimental Example 5-6. Confirmation of the effect of promoting organoid differentiation by mixing a support composition of an extracellular matrix derived from decellularized gastric tissue with an existing culture support

[0463] The effect of promoting organoid differentiation by mixing the hydrogel scaffold composition of the present invention with an existing culture scaffold was confirmed.

[0464] Specifically, to construct a culture system that could further promote the differentiation of gastric organoids while replacing the existing culture support Matrigel to the maximum extent possible, SEM hydrogel and Matrigel were mixed at a ratio of 9:1 (v / v) and tests were conducted on a new culture support. The Matrigel group was used as the control group.

[0465] As a result, the mRNA expression levels of each marker in gastric organoids cultured for 5 days from SEM / MAT (9:1) hydrogel and Matrigel were compared. Not only was the expression of Lgr5, which is related to stemness, significantly increased, but the expression levels of various differentiation markers Gif (parietal cell) and Pgc (chief cell), which are expressed in specific gastric tissue cells, were also significantly increased. The expression of Pga5 (chief cell) was also increased compared to organoids cultured from Matrigel (Figure 46 (A)).

[0466] In addition, the expression of various marker proteins was confirmed through immunostaining of gastric organoids cultured for 5 days on the SEM / MAT (9:1) hydrogel scaffold. The results showed that the differentiation of gastric organoids was promoted and well-formed through the expression of MUC5AC (gastric pit cells), CHGA (endocrine cells), HK (parietal cells), and ECAD (epithelial cells) (Figure 46 (B)).

[0467] These results suggest that mixing a small amount of Matrigel into SEM-based hydrogel makes it possible to construct a culture scaffold that can further promote the proliferation and differentiation of gastric organoids.

[0468] Experimental Example 5-7. Confirmation of the effect of enhancing the function of organoids by mixing a support composition of an extracellular matrix derived from decellularized gastric tissue with an existing culture support

[0469] The effect of enhancing the function of organoids by mixing the hydrogel scaffold composition of the present invention with an existing culture scaffold was confirmed.

[0470] Specifically, the degree of functional enhancement of gastric organoids cultured for 5 days on a hydrogel scaffold of SEM / MAT (9:1) was analyzed. Acridine Orange staining confirmed the acid secretion function, one of the important functions mentioned above. The Matrigel group was used as a control group.

[0471] As a result, as can be seen in Figure 47, the fluorescence expression after Acridine Orange staining was compared for each group. It was confirmed that the acid secretion of gastric organoids cultured from SEM / MAT (9:1) hydrogel was significantly enhanced compared to gastric organoids cultured from Matrigel. This experiment confirmed that by mixing SEM hydrogel scaffolds derived from decellularized gastric tissue, it is possible to produce gastric organoids with better functionality.

[0472] Experimental Example 5-8. Confirmation of tissue-specific effects of the support composition of extracellular matrix derived from decellularized gastric tissue

[0473] To confirm whether the stomach-specific extracellular matrix components contained in the scaffold derived from decellularized gastric tissue exhibit tissue-specific effects in the culture of gastric organoids, we performed experiments in which gastric organoids were cultured on decellularized scaffolds derived from other organs and compared them.

[0474] Specifically, gastric organoids were cultured on decellularized scaffolds derived from each tissue for five days, and then various analyses were carried out.

[0475] As a result, when culture was attempted using decellularized scaffolds derived from the stomach, intestine, skin, lymph, heart, and muscle, it was confirmed that gastric organoids were well formed on the decellularized scaffolds derived from the stomach and intestine. On the other hand, it was confirmed that gastric organoids were not well formed on the decellularized scaffolds derived from other tissues, such as skin (Figure 48(A)).

[0476] Quantitative analysis of the organoid formation efficiency confirmed that the stomach- and intestine-derived decellularized scaffolds showed similar formation efficiency, whereas the skin tissue-derived decellularized scaffold showed a decreased formation efficiency (Figure 48(B)).

[0477] By comparing the expression of marker mRNA in gastric organoids cultured for 5 days on decellularized scaffolds derived from each organ, it was confirmed that the expression level of Lgr5, which is related to stemness, was significantly reduced in organoids cultured on scaffolds derived from skin tissue (Figure 48(C)).

[0478] Through this experiment, it was possible to confirm the tissue-specific effect of the decellularized scaffold derived from gastric tissue in the culture of gastric organoids, and it was also confirmed that the decellularized scaffold derived from the intestine, which is a digestive tissue similar to the above, exhibits a similar effect.

[0479] Meanwhile, we attempted to confirm the tissue-specific effect of the scaffold derived from decellularized gastric tissue through the relative quantification of ECM proteins contained in the scaffold derived from decellularized gastric tissue (SEM) and the scaffold derived from decellularized skin tissue (SkEM).

[0480] As a result, as can be seen in Figure 49, when comparing a total of 55 extracellular matrix proteins detected in SEM and SkEM, it can be seen that 41 extracellular matrix proteins are more abundant in SEM than in SkEM. Among these, major extracellular matrix components such as fibronectin and laminin, which are known to be important for organoid development, are more abundant in SEM, which is why it was determined that SEM is more suitable for culturing gastric organoids.

[0481] Experimental Example 5-9. Analysis of differences in components of extracellular matrix support composition derived from decellularized gastric tissue according to tissue age

[0482] The differences in the components of the extracellular matrix of the support composition of the present invention according to tissue age were analyzed.

[0483] Specifically, gastric organoids were cultured from SEM hydrogels derived from the stomach tissue of young pigs (Piglet) and SEM hydrogels derived from the stomach tissue of adult pigs (Adult pig), and the differences in gene expression in gastric organoids and the differences in the extracellular matrix protein components contained in each hydrogel were analyzed.

[0484] The gene expression differences of gastric organoids cultured from each hydrogel were analyzed through qPCR experiments, and it was confirmed that gastric organoids cultured from scaffolds derived from piglet stomach tissue showed an overall increased expression of stem cell and differentiation markers compared to gastric organoids cultured from scaffolds derived from adult pig stomach tissue (Figure 50(a)).

[0485] Through quantitative comparison of the extracellular matrix protein components contained in each hydrogel, it was confirmed that the piglet stomach tissue-derived scaffold contained relatively more extracellular matrix proteins than the adult pig stomach tissue-derived scaffold. In particular, the piglet tissue-derived scaffold contained more major extracellular matrix components such as fibronectin and laminin, indicating that the scaffold made from the tissue of a young individual is more effective in the formation and differentiation of gastric organoids (Figure 50(b)).

[0486] Experimental Example 5-10. Optimization of culture medium conditions for organ culture of gastric organoids in a support composition of extracellular matrix derived from decellularized gastric tissue

[0487] Conditions for optimizing the culture medium for organ culture of gastric organoids on the support composition of the present invention were selected.

[0488] Specifically, as can be seen from FIG. 51(a), an experiment was conducted while adjusting factors (reagents marked in red) added additionally to the existing culture medium (reagents marked in black).

[0489] As a result, as can be seen in Figure 51(b), organ culture of gastric organoids was attempted in the existing culture medium (Conv), the medium supplemented with A83-01 (Conv+A), the medium supplemented with nicotinamide (Conv+Ni), and the medium supplemented with both (Conv+A+Ni). By investigating the maximum number of subcultures and analyzing the morphology of the organoids, it was found that long-term subculture was not possible under the existing culture medium conditions or the culture medium conditions supplemented with only one additional factor. However, it was confirmed that subculture was possible at least eight times under the improved culture medium conditions supplemented with both A83-01 and nicotinamide.

[0490] Experimental Example 5-11. Organ culture of gastric organoids in a support composition of extracellular matrix derived from decellularized gastric tissue

[0491] Under the culture medium conditions established in the above experimental examples, organ culture of gastric organoids was attempted in the scaffold derived from decellularized gastric tissue, and various stemness markers were analyzed. The Matrigel group was used as a control group.

[0492] After culturing for about 2 months (61 days) with continued subculture in SEM hydrogel, it was confirmed that the gastric organoids were spherical, just like those cultured from Matrigel, and that their size was maintained at a similar level throughout the culture (Figure 52(A)).

[0493] We compared the expression patterns of stemness-related mRNA in gastric organoids cultured from SEM hydrogel and Matrigel on days 5 and 11 of culture. We confirmed that the expression of Lgr5, Axin2, and Olfm4 genes, which are related to stem cell markers (stemness), was maintained at a similar level or even higher than in the Matrigel group (Figure 52(B)).

[0494] After eight passages each, on day 45, when comparing the expression levels of Lgr5, Axin2, and Olfm4 in the SEM hydrogel group and the MAT Matrigel group, it was confirmed that the expression levels were maintained at similar or even higher levels in the gastric organoids cultured from SEM hydrogel (Figure 22(C)).

[0495] Through this, it was confirmed that gastric organoids can be cultured for at least two months on a hydrogel scaffold derived from decellularized gastric tissue, and that they have similar or even improved levels of stemness compared to organoids cultured from Matrigel during the same culture period.

[0496] Experimental Example 5-11. Analysis of transcriptome expression in gastric organoids cultured from extracellular matrix support compositions derived from decellularized gastric tissue

[0497] The expression of the transcriptome of gastric organoids cultured from the support composition of the present invention was analyzed.

[0498] Specifically, gene expression in gastric organoids cultured from SEM hydrogel was confirmed through RNA sequencing, and the level of transcript expression was compared with that of organoids cultured from Matrigel. When comparing the two groups, genes with differential expression levels (Differentially Expressed Genes; DEGs) were selected and used for Gene Ontology analysis.

[0499] Among the genes expressed in both gastric organoids cultured from SEM hydrogel and Matrigel, 590 DEGs were selected using the statistical criteria of 2-fold, p-value<0.05, and FDR<0.1, and plotted in a heatmap. When comparing the two organoids, it was confirmed that the morphology of the gastric organoids cultured from each hydrogel was similar, but the expression of a large number of genes showed differences (Figure 53(A)).

[0500] Gene ontology analysis was performed to analyze the gene categories that were increased (orange) and decreased (green) in the SEM hydrogel group. It was confirmed that the expression of genes belonging to categories related to extracellular matrix, such as extracellular region, extracellular region part, proteinaceous extracellular matrix, and extracellular matrix, was significantly increased. In contrast, the most frequently observed decrease in the expression of genes related to lipid metabolism, such as cholesterol biosynthetic process, sterol biosynthetic process, and steroid metabolic process, was observed (Figure 53 (B)).

[0501] A total of 28 DEG genes whose expression levels were increased by more than 4 times in the SEM group compared to the Matrigel group were selected, and it was confirmed that most of the genes were included in the extracellular matrix-related gene category, confirming that the expression of extracellular matrix-related genes was significantly increased. In particular, 25 of the 28 increased genes were included in the extracellular region category, confirming that the largest increase was observed. In addition, it was confirmed that 14 genes related to the extracellular region part, 4 genes related to proteinaceous extracellular matrix, 4 genes related to extracellular matrix, and 4 genes related to regulation of cell proliferation were included, confirming that SEM hydrogel significantly increased the expression of genes related to the extracellular matrix and cell proliferation of gastric organoids compared to Matrigel (Figure 53(C)).

[0502] Meanwhile, the transcriptome of mouse stomach tissue was confirmed through RNA sequencing analysis and compared with the transcriptome of gastric organoids cultured from each hydrogel.

[0503] The ratio of genes that were increased or decreased in the transcripts of gastric organoids cultured from each hydrogel compared to mouse gastric tissue was divided by ontology, and the analysis was carried out by selecting ontology as words related to extracellular matrix and cell-matrix interactions.The expression levels of gastric organoids were decreased in most categories compared to gastric tissue, but it was confirmed that gastric organoids cultured from SEM hydrogel had a greater number of increased genes compared to gastric organoids cultured from Matrigel (Figure 54(a)).

[0504] Analysis of individual genes confirmed that the expression levels of genes such as Nid1 and Pxdn, which are involved in the extracellular matrix; Msi1, Dbn1, Chgb, and Nrg1, which are involved in gastric epithelial cells; and pyy and Cpt1c, which are related to hormone activity, were higher in gastric organoids cultured from SEM hydrogel than in gastric organoids cultured from Matrigel, and were closer to the expression levels in gastric tissue (Figure 54(b)).

[0505] Through this transcript analysis, it was confirmed that gastric organoids cultured from SEM hydrogel were more similar to gastric tissue than gastric organoids cultured from Matrigel.

[0506] Experimental Example 6: Verification of the usability of the support composition

[0507] Experimental Example 6-1. Transplantation of gastric organoids into the body using a support composition derived from decellularized gastric tissue

[0508] Animal experiments and histological analysis were conducted to utilize the hydrogel scaffold of the present invention as a material for transplantation of gastric organoids.

[0509] Specifically, in order to efficiently deliver and engraft gastric organoids into the damaged gastric tissue of a mouse gastric ulcer model, gastric organoids were transplanted using SEM hydrogel, and in order to adjust the viscosity of the hydrogel for easy injection during transplantation, SEM hydrogel was mixed at 1:20 (v / v) = SEM:culture medium. To track the organoids transplanted in vivo, gastric organoids made from stem cells extracted from EGFP-expressing mouse tissue were used for transplantation. In addition, gastric ulcers were induced in the mouse model by treating the stomach wall of the mouse with 100% acetic acid for 30 seconds using a capillary tube, and gastric organoids were transplanted into the damaged gastric tissue site using SEM hydrogel, and the degree of engraftment of the organoids was observed (Figure 55(a)).

[0510] Gastric organoids labeled with DiI fluorescent dye were transplanted into a mouse gastric ulcer model using a decellularized SEM hydrogel scaffold, and the engraftment was observed one day after transplantation through fluorescence expression. It was confirmed that the transplanted organoids were present throughout the gland of the stomach tissue. No fluorescence was observed in the damaged stomach tissue where the organoids were not transplanted (Figure 55(b)).

[0511] Gastric organoids expressing EGFP were transplanted into a mouse gastric ulcer model using a decellularized SEM hydrogel scaffold, and the engraftment was observed one day later through fluorescence expression. It was confirmed that the transplanted organoids were well engrafted and present in the damaged tissue area (Figure 55(c)).

[0512] Experimental Example 6-2. Verification of the possibility of culturing gastric organoids using a support composition stored for a long period of time

[0513] The culturing ability of gastric organoids in the support composition of the present invention that had been stored for a long period of time was verified.

[0514] Specifically, the SEM solution was frozen and stored at -80°C for a long period of time, and the SEM hydrogel produced by thawing it was used to culture gastric organoids, confirming the feasibility and safety of long-term storage (Figure 56(A)).

[0515] As a result, as can be seen from Figure 56(B), it was confirmed that gastric organoids could be cultured well even in SEM hydrogel thawed after long-term frozen storage (image of organoids on the 5th day of culture), and the organoid formation efficiency was maintained similarly to that of SEM hydrogel and Matrigel freshly prepared just before culture. In addition, there was no significant difference in the mRNA expression levels of stem cell markers and differentiation markers. Through this, it was confirmed that the scaffold derived from decellularized gastric tissue can be stably maintained without denaturation even when stored for a long period of time, for a minimum of 6 months, under frozen conditions (-80℃), and can be used for the culture of gastric organoids. It was verified that the scaffold can be developed into a product with a long shelf life by being able to be stored for a long period of time.

[0516] On the other hand, the SEM solution was stored in a refrigerator at 4°C and used to culture gastric organoids to confirm whether it could be stored for a long period of time, whether it would denature, and its safety (Figure 57(A)).

[0517] As a result, as can be seen from Figure 57(B), it was confirmed that gastric organoids were formed at a similar level well in the hydrogel made with refrigerated SEM solution compared to SEM hydrogel and Matrigel made freshly just before culturing, and that they could be well maintained and cultured (image of organoids on the 5th day of culturing). Through this, it was confirmed that the scaffold derived from decellularized gastric tissue maintained its safety without denaturation for at least one month even when stored refrigerated at 4℃ in solution form, and that it could be used for culturing gastric organoids after refrigerated storage. It was also verified that the long-term storage possible means that it can be developed into a product with a long shelf life.

[0518] Experimental Example 6-3. Verification of the possibility of culturing gastric cancer organoids using the support composition

[0519] The possibility of culturing gastric cancer organoids using the support of the present invention was confirmed.

[0520] Specifically, when MKN-74 and NCI-N87 gastric cancer cell lines were cultured in 3D in SEM hydrogel, it was confirmed that cancer organoids were formed (images of organoids on the 10th day of culture and analysis of immunostaining).

[0521] As a result, as can be seen in Figure 58, it was confirmed through observation with an optical microscope that organoids were well formed in both gastric cancer cell lines within the SEM hydrogel, and immunostaining was used to stain Ki67 protein, which is associated with cell proliferation, confirming that cells within the organoids were actively proliferating.

[0522] Through this experiment, it was confirmed that the SEM hydrogel scaffold derived from decellularized gastric tissue is capable of culturing not only stem cell-derived gastric organoids, but also gastric cancer organoids, confirming the possibility that SEM hydrogel may be utilized as a core technology of a culture platform for constructing an in vitro model of gastric cancer.

[0523] Experimental Example 6-4. Confirmation of the feasibility of mass culturing of gastric organoids using a support composition and a microfluidic chip

[0524] The possibility of constructing a culture platform for gastric organoids based on SEM hydrogel was confirmed using a microfluidic chip that enables mass culture of organoids and screening of multiple drugs. The microfluidic chip can provide a fine flow of culture medium to the organoids using a simple method using a rocker. In addition, the chip has many chambers, making it possible to mass-produce organoids (Figure 59(a)).

[0525] In the above device, gastric organoids were cultured using the support of the present invention.

[0526] When SEM hydrogel containing gastric organoids was cultured in a microfluidic chip, it was confirmed that when the organoids were cultured for 5 days with a fine flow of culture medium in the culture chamber, the gastric organoids were well cultured and grew to a size of 1 mm (Figure 59(b)).

[0527] These results suggest that gastric organoids cultured in 3D within a hydrogel scaffold derived from decellularized gastric tissue can be grafted with microfluidic chip technology to combine the tissue-specific extracellular matrix microenvironment with precise dynamic culture of microfluidic flow, enabling efficient mass production of organoids.

[0528] In the present invention, a decellularized liver tissue-derived scaffold for the culture and transplantation of hepatic organoids and a manufacturing method thereof are described. The liver tissue is decellularized to remove all cellular components, and decellularized liver tissue is produced in which liver-specific extracellular matrix components are preserved. A three-dimensional hydrogel based on this is then applied to the culture of hepatic organoids.

[0529] The decellularized liver tissue-derived scaffold developed in the present invention is composed only of extracellular matrix components of pure water from which cellular antigens have been removed, so it does not cause inflammatory reactions or immune rejection of tissues when transplanted and has excellent biocompatibility. It is easy to fabricate and has a low manufacturing cost, so it is more economical than Matrigel and can be used as a safe culture and transplantation material.

[0530] In fact, protein analysis confirmed that the scaffold derived from decellularized liver tissue contains various extracellular matrices and factors specific to liver tissue. It was confirmed that stem cell-derived liver organoids could develop and grow in the hydrogel scaffold derived from the decellularized liver tissue produced, and various concentrations of the decellularized liver tissue scaffold were tested to select the hydrogel concentration condition that is optimal for culturing liver organoids.

[0531] When comparing hepatic organoids cultured on the developed scaffold derived from decellularized liver tissue with organoids cultured on the control group, Matrigel, it was confirmed that the differentiation ability and liver functionality (albumin secretion, cytochrome activity, element synthesis, etc.) were similarly maintained or improved. This verified that the scaffold derived from decellularized liver tissue can replace the existing Matrigel for culturing hepatic organoids.

[0532] Through these results, it was confirmed that liver organoids cultured from the support developed in the present invention can more accurately embody the structure and function of liver tissue than organoids cultured from the existing matrix (Matrigel).

[0533] In the present invention, liver injury was induced in a mouse model, and liver organoids were transplanted using a hydrogel scaffold derived from decellularized liver tissue. It was confirmed that the organoids were successfully engrafted at the damaged site, enabling efficient transplantation, and the feasibility of using liver organoids as a transplant recipient was also confirmed.

[0534] In the following, preferred examples are presented to aid in understanding the present invention. However, the following examples are merely provided to facilitate understanding of the present invention, and the contents of the present invention are not limited to the following examples.

[0535] Example 3: Preparation of a support composition comprising extracellular matrix derived from decellularized liver tissue

[0536] A support composition comprising an extracellular matrix derived from decellularized liver tissue was prepared as follows (see Figure 60(A)).

[0537] Example 3-1. Preparation of extracellular matrix (LEM) derived from decellularized liver tissue

[0538] Pig liver tissue was prepared by isolating and cutting into pieces, and the liver tissue was stirred with 1% Triton X-100 and 0.1% ammonium hydroxide to remove only the liver tissue cells, producing decellularized liver tissue.

[0539] The decellularized liver tissue was then freeze-dried and crushed to produce a decellularized liver tissue-derived extracellular matrix (LEM).

[0540] Example 3-2. Preparation of support composition

[0541] 10 mg of the extracellular matrix derived from the decellularized liver tissue 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. The mixture was mixed uniformly with 10X PBS and 1M NaOH at neutral pH and in the electrolyte state of 1X PBS buffer, and gelled at 37°C for 30 minutes to prepare a hydrogel-type support composition (Figure 60(B)).

[0542] Experimental Example 7: Analysis of extracellular matrix (LEM) derived from decellularized liver tissue

[0543] The extracellular matrix derived from the decellularized liver tissue of Example 3-1 was analyzed as follows.

[0544] Experimental Example 7-1. Analysis of the characteristics of extracellular matrix derived from decellularized liver tissue

[0545] The characteristics of the extracellular matrix derived from the decellularized liver tissue of Example 3-1 were analyzed.

[0546] Specifically, DNA was extracted from the decellularized liver tissue scaffold and liver tissue that had not been subjected to the decellularization process, and a quantitative comparison was performed. The GAG ​​content in the scaffold was measured using a dye-binding assay using chondroitin sulfate and dimethylmethylene blue (Figure 61(A)).

[0547] For H&E staining, the decellularized liver tissue scaffold and tissue that had not undergone the decellularization process were thinly cut, and the cell nuclei were stained with hematoxylin and the cytoplasm with eosin to analyze the degree of removal of cell nuclei in the liver tissue scaffold that had undergone the decellularization process (Figure 61(B)).

[0548] Decellularized liver tissue scaffolds were prepared according to concentration, and the internal structure of the hydrogel was analyzed using a scanning electron microscope, which scans an electron beam to form an image (Figure 61(C)).

[0549] A quantitative comparison of DNA before and after the decellularization process of the decellularized liver tissue scaffold confirmed that most of the cellular components were removed by the decellularization process. A quantitative analysis of glycosaminoglycan (GAG), a representative component of the extracellular matrix, confirmed that GAG was well preserved and remained within the decellularized liver tissue (Figure 61(A)).

[0550] H&E staining confirmed that the structure of the prepared decellularized liver tissue matrix was well maintained and all cellular components were removed (Figure 61(B)).

[0551] Through analysis using a scanning electron microscopy (SEM), it was confirmed that the hydrogel scaffolds derived from decellularized liver tissue prepared at different concentrations had an internal structure in the form of nanofiber bundles (Figure 61 (C)), thereby confirming that it is possible to provide a three-dimensional microenvironment suitable for culturing liver organoids.

[0552] Experimental Example 7-2. Analysis of physical properties of extracellular matrix of decellularized liver tissue according to concentration

[0553] To investigate the difference in physical properties depending on the concentration of the scaffold derived from decellularized liver tissue, hydrogel formation was induced under four concentration conditions (2, 4, 6, and 8 mg / ml), and the mechanical properties were measured through rheological analysis.

[0554] As a result, as can be seen in Figure 62, the storage modulus (G') value was consistently higher than the loss modulus (G'') value under all LEM scaffold concentration conditions, confirming that a stable polymer network was formed through cross-linking within the hydrogel. As the LEM concentration increased, the physical properties also increased, and the physical properties were generally lower compared to the control Matrigel (MAT) group. The high-concentration (8mg / ml) LEM hydrogel had physical properties similar to Matrigel.

[0555] Experimental Example 7-3. Analysis of extracellular matrix proteins derived from decellularized liver tissue

[0556] In order to understand the components of the support derived from decellularized liver tissue, proteomics analysis using a mass spectrometer was performed, and it was confirmed that the LEM contains a variety of extracellular matrices (collagens, ECM glycoproteins, proteoglycans) and growth factor proteins specific to liver tissue.

[0557] As a result, as shown in Figure 63, it was confirmed that the existing commonly used scaffold, Matrigel (MAT), is mostly composed of ECM glycoproteins, while the decellularized liver tissue matrix (LEM) is composed of various components, in the order of collagens and ECM glycoproteins, followed by proteoglycans and ECM regulators. Among the top 10 extracellular matrix (ECM) proteins, Biglycan (BGN), Lumican (LUM), and Asporin (ASPN), which are specifically present in LEM, are major proteins involved in ECM remodeling during liver tissue development, and PRELP is known to play an important role in maintaining normal liver cell structure. It was confirmed that the decellularized liver tissue-derived extracellular matrix (LEM) scaffold created through this contains various extracellular matrix proteins present in actual liver tissue that play an important role in liver structure, development, and function compared to Matrigel.

[0558] Furthermore, as can be seen in Figure 64, when proteins known to be highly expressed in liver tissue were detected and compared in Matrigel (MAT) and the scaffold derived from decellularized liver tissue (LEM), more matrisome and non-matrisome proteins associated with liver tissue were detected in LEM than in Matrigel (Figure 64(a)).

[0559] When a Gene Ontology analysis was conducted to investigate the functions of the protein components other than the extracellular matrix (ECM) contained in each matrix, it was confirmed that the non-matrisome components contained in Matrigel are mainly responsible for functions related to the biosynthesis and metabolism of peptides and amides, whereas the non-matrisome components contained in the LEM scaffold prepared according to the present invention are mainly involved in roles related to small molecules, various organic acids, and cell metabolism (Figure 64(b)).

[0560] In other words, it is believed that the non-matrisome protein components other than the ECM contained in the LEM scaffold play an important role in the formation of highly functional liver organoids in which organic acid metabolism, an important function of liver cells, and various cellular metabolic capabilities are enhanced.

[0561] Experimental Example 8: Analysis of scaffold compositions containing extracellular matrix derived from decellularized liver tissue

[0562] Experimental Example 8-1. Selection of the concentration of hydrogel scaffold derived from decellularized liver tissue and the culture of liver organoids (cholangiocyte-derived liver organoids; CLO)

[0563] Hydrogels were prepared with different concentrations of LEM derived from decellularized liver tissue, and hepatic organoids (CLOs) were cultured in the hydrogels. The formation efficiency was compared to select the most suitable concentration for organoid culture. Matrigel, a commonly used culture scaffold, was used as the control. Hepatic organoids were produced using bile duct cells extracted from mouse liver tissue.

[0564] As shown in Figure 65, when LEM hydrogel derived from decellularized liver tissue was applied under various LEM concentration conditions for the cultivation of liver organoids, it was confirmed that liver organoids with a similar morphology to the control group, Matrigel (MAT), were well formed under all concentration conditions (2, 4, 6, 8 mg / ml) (Figure 65(A)). When comparing the formation efficiency of liver organoids depending on the concentration of LEM hydrogel scaffold derived from decellularized liver tissue on the 7th day of cultivation, it was confirmed that the formation efficiency was highest under conditions of 4 mg / ml and 6 mg / ml (Figure 65(B)).

[0565] Experimental Example 8-2. Analysis of the difference in differentiation ability of hepatic organoids (bile duct cell-derived hepatic organoids, CLO) depending on the LEM concentration of hydrogel scaffolds derived from decellularized liver tissue

[0566] LEM hydrogel scaffolds derived from decellularized liver tissue were prepared in various concentrations and used to culture mouse liver organoids, and the expression of differentiation-related genes in organoids cultured for 7 days under each concentration condition was compared and analyzed using quantitative PCR (qPCR). Matrigel (MAT), a commonly used culture scaffold, was used as a control group.

[0567] As shown in Figure 66, when comparing gene expression according to LEM concentration conditions, the gene related to stemness (LGR5) was slightly decreased in liver organoids cultured from LEM hydrogel, but the expression of liver differentiation-related markers (Krt19, Krt18, Hnf4a, Hnf1b, Foxa3) tended to increase. Considering that the difference in the formation efficiency of liver organoids compared to Matrigel at 6mg / ml among the LEM concentration conditions was not large and the expression of liver differentiation markers was further increased, the LEM hydrogel concentration was determined to be 6mg / ml LEM condition and then applied to the liver organoid experiment.

[0568] Experimental Example 8-3. Analysis of hepatic organoids (cholangiocyte-derived hepatic organoids, CLO) cultured from hydrogel scaffolds derived from decellularized liver tissue

[0569] We performed immunostaining of hepatic organoids cultured on Matrigel, the most widely used scaffold for culturing organoids, and LEM hydrogel (6mg / ml) scaffold derived from decellularized liver tissue. Hepatic organoids were produced using bile duct cells extracted from mouse liver tissue, and were compared through immunostaining on the 7th day of culture.

[0570] As shown in Figure 67, immunostaining for liver-specific markers confirmed that liver organoids cultured on the LEM hydrogel scaffold derived from decellularized liver tissue also expressed liver tissue-specific markers at a similar level to the Matrigel group, compared to the control group of liver organoids cultured on Matrigel. * KRT19 (cholangiocyte marker), ECAD (cell-cell junction marker), SOX9 (cholangiocyte progenitor marker), Ki67 (proliferative cell marker), ALB (mature hepatocyte marker)

[0571] Experimental Example 8-4. Analysis of the functionality of hepatic organoids (cholangiocyte-derived hepatic organoids, CLO) cultured from hydrogel scaffolds derived from decellularized liver tissue

[0572] Hepatic organoids derived from bile duct cells were cultured and liver functionality was evaluated on day 7. Matrigel, a commonly used culture support, was used as a control group, and LEM hydrogel was applied at a concentration of 6 mg / ml.

[0573] As shown in Figure 68, PAS staining to analyze the storage capacity of polysaccharides such as glycogen revealed that the hepatic organoids cultured from LEM hydrogel had a similar level of polysaccharide storage capacity to the control group (MAT) (Figure 68(A)). When various liver function analyses (albumin secretion, element synthesis capacity, cytochrome activity) were carried out, it was confirmed that the CLO-type hepatic organoids cultured from LEM hydrogel showed similar functionality to the hepatic organoids cultured from Matrigel (Figure 68(B)).

[0574] Experimental Example 8-5. Organ culture of hepatic organoids (bile duct cell-derived hepatic organoids, CLO) using hydrogel scaffolds derived from decellularized liver tissue

[0575] We attempted organ culture of mouse bile duct cell-derived hepatic organoids in the LEM hydrogel scaffold derived from the decellularized liver tissue established above, and performed liver-specific differentiation marker analysis. The Matrigel group was used as a control group.

[0576] As shown in Figure 69, when the hepatic organoids were cultured for one month while continuing subculture in LEM hydrogel, the image of hepatic organoid morphology was observed and it was confirmed that the pattern and size were similar to those of hepatic organoids cultured from Matrigel (Figure 69(A)). Even when the hepatic organoids were cultured for more than one month, it was confirmed that the expression of hepatic differentiation markers was increased in the hepatic organoids cultured from the LEM hydrogel scaffold derived from decellularized liver tissue compared to the Matrigel-based organoids, and it was found that the longer the culture period, the more the differentiation of the organoids was promoted in the LEM hydrogel (Figure 69(B)).

[0577] Experimental Example 8-6. Cultivation and analysis of human hepatic organoids (human cholangiocyte-derived hepatic organoids - hCLO) using hydrogel scaffolds derived from decellularized liver tissue

[0578] We confirmed whether it is possible to culture hepatic organoids derived from human liver tissue using LEM hydrogel scaffolds (6mg / ml) derived from decellularized liver tissue. We isolated hepatic cholangiocytes and cultured hepatic organoids derived from human cholangiocytes, and used Matrigel as a control group.

[0579] As shown in Figure 70, human-derived hepatic organoids were well formed on the LEM hydrogel scaffold derived from decellularized liver tissue, as was the case with Matrigel (MAT), and it was confirmed that long-term subculture for more than 50 days was possible (Figure 11(A)). When the expression of liver tissue markers was analyzed through quantitative PCR analysis on the 7th day of culture, the expression of the LGR5 gene, which is related to stemness, and liver differentiation-related marker genes (HNF4A, SOX9, FOXA2) in the hepatic organoids cultured from the LEM hydrogel was similar to that in the organoids cultured from Matrigel, or showed a tendency to be slightly increased (Figure 70(B)).

[0580] In addition, LEM hydrogel scaffolds derived from decellularized liver tissue were prepared in different concentrations and applied to the culture of human liver organoids, and the expression of differentiation-related genes in organoids cultured for 14 days under each concentration condition was compared and analyzed using quantitative PCR (qPCR). Matrigel (MAT), a commonly used culture scaffold, was used as the control group.

[0581] As a result, as can be seen in Figure 71, when comparing gene expression according to LEM concentration conditions, it was confirmed that there was no significant difference between the LEM hydrogel and Matrigel (MAT) groups in the gene related to stemness (LGR5) and liver differentiation-related markers (SOX9, HNF4A, KRT19, FOXA2), and they showed similar expression levels. Considering that 6mg / ml of hydrogel among the LEM concentration conditions showed good liver organoid formation efficiency and slightly increased expression of KRT19 and HNF4A differentiation markers, the LEM hydrogel concentration was determined to be 6mg / ml LEM, and was then applied to the liver organoid experiment.

[0582] We also performed immunostaining of hepatic organoids cultured on Matrigel (MAT) and LEM hydrogel (6mg / ml) scaffolds derived from decellularized liver tissue. Hepatic organoids were created using bile duct cells extracted from human liver tissue, and were compared through immunostaining on the 14th day of culture.

[0583] Through immunostaining for liver-specific markers, it was confirmed that liver organoids cultured on the LEM hydrogel scaffold derived from decellularized liver tissue expressed liver tissue-specific markers at a similar level to the Matrigel group, compared to the control group of Matrigel (MAT) (Figure 72(A)).

[0584] Proceeding with liver function analysis (ability to synthesize elements, albumin secretion) confirmed that human liver organoids cultured from LEM hydrogel showed similar or slightly higher functionality than liver organoids cultured from Matrigel (Figure 72(B)).

[0585] * KRT19 (cholangiocyte marker), ECAD (cell-cell junction marker), SOX9 (cholangiocyte progenitor marker), Ki67 (proliferative cell marker), F-actin (filamentous actin marker)

[0586] Experimental Example 8-7. Cultivation of hepatic organoids (hepatocyte-derived liver organoids; HLO) with different concentrations of hydrogel scaffolds derived from decellularized liver tissue and selection of the optimal concentration

[0587] Hydrogels were prepared with different concentrations of decellularized liver scaffolds, and liver organoids were cultured and analyzed for formation efficiency and gene expression. Matrigel, a commonly used culture scaffold, was used as the control group. Liver organoids were prepared using purified hepatocytes extracted by perfusion of mouse liver tissue.

[0588] As a result, as shown in Figure 73, when hepatocyte-derived hepatic organoids were cultured in the decellularized LEM hydrogel scaffold for 14 days, hepatic organoids with a similar pattern to the control group (MAT) were formed (Figure 73(A)). The formation efficiency of hepatic organoids according to the concentration of the decellularized scaffold was compared with the control group (MAT) on the 7th day of culture (Figure 73(B)). When the expression of liver tissue markers of organoids cultured from the LEM hydrogels prepared according to the concentration was analyzed through quantitative PCR analysis, it was confirmed that the hepatic differentiation markers (Krt18, Hnf4a, Cdh1) tended to increase further in the decellularized LEM scaffold group (Figure 73(C)). Since the formation efficiency was stable and the expression level of hepatic differentiation markers increased at a concentration of 4mg / ml, it was determined that this was an appropriate condition, and a concentration of 4mg / ml was then applied for HLO culture.

[0589] Experimental Example 8-8. Comparison of the growth of hepatic organoids (HLO) formed from hydrogel scaffolds (LEM) derived from decellularized liver tissue and existing culture scaffolds (MAT)

[0590] Hepatic organoids were prepared using purified hepatocytes extracted by perfusion of mouse liver tissue, and cultured on decellularized LEM scaffold (4mg / ml) and on Matrigel, a commonly used culture scaffold, to compare the growth rates of the organoids.

[0591] As a result, as shown in Figure 74, when comparing hepatic organoids derived from mouse hepatocytes grown on Matrigel, which is the most widely used material for organoid culture, and LEM hydrogel (4 mg / ml) scaffolds derived from decellularized liver tissue, it was confirmed that the hepatic organoids formed from each scaffold continued to grow and could be successfully subcultured.

[0592] Experimental Examples 8-9. Analysis of hepatic organoids (HLO) derived from hepatocytes cultured from decellularized liver tissue-derived hydrogel scaffolds (LEM)

[0593] Hepatic organoids were cultured for 20 days using decellularized liver scaffold hydrogel (4mg / ml), and immunohistochemical analysis and comparative analysis of liver functionality were performed. Matrigel, a commonly used culture scaffold, was used as a control. Hepatic organoids were produced using purified hepatocytes extracted by perfusion of mouse liver tissue.

[0594] As shown in Figure 75, when comparing the expression of various markers through immunostaining with organoids cultured from the control group of Matrigel (MAT), it was confirmed that liver tissue-specific markers were well expressed in the decellularized LEM scaffold group as well (Figure 75(A)).

[0595] * ALB (hepatocyte marker), ECAD (tight junction marker), F-actin (cytoskeleton marker)

[0596] PAS (Periodic Acid Schiff) staining, which confirms the storage capacity of polysaccharides such as glycogen, confirmed that both groups had the ability to store glycogen (Figure 75(B)). Comparing the element synthesis capacity and albumin secretion amount, it was confirmed that the functionality was increased in the decellularized LEM scaffold group compared to the control group (MAT) (Figure 75(C)).

[0597] Experimental Example 9: Confirmation of the feasibility of using hydrogel scaffolds derived from decellularized liver tissue

[0598] Experimental Example 9-1. Cultivation of hepatic organoids derived from human-induced pluripotent stem cells (hiPSCs) using a hydrogel scaffold derived from decellularized liver tissue

[0599] To adjust the physical properties of the hydrogel suitable for the formation of hepatic organoids from human induced pluripotent stem cells, a hydrogel layer was created by mixing LEM and culture solution at a ratio of 1:1 (v / v), and then cells required for the formation of hepatic organoids (human induced pluripotent stem cell-derived hepatic endoderm cells, vascular endothelial cells, and mesenchymal stem cells) were applied on top of the hydrogel layer. Within 72 hours, the cells formed into hepatic organoids through self-organization. Protein expression was comparatively analyzed through immunostaining of hepatic organoids cultured on the decellularized hepatic scaffold hydrogel (LEM). Matrigel (MAT), a commonly used culture scaffold, was used as a control group.

[0600] As shown in Figure 76, when hepatic endoderm cells + endothelial cells + mesenchymal stem cells derived from induced pluripotent stem cells were cultured from Matrigel (MAT) and hydrogel (LEM) layers derived from decellularized liver tissue, the cells solidified over time and formed 3D organoids within 72 hours in both groups (Figure 76(A)). When immunostaining was performed, it was confirmed that liver-related markers (ALB, AFP) and vascular cell marker (CD31) were well expressed in both groups (Figure 76(B)).

[0601] * ALB (mature hepatocyte marker), AFP (early hepatocyte marker), CD31 (vascular endothelial marker).

[0602] Experimental Example 9-2. Analysis of human induced pluripotent stem cell-derived hepatic organoids (hiPSC-LO) cultured on hydrogel scaffolds derived from decellularized liver tissue

[0603] Human induced pluripotent stem cell-derived hepatic organoids were cultured for 7 days using decellularized liver scaffold hydrogel, and functionality and gene expression were analyzed. Matrigel (MAT), a commonly used culture scaffold, was used as a control. Hepatic organoids were produced through the self-organization of human induced pluripotent stem cell-derived hepatic endoderm cells, vascular endothelial cells, and mesenchymal stem cells.

[0604] As shown in Figure 77, when PAS staining related to liver functionality was performed, it was confirmed that both the Matrigel group and the LEM hydrogel group had good glycogen storage. In the case of element synthesis ability, it was confirmed that it was more highly expressed in organoids cultured from decellularized matrix (Figure 77 (A)). When the expression of liver tissue markers was analyzed through quantitative PCR analysis, the expression of OCT4 gene, a marker related to pluripotency, of liver organoids cultured from LEM hydrogel was slightly decreased, but the expression of liver differentiation-related marker genes (SOX17, HNF4A) and blood vessel-related marker genes (PECAM1, CD34) tended to be higher than that of organoids cultured from Matrigel (Figure 77 (B)).

[0605] Experimental Example 9-3. Creation of a liver fibrosis model using hepatic organoids (CLOs) cultured from decellularized liver tissue-derived hydrogel scaffolds (LEM)

[0606] For disease modeling using scaffold-based hepatic organoids derived from decellularized liver tissue, TGF-beta was screened at various concentrations to confirm its potential for inducing liver fibrosis. The model was induced using mouse cholangiocyte-derived hepatic organoids (CLOs) cultured for 7 days. To create a liver fibrosis model, organoids were treated with various concentrations of TGF-beta for 48 hours and observed.

[0607] As shown in Figure 78, unlike the organoids that were not treated with TGF-β, the organoids that were treated with TGF-β showed changes in morphology and poor growth (scale bars = 100 μm) (Figure 78 (A)). To confirm liver fibrosis, SMA (Smooth Muscle Actin) and COL1A1 were stained to confirm the accumulation of extracellular matrix (ECM) around the organoids (scale bars = 100 μm) (Figure 78 (B)). Quantitative PCR analysis confirmed that the expression of liver fibrosis markers was highest in the group treated with 4 ng / ml TGF-β (Figure 78 (C)). Under normal conditions without TGF-β treatment, the expression of liver fibrosis marker (Col1a1) was decreased and the expression of bile duct cell marker (Krt19) was increased in the scaffold group derived from decellularized liver tissue compared to the control Matrigel group. However, when fibrosis was induced in hepatic organoids cultured on a scaffold derived from decellularized liver tissue through TGF-β treatment, the expression of fibrosis markers was significantly increased and the expression of bile duct cell markers was significantly decreased (Figure 78(D)). This experiment confirmed the feasibility of creating a fibrosis model using hepatic organoids cultured on a scaffold of extracellular matrix derived from decellularized liver tissue.

[0608] Meanwhile, for disease modeling using human cholangiocyte-derived hepatic organoids (hCLOs) cultured on a support derived from decellularized liver tissue, TGF-β was tested at various concentrations, and the most suitable concentration (80ng / ml) was treated to confirm the possibility of inducing liver fibrosis.

[0609] As shown in Figure 79, to create a liver fibrosis model, TGF-β was treated at different concentrations and the same organoids were observed after 3 days. It was confirmed that the untreated liver organoids grew for 3 days, but the treated liver organoids all changed in morphology and did not grow well (Figure 79(A)). To verify the liver fibrosis model, SMA (Smooth Muscle Actin) was stained to confirm the accumulation of extracellular matrix (ECM) around the organoids. Through this experiment, it was confirmed that a human liver fibrosis model can be created using liver organoids cultured from the decellularized liver scaffold (Figure 79(B)).

[0610] Experimental Example 9-4. Transplantation of hepatic organoids into the body using hydrogel scaffolds derived from decellularized liver tissue

[0611] Animal experiments and histological analysis were conducted to confirm the use of LEM hydrogel scaffolds derived from decellularized liver tissue as a transplantation material for liver organoids. Liver organoids were transplanted using LEM hydrogel to efficiently deliver and engraft them into the liver tissue of a mouse liver toxicity injury model. To adjust the viscosity of the LEM hydrogel for easy injection during transplantation, the LEM hydrogel was mixed at 1:10 (v / v) = LEM:culture medium.

[0612] As shown in Figure 80, a liver injury model was induced by intraperitoneal injection of acetaminophen (24 hours) and the injury model was verified through H&E histology analysis (Figure 80(A)). Bile duct cell-derived liver organoids (CLOs) labeled with DiI fluorescent reagent were transplanted into the mouse liver injury model. The liver organoids were transplanted using Matrigel and decellularized LEM hydrogel as a transplantation support, and the engraftment was observed through fluorescence expression one day after transplantation. It was confirmed that the liver organoids transplanted into the injury site survived well and existed within the liver tissue, confirming the potential of LEM hydrogel as a biomaterial for organoid transplantation (Figure 80(B)).

[0613] Experimental Example 9-5. Verification of the possibility of long-term storage of hydrogel derived from decellularized liver tissue

[0614] The LEM hydrogel derived from decellularized liver tissue was stored for a long period of time at -80℃ in a frozen state, and then used to culture mouse cholangiocyte-derived hepatic organoids (CLOs). Matrigel was used as a control.

[0615] As a result, as can be seen in Figure 81, when liver organoids were cultured using the control Matrigel, LEM hydrogel freshly prepared immediately before organoid culture, and LEM hydrogel stored for a long period of time at -80°C, organoids were well formed in all groups (Figure 81(A)).

[0616] Compared to the Matrigel and LEM hydrogels prepared immediately before culturing the hepatic organoids, the hepatic organoids were cultured well in the hydrogels prepared with the LEM solution that had been frozen for one or two months, and all of the hepatic differentiation markers increased compared to the Matrigel group, and it was confirmed that there were no significant differences between the decellularized LEM hydrogels, showing similar expression levels (Figure 81(B)).

[0617] The LEM hydrogel derived from decellularized liver tissue was stored for a long period of time under refrigerated conditions at 4°C, and then used to culture human cholangiocyte-derived hepatic organoids (hCLOs). Matrigel was used as a control.

[0618] As a result, as can be seen in Figure 82, compared to Matrigel and LEM hydrogels prepared immediately before culture, human liver organoids were cultured well in hydrogels prepared with LEM solution stored in a refrigerator for two months, and it was confirmed that there was no significant difference in either stem cell potential markers or liver differentiation markers, showing similar or high expression levels (Figure 82(A)).

[0619] When liver function analysis (element synthesis ability, albumin secretion) was performed, it was confirmed that human liver organoids maintained liver functionality well even in LEM hydrogel stored for a long time, and showed similar or slightly higher functionality than liver organoids cultured from the Matrigel group (Figure 82 (B)).

[0620] Through this, it was confirmed that the LEM hydrogel scaffold derived from decellularized liver tissue maintains its safety without denaturation even when stored in a solution state at 4℃ for more than two months, and can be used for culturing hepatic organoids.

[0621] Experimental Example 9-6. Cultivation of hepatoma organoids using hydrogel scaffolds derived from decellularized liver tissue

[0622] The feasibility of culturing human hepatoma organoids using the LEM hydrogel scaffold derived from decellularized liver tissue was confirmed. Human hepatic organoids derived from normal liver tissue and hepatic organoids derived from hepatocellular carcinoma (HCC) patient tissue were cultured on Matrigel and LEM hydrogel derived from decellularized liver tissue for 20 days, and then analyzed.

[0623] As a result, as can be seen from Figure 83, it was confirmed that both normal liver organoids and liver cancer organoids were formed in the same way on the Matrigel group and the LEM hydrogel scaffold derived from decellularized liver tissue (Figure 83(A)). Immunostaining was performed on liver cancer organoids cultured from Matrigel and liver cancer organoids cultured from LEM hydrogel, and it was confirmed that liver cancer-related markers (SMA, AFP) proteins were stained in both groups (Figure 83(B)). When gene expression of normal liver organoids and liver cancer organoids cultured from LEM hydrogel derived from decellularized liver tissue was compared through qPCR analysis, it was confirmed that the expression level of LGR5, which is related to stemness, was decreased in liver cancer organoids, and the expression of TNFa and HES1, which are related to inflammatory responses, and the expression of liver cancer-related AFP and PLIN2 markers were increased (Figure 83(C)).

[0624] Through this, it was confirmed that the LEM hydrogel scaffold derived from decellularized liver tissue can be applied to the cultivation of not only normal liver organoids but also liver cancer organoids, confirming its potential use as a culture platform for constructing an in vitro model of liver cancer.

[0625] Verification of the superiority of the scaffold derived from decellularized liver tissue constructed in the present invention through comparison with the liver tissue decellularization protocol in Experimental Example 9-7.

[0626] We compared the decellularization method developed in this study (Protocol 1) with the decellularization method reported in the existing literature (Protocol 2). Protocol 2 involved treating the tissue with 4% sodium deoxycholate for 4 hours, followed by DNase I (3 hours) to remove DNA, while the decellularization method developed in this study induced decellularization using only a mixed solution of 1% Triton X-100 and 0.1% ammonium hydroxide, which is an even milder condition to reduce damage to proteins in the tissue.

[0627] As a result, as can be seen in Figure 84, H&E histology analysis confirmed that while cells were successfully removed by both methods, ECM components were more preserved in tissues treated with Protocol 1 (Figure 84(A)).

[0628] After the decellularization process, DNA was significantly reduced in both tissues treated with both protocols, but GAG quantitative analysis confirmed that the amount of GAG remaining in the tissue treated with protocol 2 was significantly less than that in the tissue treated with protocol 1. When the mechanical properties (elastic modulus) of the hydrogels derived from the decellularized liver tissue prepared with each protocol were measured, it was confirmed that the hydrogel derived from the tissue decellularized through protocol 2 had even lower properties (Figure 84(B)).

[0629] These results indicate that Protocol 1 is a more efficient method that can induce efficient decellularization while reducing damage to the extracellular matrix (ECM).

[0630] We also compared the decellularization method developed in the present invention (Protocol 1) with a commonly used decellularization method (Protocol 3). In Protocol 3, the decellularization method developed in the present invention was applied, followed by an additional 24-hour treatment with 3% sodium dodecyl sulfate reagent.

[0631] As a result, as can be seen in Figure 85, H&E histology analysis showed that while cells were well removed by both methods, ECM components were more preserved in tissues treated with Protocol 1 (Figure 85(A)).

[0632] After the decellularization process, DNA was significantly reduced in both tissues treated with both protocols, but quantitative GAG ​​analysis confirmed that the amount of GAG remaining in tissue treated with protocol 3 was significantly less than that in tissue treated with protocol 1. When the mechanical properties (elastic modulus) of the decellularized liver tissue-derived hydrogels prepared with each protocol were measured, it was confirmed that there was no significant difference in the properties of the hydrogels prepared with both protocols (Figure 85(B)).

[0633] When proteomics was performed on the liver tissue-derived LEM scaffolds prepared using each decellularization protocol, it was confirmed that the LEM scaffold prepared using Protocol 1 contained liver tissue-specific Collagen α1 (XVIII) and Kininogen 1 proteins, and a greater number of extracellular matrix proteins were also detected (Figure 85(C)).

[0634] Therefore, it was confirmed that Protocol 1 can induce a more efficient decellularization process that can reduce damage to liver tissue-specific extracellular matrix components compared to Protocol 3.

[0635] Experimental Example 9-8. Verification of the superiority of the decellularized liver tissue-derived support constructed in the present invention through comparison of liver tissue decellularization protocols (organoid culture experiment)

[0636] Mouse bile duct cell-derived hepatic organoids (CLOs) were cultured and compared using hydrogels derived from decellularized liver tissues produced using the decellularization method developed in this invention (Protocol 1), a decellularization method reported in existing literature (Protocol 2), and a commonly used existing decellularization method (Protocol 3).

[0637] It was confirmed that liver organoids were well formed from the LEM hydrogels derived from decellularized liver tissues prepared by protocols 1, 2, and 3 (Day 7) (Figure 86(A)). When gene expression was compared using quantitative PCR (qPCR) on Day 7 for liver organoids cultured from LEM hydrogels prepared by each protocol, it was confirmed that the expression of stem cell potential (Lgr5) markers and hepatic differentiation markers (Afp, Foxa3) was significantly decreased in liver organoids cultured from hydrogels prepared by protocols 2 and 3 compared to protocol 1 (Figure 86(B)). When comparing the formation efficiency of liver organoids from the existing culture scaffold Matrigel (MAT) and LEM hydrogels prepared by each decellularization protocol, it was confirmed that the formation efficiency of liver organoids was slightly decreased in the group of protocol 1, but was significantly decreased in the hydrogels prepared by protocols 2 and 3 (Figure 86(C)).

[0638] These results indicate that the LEM hydrogel prepared by Protocol 1 is more efficient in the formation of hepatic organoids and can provide a more favorable environment for the growth and differentiation of hepatic organoids. In other words, the scaffold derived from decellularized liver tissue prepared by Protocol 1 constructed in the present invention is a more excellent scaffold for culturing hepatic organoids.

[0639] Experimental Example 9-9. Interspecies comparative analysis of decellularized liver tissue-derived hydrogel scaffolds (pig vs. human)

[0640] We conducted protein analysis of Matrigel, a hydrogel scaffold derived from porcine decellularized liver tissue (Porcine LEM), and a scaffold derived from human decellularized liver tissue (Human LEM), which was produced by decellularizing human liver tissue. Through a comparison of the protein analysis of the three hydrogels, we verified the suitability of the hydrogel derived from porcine decellularized liver tissue developed in this invention for culturing human liver organoids.

[0641] The majority of the components of the extracellular matrix in the control Matrigel were ECM glycoproteins, followed by other ECM regulators and proteoglycans, whereas the LEM scaffolds derived from porcine and human decellularized liver tissues contained the most proteoglycans, with collagens and ECM glycoproteins present at similar levels. In addition, when the top 10 ECM proteins were examined, most of the components were found to be similar in porcine LEM and human LEM, which were very different from the control Matrigel. Therefore, the hydrogel scaffolds derived from porcine decellularized liver tissues can provide a microenvironment similar to that of human liver tissue, and are therefore more suitable for culturing human liver organoids than Matrigel. When analyzing the extracellular matrix protein components that were specifically present in each culture matrix, it was confirmed that the most abundant ECM proteins were detected in the LEM scaffold derived from human liver tissue, and that even more ECM protein components were detected in the LEM scaffold derived from pig liver tissue than in the control Matrigel group (Figure 87(A)).

[0642] In order to investigate the functions of the non-matrisome protein components, excluding the extracellular matrix (ECM) contained in each scaffold, a Gene Ontology analysis was performed. It was confirmed that the non-matrisome components of Matrigel are mainly responsible for functions related to the biosynthesis and metabolism of peptides and amides, whereas the non-matrisome components of human and porcine LEM scaffolds are mainly involved in roles related to small molecules, various organic acids, and cell metabolism (Figure 87(B)). In other words, in addition to the ECM contained in the human and porcine LEM scaffolds, the non-matrisome protein components are considered to play an important role in the formation of functionally mature liver organoids with enhanced organic acid metabolism and various cell metabolisms, which are important functions of hepatocytes.

[0643] Experimental Examples 9-10. Comparison of human hepatic organoids (human cholangiocyte-derived hepatic organoids - hCLO) cultured from hydrogel scaffolds derived from human and porcine decellularized liver tissue

[0644] Human hepatic organoids were cultured on Matrigel (MAT) and LEM hydrogel scaffolds derived from human and porcine decellularized liver tissues prepared above. Hepatic organoids were generated using cholangiocytes extracted from human liver tissue, and gene expression and factor synthesis ability were compared through qPCR on the 14th day of culture.

[0645] Compared to the hepatic organoids cultured from the control Matrigel (MAT), the hepatic organoids cultured from the LEM hydrogel scaffolds derived from decellularized liver tissues from two different species were also able to form organoids at a similar level to the Matrigel group (Figure 88(A)).

[0646] When gene expression was compared through qPCR analysis of liver organoids cultured from each matrix, it was confirmed that stemness-related markers (LGR5) were maintained at similar levels, while differentiation markers (KRT19, KRT7, SOX9) were increased in the decellularized liver tissue LEM hydrogel group. When liver functionality (ability to synthesize elements) was analyzed, it was confirmed that liver organoids cultured from LEM hydrogels derived from human and porcine liver tissue had similar or slightly higher levels of functionality than liver organoids cultured from Matrigel (Figure 88(B)).

[0647] This shows that the LEM scaffold derived from porcine tissue has the same performance as the LEM scaffold derived from human liver tissue, and therefore can be used to culture hepatic organoids by replacing Matrigel and matrices derived from human liver tissue.

[0648] In the present invention, a scaffold derived from decellularized lung tissue for the culture and transplantation of pulmonary organoids and a method for producing the same are disclosed. A large amount of decellularized tissue is obtained by chemically treating porcine lung tissue, and a hydrogel scaffold is produced based on the scaffold and applied to the culture of pulmonary organoids. The newly developed decellularized culture scaffold can be produced with a simple process and low cost, and is highly economical, which is advantageous for commercialization. In addition, the developed decellularized scaffold is in a state where all immunogenic cells have been removed, and contains components of the extracellular matrix specific to lung tissue and various factors, which can improve the culture of pulmonary organoids.

[0649] In order to find the optimal conditions for culturing pulmonary organoids, we conducted organoid culture experiments using hydrogel scaffolds derived from decellularized lung tissue at various concentrations. It was confirmed that the differentiation of pulmonary organoids cultured on the optimized decellularized lung tissue scaffold was promoted. Compared to the commonly used scaffold, Matrigel, the expression of various lung tissue cell-specific proteins was also observed. It was also confirmed that organ culture was possible over many passages. It was confirmed that the decellularized scaffold produced through this could be used as a substitute for Matrigel as a culture matrix for pulmonary organoids.

[0650] In the present invention, it was confirmed that it is possible to culture not only mouse-derived lung organoids but also human-derived lung organoids using the decellularized lung tissue scaffold. It was also confirmed that human lung organoids cultured from the decellularized scaffold well expressed lung tissue cell-specific proteins, making organ culture possible.

[0651] In the present invention, in addition to the method of three-dimensionally culturing pulmonary organoids in a decellularized scaffold, a method of culturing pulmonary organoids without a culture scaffold was additionally constructed by adding a decellularized scaffold dissolved in a culture medium. It was confirmed that the efficiency of organoid formation was greatly increased and differentiation into alveolar-specific cells was promoted when a decellularized scaffold solution was added to the culture medium. If organoids can be cultured without a hydrogel scaffold, unlike the method of culturing organoids from an existing scaffold, desired factors and stimuli can be added directly to the organoids, and sample separation and collection are easy, which can have many advantages in organoid research.

[0652] Furthermore, the present invention has confirmed the possibility of producing a disease model using lung organoids. By adding fine dust particles at different concentrations, a lung organoid model for studying the harmfulness of fine dust can be produced, and a drug that induces pulmonary fibrosis can be treated to model pulmonary fibrosis. Such a disease model platform is expected to be useful for pulmonary fibrosis-related research and drug screening.

[0653] In the present invention, we have also established a technique for culturing alveolar-specific organoids. The alveolus is a unit that performs the role of gas exchange in the lungs and is one of the most important lung tissues. It is expected that the use of alveolar organoids will enable more diverse lung disease research.

[0654] In the following, preferred examples are presented to aid in understanding the present invention. However, the following examples are merely provided to facilitate understanding of the present invention, and the contents of the present invention are not limited to the following examples.

[0655] Example 4: Preparation and analysis of decellularized lung tissue-derived extracellular matrix (Lung Extracellular Matrix, LuEM) for the culture of lung organoids

[0656] Example 4-1. Preparation of extracellular matrix support (Lung Extracellular Matrix, LuEM) derived from decellularized lung tissue

[0657] The lung tissue was cut into small pieces and treated with a decellularization solution of 1% Triton X-100 and 0.1% ammonium hydroxide for 48 hours under refrigerated conditions, after which the extracellular matrix of decellularized lung tissue in powder form was produced through freeze-drying.

[0658] Example 4-2. Preparation of support composition

[0659] The dried powdered tissue can be stored for a long period of time. It was dissolved in 4 mg / mL pepsin solution (4 mg of pepsin powder derived from porcine gastric mucosa dissolved in 1 mL of 0.02 M HCl) for 48 hours, and then uniformly mixed with 10X PBS and 1 M NaOH at neutral pH and in the electrolyte state of 1X PBS buffer, and gelled at 37°C for 30 minutes to prepare a hydrogel-type support composition.

[0660] Experimental Example 10: Analysis of Lung Extracellular Matrix (LuEM) derived from decellularized lung tissue

[0661] Experimental Example 10-1. Analysis of the characteristics of the extracellular matrix support (Lung Extracellular Matrix, LuEM) derived from decellularized lung tissue

[0662] We fabricated a lung extracellular matrix (LuEM) scaffold from decellularized lung tissue of pigs and analyzed its properties.

[0663] Specifically, to confirm whether the cells were sufficiently removed from the fabricated decellularized scaffold and whether the extracellular matrix components were sufficiently remaining, DNA and GAG (Glycosaminoglycans) were quantified. After the decellularization process, it was confirmed that most of the DNA was removed, and GAG was present at a similar level to that of actual lung tissue (**p<0.01 versus Before) (Figure 89(A)).

[0664] In addition, H&E histological analysis of the tissue after the decellularization process confirmed that most of the purple-stained cell nuclei had been removed, while the characteristic structure of lung tissue was maintained (Figure 89(B)).

[0665] In addition, to confirm the internal structure of the three-dimensional hydrogel fabricated based on the components of the extracellular matrix derived from decellularized lung tissue, a scanning electron microscopy (SEM) was used for analysis. The results showed that it had a porous structure based on extracellular matrix nanofibers, and had structural characteristics and an environment suitable for material exchange for the cultivation of organoids (Figure 89(C)).

[0666] Experimental Example 10-2. Analysis of the physical properties of decellularized lung tissue-derived extracellular matrix (Lung Extracellular Matrix, LuEM) for the culture of lung organoids

[0667] An experiment was conducted to confirm whether the extracellular matrix support derived from decellularized lung tissue has suitable physical properties for culturing lung organoids.

[0668] Specifically, hydrogels were prepared based on the components of the extracellular matrix of decellularized lung tissue at various concentrations, and their physical properties were measured through rheological analysis.

[0669] As a result, as can be seen from Figure 90, the physical properties tended to increase as the LuEM concentration increased. Matrigel, which is widely used as an existing culture scaffold, has also been reported to have physical properties of less than 100 Pa, and it was confirmed that the physical properties of LuEM hydrogel are at a level suitable for the culture of lung organoids.

[0670] Experimental Example 10-3. Analysis of protein content of decellularized lung tissue-derived extracellular matrix (Lung Extracellular Matrix, LuEM) for the culture of lung organoids

[0671] To identify the components of the extracellular matrix contained in the LuEM scaffold derived from decellularized lung tissue, protein analysis was performed using a mass spectrometer.

[0672] As a result, as can be seen in Figure 91, it was confirmed that many extracellular matrix proteins and glycoproteins, such as various collagens, proteoglycans, and glycoproteins, are contained in the LuEM scaffold derived from decellularized lung tissue. Through this, it is expected that such lung tissue-specific factors present in actual lung tissue will also play a positive role in the culture of lung organoids.

[0673] Experimental Example 10-4. Kinds and quantitative analysis of matrisome proteins in extracellular matrix support (Lung Extracellular Matrix, LuEM) derived from decellularized lung tissue

[0674] The matrisome proteins contained in LuEM were detected through protein analysis using a mass spectrometer, classified by type, and a relative quantitative analysis was performed.

[0675] As a result, it was confirmed that various types of extracellular matrix matrisome proteins, such as collagen, glycoprotein, and proteoglycan, were uniformly present within the LuEM scaffold, and it was predicted that such components present in actual lung tissue may promote the formation and development of lung organoids (Figure 92(A)(B)).

[0676] By quantitative protein analysis, the 10 most abundant matrisome proteins in the LuEM scaffold were identified, and it was confirmed that various types of collagen, decorin, elastin, fibrinogen (alpha), biglycan, and other proteins were abundantly contained. In addition, when matrisome proteins known to be specifically abundant in actual lung tissue were identified in the LuEM scaffold, it was confirmed that four of these proteins were present (Figure 92(C)). It was predicted that such diverse matrisome components contained in LuEM could promote the formation, development, and differentiation of lung organoids.

[0677] Therefore, it was predicted that LuEM hydrogels with such diverse protein compositions could be used as effective supports for the culture of lung organoids by providing a lung tissue-specific extracellular matrix microenvironment.

[0678] Experimental Example 10-5. Analysis of non-matrisome proteins in the extracellular matrix support (Lung Extracellular Matrix, LuEM) derived from decellularized lung tissue

[0679] The non-matrisome proteins in the extracellular matrix support (Lung Extracellular Matrix, LuEM) derived from decellularized lung tissue of the present invention were analyzed.

[0680] As a result, it was confirmed that LuEM contains not only matrisomes but also a large amount of non-matrisome proteins (Figure 93(A)). Furthermore, when checking the non-matrisome proteins that are known to be abundant in actual lung tissue, it was confirmed that LuEM contains seven diverse non-matrisome proteins (Figure 93(B)).

[0681] On the other hand, we analyzed non-matrisome proteins using the gene ontology biological process (GOBP) method to determine what biological processes they contain.

[0682] As a result, it was confirmed that the sample contained a particularly large amount of proteins responsible for functions related to tissue formation and development, such as cellular component organization or biogenesis, organelle organization, and cytoskeleton organization (Figure 93(C)).

[0683] Therefore, it was predicted that LuEM supports containing such non-matrisome proteins might improve the formation and development of lung organoids.

[0684] Experimental Example 10-6. Comparative analysis of lung tissue decellularization protocols

[0685] An experiment was conducted to compare the decellularization method of the present invention (Protocol 1) with the decellularization method reported in the existing literature (Protocol 2). The existing literature used DNase I after treating with 4% sodium deoxycholate for 4 hours to remove DNA, whereas the present invention used only a solution of 1% Triton X-100 and 0.1% ammonium hydroxide, which is an even milder condition, to minimize damage to proteins in the tissue.

[0686] As a result, when comparing the amount of remaining DNA after the decellularization process, it was confirmed that both protocols were able to successfully remove DNA. However, when comparing the remaining extracellular matrix components through GAG quantification, it was confirmed that the GAG ​​components were well preserved in the tissue treated with protocol 1, whereas the remaining GAG components were significantly reduced in the tissue treated with protocol 2 (Figure 94(a)).

[0687] Furthermore, after producing a hydrogel using LuEM derived from the decellularized lung tissue, the physical properties were measured, and it was confirmed that the physical properties of the hydrogel produced using LuEM derived from the decellularized tissue processed by Protocol 2 were somewhat reduced (Figure 94(b)).

[0688] Furthermore, a comparison of H&E histology analysis confirmed that while both methods effectively removed cells, more ECM proteins remained in tissues treated with protocol 1 (Figure 94(c)).

[0689] Furthermore, protein analysis of the LuEM supports prepared by each decellularization protocol confirmed that tissue treated with protocol 1 contained a greater number of extracellular matrix proteins than tissue treated with protocol 2 (Figure 94(d)).

[0690] Through this, it was predicted that the decellularization method established in this invention is more effective in preserving extracellular matrix proteins of lung tissue than existing methods, and therefore the LuEM hydrogel prepared in Protocol 1 can be applied as a more suitable support for the culture of lung organoids.

[0691] In addition, an experiment was conducted to compare the decellularization method of the present invention (Protocol 1) with a commonly used existing decellularization method (Protocol 3). Specifically, in Protocol 3, after the decellularization method of the present invention, the cells were treated with 3% sodium dodecyl sulfate, a commonly used decellularization reagent, for an additional 24 hours.

[0692] As a result, when comparing the amount of DNA remaining after the decellularization process, it was confirmed that both protocols were able to successfully remove DNA. However, when comparing the remaining extracellular matrix components through GAG quantification, it was confirmed that the GAG ​​components were well preserved in the tissue treated with protocol 1, whereas the remaining GAG components were significantly reduced in the tissue treated with protocol 3 (Figure 95(a)).

[0693] In addition, after preparing hydrogels using LuEM derived from the decellularized lung tissue, the physical properties were measured, and it was confirmed that there was no significant difference in the physical properties of the hydrogels prepared using each protocol (Figure 95(b)).

[0694] Furthermore, a comparison of H&E histology analysis confirmed that while both methods effectively removed cells, more ECM proteins remained in tissues treated with protocol 1 (Figure 95(c)).

[0695] In addition, protein analysis of the LuEM supports prepared by each decellularization protocol confirmed that tissue treated with protocol 1 contained slightly more extracellular matrix proteins than tissue treated with protocol 3 (Figure 95(d)).

[0696] Through this, it was predicted that the decellularization method established in this invention is more effective in preserving extracellular matrix proteins of lung tissue than the method using sodium dodecyl sulfate (SDS) reagent, and therefore the LuEM hydrogel prepared in Protocol 1 can be applied as a more suitable support for the culture of lung organoids.

[0697] Experimental Example 11: Analysis of scaffold compositions containing extracellular matrix derived from decellularized lung tissue

[0698] Experimental Example 11-1. Analysis of mouse lung organoid formation according to the concentration of extracellular matrix support (Lung Extracellular Matrix, LuEM) derived from decellularized lung tissue

[0699] A hydrogel derived from decellularized lung tissue was prepared using various concentrations of LuEM components, and lung organoids were cultured in the hydrogel. On the seventh day of culture, the formation efficiency of lung organoids under each condition was compared and analyzed. Matrigel (MAT), a commonly used culture scaffold, was used as the control group, and lung organoids were generated using stem cells extracted from mouse lung tissue.

[0700] We tried culturing lung organoids under four LuEM hydrogel concentrations. At concentrations of 3 mg / ml or higher, it was confirmed that organoids were formed in a pattern similar to that of the MAT condition, but at a concentration of 1 mg / ml, most of the organoids sank due to their low physical properties and grew on the bottom of the culture dish (Figure 96(A)). Therefore, it was confirmed that the 1 mg / ml LuEM concentration is not suitable as a culture support for lung organoids, and in subsequent experiments, the 1 mg / ml condition was removed.

[0701] Comparing the formation efficiency under each hydrogel condition, it was found that the formation efficiency was somewhat lower in hydrogels with LuEM concentrations of 3 mg / ml or more than in the control group, Matrigel. However, most of the LuEM conditions induced organoid formation efficiencies of 75% or more, verifying that LuEM hydrogel can be used for the culture of lung organoids without any problems (**p<0.01 versus MAT) (Figure 96(b)).

[0702] Experimental Example 11-2. Analysis of the differentiation degree of mouse lung organoids according to the concentration of extracellular matrix support (Lung Extracellular Matrix, LuEM) derived from decellularized lung tissue

[0703] Based on various concentrations of LuEM components, decellularized lung tissue-derived hydrogels were prepared and applied to the culture of lung organoids. On the seventh day of culture, the expression of four pulmonary bronchiolar tissue-related genes was comparatively analyzed, and the commonly used culture scaffold, Matrigel (MAT), was used as a control group.

[0704] As a result, as can be seen in Figure 97, it was confirmed that Krt5, a basal cell-related gene, showed a significantly higher level of expression in lung organoids cultured from LuEM hydrogel compared to the control group. There was no significant difference in expression levels between the 3, 5, and 7 mg / ml LuEM hydrogel groups.

[0705] Furthermore, it was confirmed that Muc5ac, a gene related to Goblet cells, and Scgb1a1, a gene expressed from Club cells, showed the highest expression in LuEM hydrogel with a concentration of 7 mg / ml.

[0706] Foxj1, a gene related to ciliated cells, was found to have similar levels of expression in all hydrogel conditions, including the control group (*p<0.05 and**p<0.01 versus MAT).

[0707] Therefore, it was confirmed that the culture of lung organoids in LuEM hydrogel promoted or maintained the expression of genes related to the differentiation of pulmonary bronchioles at a similar level.Among these, the 7mg / ml LuEM hydrogel group showed the highest level of overall gene expression, so it was predicted that this concentration of LuEM hydrogel could be used as a support that could further promote the differentiation of organoids into pulmonary bronchiolar tissue compared to existing Matrigel, and the concentration was fixed at 7mg / ml LuEM conditions for further experiments.

[0708] Experimental Example 11-3. Comparative analysis of the expression of specific marker proteins in mouse lung organoids cultured from decellularized lung tissue-derived extracellular matrix (Lung Extracellular Matrix, LuEM) and Matrigel

[0709] The expression of various lung tissue-specific proteins in lung organoids cultured from LuEM hydrogel derived from decellularized lung tissue was analyzed by immunostaining.

[0710] Specifically, the results were compared with lung organoids cultured on the seventh day from culture using the existing culture support, Matrigel. This experiment used 7mg / ml LuEM hydrogel, which was confirmed as the most suitable condition above, and Matrigel was used as a control group.

[0711] As a result, as shown in Figure 98, various cell types present in lung tissue, Club cell (CC10), Goblet cell (MUC5AC), Basal cell (P63), were confirmed to be expressed in both organoids cultured under both conditions, and KI67 protein related to cell proliferation and Sox9, a protein expressed from lung progenitor cells, were also observed to be expressed in both organoids, confirming that cells in the lung organoids were actively proliferating. In addition, through cell-cell interaction and staining of E-cadherin (ECAD) and F-actin associated with cytoskeleton, it was confirmed that cells were organically connected to form organoids.

[0712] Experimental Example 11-4. Analysis of the functionality of mouse lung organoids cultured from decellularized lung tissue-derived extracellular matrix (Lung Extracellular Matrix, LuEM) and Matrigel

[0713] The functionality of lung organoids cultured from LuEM hydrogel derived from decellularized lung tissue was compared and analyzed by observing the swelling phenomenon caused by drugs. The swelling degree of lung organoids over time was observed after treatment with forskolin, a CFTR (cystic fibrosis transmembrane conductance regulator) antagonist, and Matrigel was used as a control.

[0714] As a result, as can be seen in Figure 99, it was confirmed that the lung organoids cultured from the LuEM hydrogel scaffold expanded over time after forskolin treatment. It was confirmed that they expanded more than the lung organoids cultured from Matrigel. This shows that the lung organoids cultured from LuEM hydrogel have a similar level of functionality to the lung organoids cultured from Matrigel.

[0715] Experimental Example 11-5. Analysis of mouse lung organoids cultured on a support made of extracellular matrix (Lung Extracellular Matrix, LuEM) derived from decellularized lung tissue

[0716] Mouse lung organoids cultured from the extracellular matrix support (Lung Extracellular Matrix, LuEM) derived from decellularized lung tissue of the present invention were analyzed.

[0717] Specifically, in decellularized LuEM hydrogel and Matrigel, as can be seen from the images of organoid morphology obtained during organ culture of lung organoids for 93 days (Figure 100(A)), it was observed that the organoids had a hollow structure at the beginning, and changed to a filled structure as the culture continued. Matrigel was used as a control group.

[0718] In addition, to confirm the cell distribution of the lung organoids cultured for 150 days while subculture was progressed up to 17 times in each culture support, immunostaining analysis was performed with CC10 (club cells), acetylated α-tubulin (ciliated cells), and P63 (basal cells). As a result, it was confirmed that the composition of various lung tissue cells was maintained within the lung organoids cultured from LuEM hydrogel for up to 150 days. In addition, it was confirmed that F-actin, a cytoskeleton marker, was also well expressed (Figure 100(B)).

[0719] On day 150 of culture, qPCR was used to compare the expression of Krt5 (basal cells) and Muc5ac (goblet cells), confirming that lung organoids cultured from LuEM hydrogel showed significantly higher levels of expression than organoids cultured from Matrigel (*p<0.05 and ***p<0.001 versus MAT) (Figure 100(C)).

[0720] These results confirmed that LuEM hydrogel can be used to culture mouse lung organoids at a similar or superior level to existing Matrigel, verifying its potential as an alternative material to Matrigel.

[0721] In addition, when comparing the expression of Krt5 (basal cells), Muc5ac (goblet cells), and Scgb1a1 (club cells) in lung organoids that had been subcultured 1, 3, and 7 times in LuEM hydrogel, it was confirmed that the expression of each marker increased as the subculture progressed. This confirmed that the differentiation of lung organoids was further promoted as the culture continued in LuEM hydrogel (Figure 101).

[0722] These results confirmed that the use of LuEM hydrogel may promote the differentiation of specific cells the longer the culture is continued, making it possible to produce lung organoids that are superior to those produced using existing culture scaffolds.

[0723] Experimental Example 11-6. Confirmation of the tissue-specific effect of the extracellular matrix support (Lung Extracellular Matrix, LuEM) derived from decellularized lung tissue

[0724] To confirm whether the tissue-specific proteins contained in LuEM hydrogel have tissue-specific effects in the culture of lung organoids, a comparative experiment was conducted in which lung organoids were cultured on decellularized scaffolds derived from other organs, and lung organoids from each tissue-derived hydrogel were cultured for 7 days and then analyzed.

[0725] Specifically, we tried culturing lung organoids using decellularized scaffolds derived from lung, muscle, and kidney. It was confirmed that, while the lung and muscle decellularized scaffolds were often cultured to a size similar to that of the control group, the Matrigel group, the kidney decellularized scaffolds formed smaller organoids (Figure 102(A)).

[0726] Quantitative analysis of the formation efficiency confirmed that the lung and muscle-derived decellularized scaffolds showed similar formation efficiency, whereas the kidney tissue-derived decellularized scaffold showed a decreased formation efficiency compared to the other decellularized scaffolds (**p<0.01 versus MAT) (Figure 102(B)).

[0727] The expression of Krt5 (basal cells), Muc5ac (goblet cells), and Scgb1a1 (club cells) was compared in lung organoids cultured for 7 days on decellularized scaffolds derived from each organ. As a result, it was confirmed that the expression of basal cells was the highest in organoids cultured from the lung-derived decellularized scaffold, and that the expression of club cells was significantly reduced in the muscle-derived decellularized scaffold (**p<0.01 versus NT) (Figure 102(C)).

[0728] Through this experiment, it was demonstrated that LuEM hydrogel is superior to other organ-derived decellularized scaffolds in culturing lung organoids, and tissue-specific effects can be confirmed.

[0729] Experimental Example 11-7. Cultivation of human lung organoids using a support of extracellular matrix (Lung Extracellular Matrix, LuEM) derived from decellularized lung tissue

[0730] Using LuEM hydrogel derived from decellularized lung tissue, we conducted experiments to determine whether it was possible to culture not only mouse lung organoids, but also human lung organoids.

[0731] Specifically, we extracted stem cells from human lung tissue and cultured lung organoids on a decellularized LuEM hydrogel scaffold. As a result, we confirmed that human lung organoids were formed from the LuEM hydrogel and grew well. Although they grew relatively slower than mouse lung organoids, we confirmed that subculture and organ culture were also possible (Figure 103(A)).

[0732] In cultured human lung organoids, the expression of lung tissue marker proteins was confirmed through immunostaining. Immunostaining for P63 protein expressed by basal cells and MUC5AC, a protein expressed by goblet cells, was performed, and cells expressing P63 and MUC5AC proteins were observed in the lung organoids. In addition, it was confirmed that the lumen of the lung organoids was well formed and the structure was well connected organically due to the expression of ZO-1 and F-actin (Figure 103(B)).

[0733] Functional analysis of human lung organoids to confirm the expansion response to forskolin demonstrated that after drug treatment, the lung organoids expanded over time and functioned normally (Figure 103(C)).

[0734] The results of this experiment confirmed that human lung organoids can also be cultured using an extracellular matrix support derived from decellularized lung tissue.

[0735] Experimental Example 12: Confirmation of the applicability of the support composition of the present invention

[0736] Experimental Example 12-1. Creation of a pulmonary fibrosis model using mouse lung organoids cultured from a support of extracellular matrix (Lung Extracellular Matrix, LuEM) derived from decellularized lung tissue

[0737] For lung disease modeling using lung organoids, we confirmed the possibility of inducing pulmonary fibrosis by treating mouse lung organoids with TGF-b.

[0738] Specifically, on the fourth day after culturing, lung organoids were treated with each concentration for three days and analyzed on the seventh day. Lung organoids that were not treated with TGF-b (NT, no treatment) were used as a control group.

[0739] As a result, in order to create a pulmonary fibrosis model, lung organoids were treated with various concentrations of TGF-b and observed three days later. It was found that lung organoids that were not treated with TGF-b grew well over the three days, but all lung organoids that were treated with TGF-b showed changes in morphology and did not grow well (Figure 104(A)).

[0740] We examined the expression of Col1a2, a pulmonary fibrosis-related marker, and found that the higher the TGF-b treatment concentration, the higher the expression of Col1a2. When treated with 40ng / ml of TGF-b, the expression of Col1a2 was statistically significantly increased (*p<0.05 versus NT) (Figure 104(B)).

[0741] The expression of fibrosis-related proteins, smooth muscle actin (SMA) and collagen type 1 (Col1), was confirmed through immunostaining, and it was found that the higher the concentration of TGF-b treatment, the higher the expression of fibrosis markers (Figure 104(C)).

[0742] Through this experiment, the feasibility of creating a pulmonary fibrosis model based on lung organoids cultured on a LuEM hydrogel scaffold was confirmed.

[0743] Experimental Example 12-2. Creation of a human pulmonary fibrosis model using human lung organoids cultured from decellularized lung tissue-derived extracellular matrix (LuEM)

[0744] To model pulmonary fibrosis using human lung organoids cultured from decellularized LuEM hydrogel scaffolds, we confirmed the possibility of inducing pulmonary fibrosis by treating human lung organoids with TGF-b.

[0745] Specifically, human lung organoids cultured from each support were treated with TGF-b for 3 days and then analyzed.

[0746] The expression of pulmonary fibrosis markers ACTA2, COL1A2, and TNF-α was confirmed by quantitative PCR analysis, and it was confirmed that expression was significantly increased in TGF-b-treated organoids (*p<0.05 and **p<0.01 versus NT) (Figure 105(A)).

[0747] Three days after treatment with TGF-b, the expression of pulmonary fibrosis markers smooth muscle actin (SMA), collagen type 1 (COL1), and vimentin (VIM) was confirmed by immunostaining. It was confirmed that the expression of all markers was increased in TGF-b-treated lung organoids (Figure 105(B)).

[0748] These experimental results confirmed the feasibility of creating a human pulmonary fibrosis model using human lung organoids cultured on a decellularized lung tissue-derived extracellular matrix (LuEM) scaffold.

[0749] Experimental Example 12-3. Analysis of the functionality of a human pulmonary fibrosis model constructed from decellularized lung tissue-derived extracellular matrix (Lung Extracellular Matrix, LuEM)

[0750] Human lung organoids cultured from decellularized LuEM hydrogel scaffolds were treated with TGF-β to create a pulmonary fibrosis model, and functionality was confirmed by analyzing the degree of swelling induced by the drug forskolin.

[0751] As a result, as can be seen from Figure 106, normal organoids swell normally in response to drugs, but when pulmonary fibrosis is induced, the pulmonary organoids do not swell even when treated with drugs.

[0752] The results of this experiment confirmed a decrease in the functionality of a human pulmonary fibrosis model cultured on a decellularized lung tissue-derived extracellular matrix (LuEM) scaffold.

[0753] Experimental Example 12-4. Creation of a fine dust disease model using mouse lung organoids cultured from a support of extracellular matrix (Lung Extracellular Matrix, LuEM) derived from decellularized lung tissue

[0754] The effect of fine dust on lung organoids cultured in decellularized LuEM supports was analyzed by treating them with fine dust.

[0755] Specifically, when preparing hydrogel using LuEM components derived from decellularized lung tissue, fine dust (Particulate Matter, PM10) with a size of approximately 10 μm was mixed in to ensure that the fine dust could effectively come into contact with the lung organoids cultured within the hydrogel. Gene expression analysis was carried out on the third day after culturing the organoids with the fine dust.

[0756] As a result, when observing the image of the lung organoids cultured for 3 days from the LuEM support treated with fine dust, it was confirmed that black substances (arrows) presumed to be fine dust were observed (Figure 107(A)).

[0757] In lung organoids treated with fine dust, it was confirmed that the expression of iNOS, a gene involved in inflammatory responses, increased when treated with fine dust at a concentration of 4 mg / ml (Figure 107(B)).

[0758] These experimental results confirmed the possibility of modeling lung diseases caused by fine dust by adding fine dust while culturing lung organoids on a decellularized LuEM hydrogel scaffold.

[0759] Experimental Example 12-5. Verification of the possibility of long-term storage of extracellular matrix support (Lung Extracellular Matrix, LuEM) derived from decellularized lung tissue

[0760] LuEM solution derived from decellularized lung tissue was refrigerated at 4°C for a long period of time and used to culture mouse lung organoids, confirming the feasibility and safety of long-term storage of LuEM hydrogel (Figure 108(A)).

[0761] It was confirmed that lung organoids were formed and cultured well from hydrogels made with LuEM that had been refrigerated for 7 and 31 days, as well as from LuEM hydrogels (Fresh LuEM) and Matrigel, which were freshly prepared just before culture (image of organoids on the 7th day of culture). In addition, when comparing the mRNA expression levels of various lung-specific markers through qPCR analysis on the 7th day of culture, it was confirmed that the expression was higher than that of organoids cultured from Matrigel (MAT group), and was expressed at a similar level to organoids cultured from freshly prepared LuEM hydrogel (Fresh group) (Figure 108 (B)).

[0762] Through this, it was confirmed that the extracellular matrix scaffold derived from decellularized lung tissue can be used for culturing lung organoids after refrigerated storage, maintaining its safety without denaturation for at least one month even when stored in a solution at 4℃. It was also verified that the long-term storage possible means it can be developed into a product with a long shelf life.

[0763] Experimental Example 12-6. Confirmation of the possibility of forming lung organoids under support-free culture conditions using components of extracellular matrix (Lung Extracellular Matrix, LuEM) derived from decellularized lung tissue

[0764] An experiment was conducted to confirm the possibility of forming lung organoids by adding a solution of extracellular matrix (LuEM) derived from decellularized lung tissue to the culture medium without a culture support.

[0765] Specifically, when culturing stem cells isolated from mouse lung tissue, LuEM solution was added to the culture medium at different concentrations and the culture was carried out.

[0766] When observed on the 7th day of culture, it was confirmed that the larger the organoids formed, the higher the LuEM concentration. However, in organoids cultured under high-concentration (0.1 mg / ml) LuEM conditions, a partially cross-linked LuEM hydrogel-like substance was observed around the organoids (Figure 109(A)).

[0767] When the number and size of lung organoids formed on the 7th day of culture were quantified, it was confirmed that the higher the concentration of LuEM, the greater the number of organoids formed, and that lung organoids cultured under intermediate concentration (0.01 mg / ml) LuEM treatment conditions had the largest average size (Figure 109 (B)).

[0768] These results confirmed that pulmonary organoids can be cultured simply by adding LuEM solution to the culture medium, without the need for Matrigel or hydrogel scaffolds, which are essential for the formation and maintenance of pulmonary organoids. This method allows for easier and more efficient culture of pulmonary organoids, and has the advantage of making it easier to handle various factors and handle organoids, thereby expanding the scope of applications of organoids.

[0769] Experimental Example 12-7. Analysis of gene and protein expression in mouse lung organoids formed without a culture support

[0770] We compared the expression of lung tissue-specific markers in lung organoids cultured for 7 days under various concentrations of LuEM treatment.

[0771] Specifically, the expression of four genes related to pulmonary bronchiolar differentiation and alveolar-related genes was compared between the groups. As a result, it was confirmed that the 0.1mg / ml suspension group showed similar expression to organoids cultured in gel. In contrast, the no treatment, 0.001, and 0.01 suspension groups showed a significant increase in the expression of ciliated cells (Foxj1) and club cells (Scgb1a1). The expression of goblet cells (Muc5ac) was similar to that in LuEM hydrogel conditions, and the expression of basal cells (Krt5) was significantly decreased. This confirmed that culture under suspension conditions further promoted differentiation of basal cells into various cells (*p<0.05 and **p<0.01 versus no treatment, #p<0.05 and ##p<0.01 versus LuEM 7mg / ml) (Figure 110(A)).

[0772] Through immunostaining, it was confirmed that various cells such as ciliated cells (acetylated α-tubulin) and basal cells (P63) were expressed, and it was confirmed that if cultured without a culture support, polarization occurred and the inside and outside were reversed. As a result, it was confirmed that the 0.1mg / ml hydrogel concentration was high, and the polarization phenomenon did not occur as in the gel condition, and a lumen was present inside the organoid, while the other groups had a pattern that recognized the outside as a lumen (Figure 110(B)).

[0773] Through these experimental results, we have established a technology to effectively produce lung organoids by only treating them with LuEM solution without the need for a culture scaffold.In addition, it was confirmed that the organoids produced in this way are different from organoids cultured within existing scaffolds in that the polarization phenomenon occurs in the same way as in vivo conditions, and the inside and outside remain reversed.

[0774] Experimental Example 12-8. Analysis of protein expression in human lung organoids formed without a culture support

[0775] The expression of lung tissue-specific markers was compared in human lung organoids cultured for 7 days under culture conditions (final concentration 0.01 mg / ml) in which a 10 mg / ml LuEM solution was added at a ratio of 1:1000 (vs. the bulk of the culture medium).As a control group, a group that was not treated with anything (No treatment) and a group that was treated with Matrigel at a ratio of 1:100 (vs. the bulk of the culture medium) (MAT) were compared.

[0776] As a result, as shown in Figure 111, by treating with LuEM at an appropriate concentration, it was confirmed that a large number of organoids were bound together to form larger organoids. In addition, the organoids formed had the inside and outside reversed, and it was confirmed that the expression of ciliated cells (acetylated α-tubulin), basal cells (P63), and goblet cells (MUC5AC) was well-occurring.

[0777] Through these experimental results, the researchers established a technology to produce larger tissue-level human lung organoids without a culture support, simply by treating them with LuEM solution.

[0778] Experimental Example 12-9. Analysis of protein expression in human lung organoids formed without a culture support

[0779] It was confirmed that when human lung organoids cultured on the LuEM hydrogel scaffold were cultured under conditions without a culture scaffold, the inside and outside were reversed. In addition, it was confirmed that angiotensin-converting enzyme 2 (ACE2) and transmembrane protease, serine 2 (TMPRSS2), which are known to be cell surface proteins important for intracellular infection of COVID-19, were mainly expressed on the outside of the organoids, and they were cultured in a form optimized for viral infection experiments (Figure 112).

[0780] These experimental results confirmed the feasibility of using pulmonary organoids cultured without a culture support for infection research.

[0781] Experimental Example 12-10. Experiment on the treatment of fine dust from mouse lung organoids formed without a culture support

[0782] Pulmonary organoids formed through treatment with LuEM solution alone, without a culture support, were treated with fine dust at four different concentrations to observe the effects. The experiment was carried out under the condition that the culture solution contained LuEM solution at a concentration of 0.01mg / ml, which showed the best effect in the previous experiment. In this experiment, when fine dust, an external substance, was treated in the culture solution, the organoids were able to react directly to the fine dust, allowing for a more accurate analysis of the effects of fine dust.

[0783] When particulate matter (PM10), which is about 10 μm in size, was added to the culture medium at various concentrations of 20, 100, and 500 μg / ml, the more the concentration of particulate matter increased, the more black particulate matter (arrows) was observed in the culture medium. It was also confirmed that the particulate matter was either directly attached to the organoids or was distributed in large quantities around the organoids (Figure 113(A)).

[0784] Through Live / Dead staining to analyze cell viability, it was confirmed that the higher the concentration of fine dust (arrow), the more dead cells there were. In particular, at high concentrations (500 μg / ml), the organoids were disassembled due to cell death, and no structure remained (Figure 113 (B)).

[0785] These experimental results confirmed that fine dust induces the death of pulmonary organoids, and that by using a method of treating fine dust with pulmonary organoids formed without a culture scaffold, it is possible to more easily model pulmonary diseases caused by fine dust and more accurately predict the effects of fine dust.

[0786] Experimental Example 12-11. Promotion of alveolar differentiation of mouse lung organoids using extracellular matrix support (Lung Extracellular Matrix, LuEM) derived from decellularized lung tissue

[0787] In order to create organoids that specifically differentiate into alveolar cells, one of the most important functional structures in the lung, we tried to create organoids by regulating Notch signaling. To induce differentiation, we added DAPT reagent, which inhibits Notch signaling, to the existing culture medium and cultured them, and created hydrogels with different concentrations of LuEM to try to culture and induce differentiation of lung organoids. Matrigel was used as a control.

[0788] When observing the lung organoids cultured under each LuEM hydrogel concentration condition on the 14th day of culture, it was confirmed that organoids with similar patterns were well formed in all groups (Figure 114(A)).

[0789] When comparing the expression of the alveolar-specific markers Sftpc and Hopx on the 14th day of culture, it was confirmed that the expression level of Sftpc, an alveolar type 2 cell marker, increased with increasing LuEM concentration, while the expression level of Hopx, an alveolar type 1 cell marker, increased with decreasing LuEM concentration (**p<0.01 versus MAT) (Figure 114(B)).

[0790] On the 14th day of culture, we checked the expression of SPC, an alveolar type 2 cell marker, and Hopx, an alveolar type 1 cell marker. Although there were some differences, it was confirmed that each marker was well expressed in all groups. In addition, it was confirmed that the expression of CC10 and P63, which are bronchiolar markers, was hardly observed (Figure 114(C)).

[0791] These experimental results confirmed that it is possible to produce organoids in which differentiation is induced in alveolar cells using a decellularized lung tissue-derived extracellular matrix (LuEM) scaffold, and that the cellular composition of the organoids can be adjusted by adjusting the LuEM concentration.

[0792] Experimental Example 12-12. Formation and analysis of monolayers of human lung organoids cultured from decellularized lung tissue-derived extracellular matrix (LuEM)

[0793] Experiments were conducted to create monolayers from human lung organoids so that they can be easily used for various purposes such as infection experiments and drug screening.

[0794] Specifically, the lung organoids cultured from the LuEM hydrogel were disassembled into single cells through enzymatic treatment, and then cultured in a well plate to induce the formation of a single cell layer.

[0795] Immunostaining confirmed that the ciliated cells (acetylated α-tubulin), goblet cells (MUC5AC), and basal cells (P63) were well maintained in the prepared monolayer (Figure 115(A)). It was also confirmed that the cells constituting the monolayer well expressed cell surface proteins important for COVID-19 infection, such as ACE2 and TMPRSS2 (Figure 115(B)).

[0796] Through this experiment, we established a method to form single cell layers suitable for a variety of experimental applications from lung organoids cultured on a decellularized lung tissue-derived extracel...

Claims

1. A support composition comprising an extracellular matrix derived from decellularized organ tissue, The support composition has a hydrogel form, The organ tissue is an intestine or a liver; When the organ tissue is an intestine, the concentration of the extracellular matrix is ​​1 to 5 mg / mL; When the organ tissue is liver, the concentration of the extracellular matrix is ​​2 to 7 mg / mL. Support composition.

2. The support composition of claim 1, wherein the concentration of the extracellular matrix is ​​4 to 6 mg / mL when the organ tissue is liver.

3. (a) decellularizing the isolated organ tissue to produce a decellularized organ tissue; (b) drying the decellularized organ tissue to produce an extracellular matrix derived from the decellularized organ tissue; (c) gelling the dried decellularized organ tissue-derived extracellular matrix, The organ tissue is an intestine or a liver; A method for producing a support composition in the form of a hydrogel, wherein in step (c), when the organ tissue is intestine, the concentration of the extracellular matrix is ​​adjusted to 1 to 5 mg / mL, and when the organ tissue is liver, the concentration of the extracellular matrix is ​​adjusted to 2 to 7 mg / mL.

4. The method for producing a support composition in the form of a hydrogel according to claim 3, wherein in step (c), when the organ tissue is liver, the concentration of the extracellular matrix is ​​adjusted to 4 to 6 mg / mL.

5. The method for producing a support composition in the form of a hydrogel according to claim 3, wherein in step (a), the organ tissue is agitated with a decellularization solution.

6. The method for producing a support composition in the form of a hydrogel according to claim 5, wherein the decellularization solution contains 0.1 to 5% Triton X-100 and 0.01 to 0.5% ammonium hydroxide.

7. The method for producing a support composition in the form of a hydrogel according to claim 3, wherein the decellularization in step (a) removes 95 to 99.9% of organ tissue cells.

8. A method for culturing organoids on the support composition described in claim 1 or the support composition produced by the production method described in claim 3.

9. The method for culturing organoids described in claim 8, wherein the organoids are intestinal organoids or hepatic organoids.

Images