KR102708789B1 - Liver extracellular matrix-derived scaffold for culture and transplantation of liver organoid and preparing method thereof - Google Patents

Abstract

The present invention relates to a support for liver organoid culture and transplantation using decellularized liver tissue (Liver Extracellular Matrix; LEM).

Description

{LIVER EXTRACELLULAR MATRIX-DERIVED SCAFFOLD FOR CULTURE AND TRANSPLANTATION OF LIVER ORGANOID AND PREPARING METHOD THEREOF}

The present invention relates to a decellularized liver tissue-derived scaffold for liver organoid culture and transplantation and a method for producing the same.

Organoids, which have recently been in the spotlight, are tissue analogs that can be used in various clinical applications such as new drug screening, drug toxicity assessment, disease modeling, cell therapy, and tissue engineering, and are a rapidly growing technology worldwide. Organoids are not only composed of various cells that constitute specific organs and tissues of the human body within a three-dimensional structure, but can also implement interactions between them, so they can be applied as a much more accurate in vitro model platform than existing drug evaluation models such as cell line models or animal models.

There are various types of organoids depending on the organ, and numerous research teams around the world studying them have been using Matrigel as a common culture support to cultivate organoids. However, since Matrigel is a component extracted from mouse sarcoma tissue, it is difficult to maintain consistent product quality, is expensive, and has safety issues such as animal infectious bacteria and viral transfer, so Matrigel has many problems to solve as an organoid culture system. In particular, as a material derived from cancer tissue, it does not provide the optimal tissue-specific microenvironment required for specific tissue organoid culture. Some research has been conducted on the development of polymer-based hydrogels to replace Matrigel, but no material has been reported yet that can replace Matrigel.

Liver organoids are produced by extracting adult stem cells from liver tissue and culturing them, or by differentiating pluripotent stem cells such as human induced pluripotent stem cells or embryonic stem cells into hepatocytes and then culturing them. Since Matrigel cannot reproduce the complex liver tissue-specific microenvironment in vivo, improvements are needed in the differentiation efficiency and function of liver organoids. Therefore, the development of a culture system to produce more mature and functional liver organoids is urgently required.

In addition, intractable liver diseases such as liver fibrosis, hepatitis, cirrhosis, and liver cancer that cause massive cell loss and decreased liver function are difficult to treat with drugs, so treatment technologies that can essentially regenerate liver tissue are needed. Since liver organoids contain various liver tissue-forming cells, including stem cells that exist in actual liver tissue, organoid-based liver tissue engineering and cell therapy technologies are receiving a lot of attention. However, when transplanting large-sized tissue analogues such as organoids, the development of a support that can help with the efficient transplantation and engraftment of organoids in the diseased area is also required.

To solve the technical problems in the current liver organoid culture and transplantation, the present invention proposes a new platform for producing a liver tissue-derived decellularized scaffold through a decellularization process from liver tissue and utilizing the same for liver organoid culture. The developed decellularized liver tissue-derived hydrogel scaffold is rich in various liver tissue-specific extracellular matrices and growth factors, and enhances the differentiation, maturity, and functionality of liver organoids compared to when Matrigel was used. Furthermore, the decellularized liver tissue-derived hydrogel shows potential as a transplantation scaffold that enables efficient transplantation of liver organoids into the body.

Republic of Korea Patent Publication No. 10-2017-0143465

The present invention is to produce decellularized liver tissue by decellularizing liver tissue to remove all cellular components and preserve liver-specific extracellular matrix components, and to apply a three-dimensional hydrogel based on the decellularized liver tissue to liver organoid culture.

However, the technical problems to be solved by the present invention are not limited to the problems mentioned above, and other problems not mentioned will be clearly understood by those skilled in the art from the description below.

Hereinafter, the present invention will be described with reference to the attached drawings. However, the present invention can be implemented in various different forms, and therefore is not limited to the embodiments described herein. When a part is said to "include" a certain component, this does not mean that other components are excluded, but that other components can be additionally provided, unless specifically stated otherwise.

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

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.

Various scientific dictionaries containing terms included in the present disclosure are well known and available in the art. Any methods and materials similar or equivalent to those described herein are found to be useful in the practice or testing of the present invention, but some methods and materials are described. The invention is not limited to specific methodologies, protocols, and reagents, as these can be used in a variety of ways depending on the context in which they are used by those skilled in the art. The invention is described in more detail below.

One aspect of the present invention provides a support composition comprising a decellularized liver tissue-derived extracellular matrix (LEM).

The above “extracellular matrix” refers to a natural support for cell growth prepared by decellularization of tissues found in mammals and multicellular organisms. The extracellular matrix may be further processed by dialysis or crosslinking.

The extracellular matrix may 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.

The above extracellular matrix can contain about 90% collagen in various forms in mammals. The extracellular matrix derived from various biological tissues can have different overall structures and compositions because of the unique roles required for each tissue.

The above “derived” and “derived” mean a component obtained from the mentioned source by a useful method.

In addition, in one specific embodiment of the present invention, the extracellular matrix derived from the decellularized liver tissue may be contained in an amount 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 contained in a concentration outside the above range, the intended effect of the present invention may not be obtained, or the composition may be unsuitable for manufacture or use.

In one specific example of the present invention, the composition may have an elastic modulus of 20 to 100 Pa at 0.1 to 10 Hz, and the composition may form a stable polymer network by having an elastic modulus within the above range.

The above support composition comprises a three-dimensional hydrogel manufactured based on a liver tissue matrix composition obtained by decellularization, and can be effectively utilized for liver organoid culture.

The above decellularized liver tissue contains actual tissue-specific extracellular matrix components, so it can provide physical, mechanical, and biochemical environments for the tissue, and is very efficient in promoting differentiation into liver tissue cells and tissue-specific functionality.

The above “organoid” refers to an ultra-small living organ produced in the form of an artificial organ by culturing cells derived from tissues or pluripotent stem cells in a 3D form.

The above organoids are three-dimensional tissue analogues containing organ-specific cells that originate from stem cells and self-organize (or self-pattern) in a manner similar to the in vivo state, and can develop into specific tissues by patterning with limited factors (e.g., growth factors).

The organoids can have an anatomical structure that mimics the original state of the cell mixture (including not only limited cell types but also residual stem cells and a close physiological niche) while retaining the original physiological characteristics of the cells. The organoids can have a functional organ-like morphology and tissue-specific functions with better arrangement of cells and cell functions through three-dimensional culture methods.

Another aspect of the present invention provides a method for producing a support composition, comprising: (a) a step of decellularizing isolated liver tissue to produce decellularized liver tissue; (b) a step of drying the decellularized liver tissue to produce decellularized liver tissue-derived extracellular matrix (LEM); and (c) a step of gelling the dried decellularized liver tissue-derived extracellular matrix.

The above step (a) is a step of producing decellularized liver tissue by decellularizing the separated liver tissue.

In one specific embodiment of the present invention, the liver tissue separated in step (a) may further include a step of dicing before decellularization. The present invention includes a step of dicing the liver tissue before decellularization, so that the decellularization process is more efficient and complete cell removal is possible. The method of dicing the separated liver tissue can be performed using a known method (apparatus) and size.

In one specific embodiment of the present invention, in step (a), the liver tissue may be stirred in a decellularization solution.

The above decellularization solution may contain various components for removing cells from liver tissue, and for example, may contain hypertonic saline, peracetic acid, Triton-X, SDS or other detergent components, but in one 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, more specifically, 1% Triton X-100 and 0.1% ammonium hydroxide. By using the above decellularization solution, decellularization is performed under relaxed conditions compared to the existing process, thereby effectively removing DNA in the manufactured support, and at the same time, various proteins in the liver tissue can be preserved in greater amounts.

The above stirring may be performed for 24 to 72 hours, more specifically 36 to 60 hours, most specifically 40 to 56 hours, or as an example 48 hours, and through this stirring (decellularization) process, 95 to 99.9%, more specifically 96 to 98%, and most specifically 97.18% of liver tissue cells may be removed. If decellularization is performed for a time period outside the above range or at a level of liver tissue cell removal, there may be a problem that the quality of the manufactured support composition deteriorates or the process economy is reduced.

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

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

In one specific embodiment of the present invention, after step (b), the extracellular matrix derived from the decellularized liver tissue may be contained in an amount of 0.01 to 10 mg/mL, specifically 0.5 to 9 mg/mL, more specifically 1 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 contained in a concentration outside the above range, the intended effect of the present invention may not be obtained, or the composition may be unsuitable for manufacture or use.

The above dried extracellular matrix can be comminuted by a method including tearing, milling, cutting, crushing and shearing steps. The above comminuted extracellular matrix can be processed into a powder form by a method such as crushing or milling in a frozen or freeze-dried state.

The above step (c) is a step of gelling the extracellular matrix derived from the dried decellularized interstitial tissue.

Through the above gelation, a three-dimensional hydrogel-type scaffold can be produced by cross-linking the extracellular matrix derived from decellularized interstitial tissue, and the gelled scaffold can be utilized in various fields related to organoid culture as well as experiments and screening.

The above “hydrogel” is a material in which a liquid using water as a dispersion medium solidifies through a sol-gel phase transition, loses fluidity, and forms a porous structure. It can be formed by a hydrophilic polymer having a three-dimensional network structure and a microcrystalline structure swelling upon containing water.

The above gelation may be accomplished by dissolving the extracellular matrix derived from decellularized interstitial tissue in an acidic solution with a protein-decomposing enzyme such as pepsin or trypsin, adjusting the pH to neutral and the electrolyte state of 1X PBS buffer using 10X PBS and 1 M NaOH, and incubating at a temperature of 37°C for 30 minutes.

Another aspect of the present invention provides a method for culturing liver organoids in the support composition or the support composition manufactured by the manufacturing method.

The existing Matrigel-based culture system has a large difference between batches as it is an extract derived from animal cancer tissue, fails to simulate the actual liver environment, and has insufficient efficiency in differentiation and development into liver organoids. On the other hand, the support composition can create an environment similar to liver tissue, and is therefore suitable for liver organoid culture.

The above culturing refers to the process of maintaining and growing cells under suitable conditions, and suitable conditions may refer to, for example, the temperature at which cells are maintained, nutrient availability, atmospheric CO2 content, and cell density.

Suitable culture conditions for maintaining, propagating, expanding and differentiating different types of cells are known and documented in the art. Conditions suitable for organoid formation may be those that facilitate or permit cell differentiation and the formation of multicellular structures.

The decellularized liver tissue-derived artificial scaffold developed in the present invention is a novel liver organoid culture scaffold that overcomes the limitations of Matrigel, a representative existing organoid culture scaffold. It is expected that it will be utilized as an element technology for various preclinical and clinical studies, such as a large-scale new drug screening platform based on liver organoids or cell therapy for tissue regeneration, and that it will create high added value in industrial and economic aspects and promote the development of new medical industries.

The decellularized liver tissue-derived scaffold developed in the present invention can be used to produce advanced liver organoids compared to existing culture methods, and thus can be utilized as an economical and more accurate platform that surpasses existing in vitro models for drug testing. Since liver toxicity is an essential indicator that must be analyzed in new drug development and drug evaluation, it is expected that the advanced liver organoid-based in vitro model platform will greatly increase the success rate of new drug development and greatly reduce the cost and time required, thereby greatly contributing to the development of the medical industry.

The artificial matrix scaffold derived from decellularized liver tissue is expected to be widely used in various fields such as disease modeling research to implement various intractable liver diseases (cirrhosis, liver fibrosis, non-alcoholic hepatitis, etc.) in vitro and to elucidate their mechanisms, and construction of transplantation treatment platforms. Since the prevalence of these intractable liver diseases has recently increased significantly and requires much research, it is also possible to generate revenue as a research reagent.

The artificial support developed in the present invention can be applied not only to the culture of tissue stem cell-derived liver organoids but also to the culture of liver cancer organoids, and thus can be utilized as a precision medicine platform technology by contributing to the establishment of disease models tailored to patients with intractable diseases and cancer. Considering the size of the precision medicine market, which has been rapidly growing recently, it is expected that enormous added value can be created.

As described above, for the application of liver organoids, a culture support called Matrigel is basically essential. Compared to Matrigel, the artificial support developed in the present invention shows functionality superior to Matrigel as a culture system and is verified to have a very advantageous advantage in terms of safety and cost. Therefore, it is predicted that this Matrigel replacement effect alone will generate enormous economic profits.

Figure 1 illustrates the production of a decellularized liver tissue-derived scaffold (Liver Extracellular Matrix; LEM) for liver organoid culture.
Figure 2 shows the analysis of decellularized liver tissue-derived scaffolds (LEMs) for liver organoid culture.
Figure 3 shows the analysis of the physical properties of the decellularized interstitial tissue-derived scaffold (LEM) according to its concentration.
Figures 4 and 5 show proteomic analyses of decellularized liver tissue-derived scaffolds (LEM).
Figure 6 shows the culture and concentration selection of liver organoids (Cholangiocyte-derived liver organoids; CLO) according to the concentration of the hydrogel scaffold derived from decellularized liver tissue.
Figure 7 shows an analysis of the difference in differentiation potential of liver organoids (biliary duct cell-derived liver organoids, CLO) according to the LEM concentration of the decellularized liver tissue-derived hydrogel scaffold.
Figure 8 shows the analysis of liver organoids (cholangiocyte-derived liver organoids, CLO) cultured on a hydrogel scaffold derived from decellularized liver tissue.
Figure 9 shows an analysis of the functionality of liver organoids (cholangiocyte-derived liver organoids, CLO) cultured on a hydrogel scaffold derived from decellularized liver tissue.
Figure 10 shows long-term culture of liver organoids (biliary duct-derived liver organoids, CLO) using a hydrogel support derived from decellularized liver tissue.
Figures 11 to 13 show the culture and analysis of human liver organoids (human cholangiocyte-derived liver organoids - hCLO) using a hydrogel support derived from decellularized liver tissue.
Figure 14 shows the culture of liver organoids (hepatocyte-derived liver organoids; HLO) at different concentrations of decellularized liver tissue-derived hydrogel scaffolds and selection of the optimal concentration.
Figure 15 compares the growth of liver organoids (HLO) formed on a decellularized liver tissue-derived hydrogel scaffold (LEM) and a conventional culture scaffold (MAT).
Figure 16 shows the analysis of hepatocyte-derived liver organoids (HLOs) cultured in a decellularized liver tissue-derived hydrogel scaffold (LEM).
Figure 17 shows the culture of human-induced pluripotent stem cells (hiPSC)-derived liver organoids using a hydrogel scaffold derived from decellularized liver tissue.
Figure 18 shows the analysis of human induced pluripotent stem cell-derived liver organoids (hiPSC-LO) cultured on a decellularized liver tissue-derived hydrogel scaffold.
Figure 19 shows a liver fibrosis model created using liver organoids (CLO) cultured on a decellularized liver tissue-derived hydrogel scaffold (LEM).
Figure 20 shows a liver fibrosis model created using liver organoids (hCLO) cultured on a hydrogel scaffold derived from decellularized liver tissue.
Figure 21 shows the in vivo transplantation of liver organoids using a hydrogel scaffold derived from decellularized liver tissue.
Figures 22 and 23 verify the long-term storage potential of decellularized liver tissue-derived hydrogels.
Figure 24 shows the results of culturing liver cancer organoids using a hydrogel support derived from decellularized liver tissue.
Figures 25 and 26 show the results of verifying the superiority of the decellularized liver tissue-derived scaffold of the present invention through comparison of liver tissue decellularization protocols.
Figure 27 shows the results of verifying the superiority of the decellularized liver tissue-derived scaffold of the present invention through comparison of liver tissue decellularization protocols (organoid culture experiment).
Figure 28 shows the results of an interspecies comparative analysis of decellularized interstitial tissue-derived hydrogel scaffolds (pig vs. human).
Figure 29 shows the comparative results of human liver organoids (human cholangiocyte-derived liver organoids - hCLO) cultured on hydrogel supports derived from human and porcine decellularized liver tissue.

In the present invention, liver tissue was decellularized to remove all cellular components and decellularized liver tissue with liver-specific extracellular matrix components preserved was produced, and a three-dimensional hydrogel based on this was applied to liver organoid culture.

The decellularized interstitial tissue-derived scaffold developed in the present invention is composed only of pure extracellular matrix components from which cell antigens have been removed, so it does not cause inflammatory responses or immune rejection responses in the tissue upon transplantation and has excellent biocompatibility. It is easy to manufacture and has a low manufacturing cost, so it is economical compared to Matrigel and can be applied as a safe culture and transplant material.

In fact, proteomic analysis confirmed that the decellularized liver tissue-derived scaffold contained various extracellular matrices and factors specific to liver tissue. It was confirmed that stem cell-derived liver organoids could be generated and grown within the fabricated decellularized liver tissue-derived hydrogel scaffold, and various concentrations of the decellularized liver tissue scaffold were tested to select the hydrogel concentration conditions optimized for liver organoid culture.

When liver organoids cultured on the developed decellularized liver tissue-derived scaffold were compared with organoids cultured on the control Matrigel, it was confirmed that differentiation ability and liver functionality (albumin secretion, cytochrome activity, urea synthesis, etc.) were similarly maintained or improved. This verified that the decellularized liver tissue-derived scaffold can replace the existing Matrigel for liver organoid culture.

Through these series of results, it was confirmed that liver organoids cultured on the support developed in the present invention can better embody the structure and function of liver tissue similar to reality than organoids cultured on a conventional matrix (Matrigel).

In the present invention, when liver damage 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 able to engraft in the damaged area, enabling efficient transplantation. In addition, the possibility of utilizing liver organoids as a transplant material was confirmed.

Hereinafter, preferred examples are presented to help understand the present invention. However, the following examples are provided only to help understand the present invention more easily, and the content of the present invention is not limited by the following examples.

Example 1: Preparation of a support composition comprising extracellular matrix derived from decellularized liver tissue

A support composition including extracellular matrix derived from decellularized interstitial tissue was prepared as follows (see Fig. 1 (A)).

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

Porcine liver tissue was isolated and prepared by dicing, and the liver tissue was stirred with 1% Triton X-100 and 0.1% ammonium hydroxide to remove only the cells of the liver tissue, thereby preparing decellularized liver tissue.

Afterwards, the decellularized liver tissue was freeze-dried and pulverized to produce decellularized liver tissue-derived extracellular matrix (LEM).

Example 1-2. Preparation of support composition

10 mg of the above decellularized liver tissue-derived extracellular matrix was dissolved in a 4 mg/ml pepsin solution (a solution in which 4 mg of pepsin powder was dissolved in 1 ml of 0.02 M HCl) for 48 hours. After uniformly mixing with 10X PBS and 1 M NaOH to a neutral pH and electrolyte state of 1X PBS buffer, the mixture was gelled at 37°C for 30 minutes to produce a hydrogel-type support composition (Fig. 1 (B)).

Experimental Example 1: Analysis of extracellular matrix (LEM) derived from decellularized liver tissue

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

Experimental Example 1-1. Characteristics of extracellular matrix derived from decellularized liver tissue

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

Specifically, DNA was extracted from decellularized liver tissue scaffolds and non-decellularized liver tissues, and quantitative comparisons were performed, and the GAG content in the scaffolds was measured using a dye-binding analysis method of chondroitin sulfate and dimethylmethylene blue (Fig. 2 (A)).

For H&E staining, the decellularized liver tissue scaffold and the tissue that had not undergone the decellularization process were thinly sectioned, and the cell nuclei were stained with hematoxylin and the cytoplasm was stained with eosin to analyze the degree of cell nuclei removal in the decellularized liver tissue scaffold (Fig. 2 (B)).

The decellularized interstitial tissue scaffolds were fabricated at various concentrations, and the internal structure of the hydrogels was analyzed using a scanning electron microscope that forms an image by scanning an electron beam (Fig. 2(C)).

Through quantitative comparison of DNA before and after the decellularization process of the decellularized interstitial tissue scaffold, it was confirmed that most cellular components were removed by the decellularization process. Through quantitative analysis of Glycosaminoglycan (GAG), one of the representative extracellular matrix components, it was confirmed that GAG was well preserved and remained in the decellularized interstitial tissue (Fig. 2 (A)).

The structure of the decellularized interstitial tissue matrix produced by H&E staining was confirmed to be well maintained and all cellular components were removed (Fig. 2 (B)).

Analysis using scanning electron microscopy (SEM) confirmed that the decellularized liver tissue-derived hydrogel scaffolds produced at various concentrations had an internal structure in the form of nanofiber bundles (Fig. 2 (C)), and thus could provide a three-dimensional microenvironment suitable for the culture of liver organoids.

Experimental Example 1-2. Analysis of physical properties of extracellular matrix of decellularized liver tissue by concentration

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

As a result, as confirmed in Fig. 3, the storage modulus (G') value was consistently higher than the loss modulus (G'') value under all concentration conditions of the LEM support, confirming that a stable polymer network was formed through cross-linking within the hydrogel. It was confirmed that the physical properties also increased as the LEM concentration increased, and the physical properties were confirmed to be lower overall compared to the control Matrigel (MAT) group. High-concentration (8 mg/ml) LEM hydrogel had similar physical property values to Matrigel.

Experimental Example 1-3. Proteomic Analysis of Extracellular Matrix Derived from Decellularized Liver Tissue

Proteomics using mass spectrometry was performed to identify the components of the scaffold derived from decellularized liver tissue, and it was confirmed that various liver tissue-specific extracellular matrix (collagens, ECM glycoproteins, proteoglycans) and growth factor proteins are contained in LEM.

As a result, as confirmed in Fig. 4, the existing commercialized support, Matrigel, is mostly composed of ECM Glycoproteins, whereas the decellularized liver tissue matrix (LEM) is composed of various components, with Collagens and ECM Glycoproteins being the most abundant, followed by Proteoglycans and ECM regulators. Among the top 10 extracellular matrix (ECM) proteins, Biglycan (BGN), Lumican (LUM), and Asporin (ASPN), which exist specifically 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 hepatocyte structure. Through this, it was confirmed that the fabricated decellularized liver tissue-derived extracellular matrix (LEM) support contains various extracellular matrix proteins present in actual liver tissue that play an important role in liver structure, development, and function, compared to Matrigel.

In addition, as confirmed in Fig. 5, when proteins known to be highly expressed in liver tissue were detected and compared in MAT and LEM, matrisome and non-matrisome proteins related to liver tissue were detected in much greater amounts in LEM than in MAT (Fig. 5 (a)).

When Gene Ontology analysis was performed to determine the functions of protein components excluding the extracellular matrix (ECM) included in each matrix, it was confirmed that the non-matrisome components included in the Matrigel were mainly responsible for functions related to the biosynthesis and metabolism of peptides or amides, whereas the non-matrisome components included in the LEM support produced in the present invention were mainly involved in roles related to small molecules, various organic acids, and cell metabolism (Fig. 5 (b)).

That is, it is thought that non-matrisome protein components other than ECM contained in the LEM scaffold will play an important role in the formation of highly functional liver organoids with enhanced organic acid metabolism and various cell metabolic capabilities, which are important functions of hepatocytes.

Experimental Example 2: Analysis of a Support Composition Containing Extracellular Matrix Derived from Decellularized Liver Tissue

Experimental Example 2-1. Culturing and concentration selection of liver organoids (Cholangiocyte-derived liver organoids; CLO) according to the concentration of hydrogel scaffold derived from decellularized liver tissue

Hydrogels were prepared by different concentrations of LEM derived from decellularized liver tissue, and liver organoids (CLO) were cultured and the formation efficiency was compared to select the appropriate concentration conditions for organoid culture. Matrigel, a commercialized culture support, was used as a control. Liver organoids were prepared using cholangiocytes extracted from rat liver tissue.

As shown in Fig. 6, when the decellularized liver tissue-derived LEM hydrogel was applied under various LEM concentration conditions for liver organoid culture, it was confirmed that liver organoids with a morphology similar to that of the control group, MATRIGEL, were well formed in all concentration conditions (2, 4, 6, and 8 mg/ml) (Fig. 6 (A)). When the liver organoid formation efficiency according to the concentration of the decellularized liver tissue-derived LEM hydrogel scaffold on the 7th day of culture was compared, it was confirmed that the formation efficiency was the highest in the 4 mg/ml and 6 mg/ml conditions (Fig. 6 (B)).

Experimental Example 2-2. Analysis of the difference in differentiation potential of liver organoids (biliary tract-derived liver organoids, CLO) according to LEM concentration of hydrogel scaffolds derived from decellularized liver tissue

Decellularized liver tissue-derived LEM hydrogel supports were prepared at various concentrations and applied to mouse liver organoid culture. The differentiation-related gene expression of organoids cultured for 7 days under each concentration condition was compared and analyzed using quantitative PCR (qPCR) method. A commercialized culture support, MATRIGEL, was used as a control.

As shown in Fig. 7, when gene expression was compared by LEM concentration condition, the stemness-related gene (LGR5) slightly decreased in liver organoids cultured in LEM hydrogel, but liver differentiation-related markers (Krt19, Krt18, Hnf4a, Hnf1b, Foxa3) showed a tendency to increase in expression. Considering that the liver organoid formation efficiency of 6 mg/ml LEM hydrogel among the LEM concentration conditions was not significantly different from that of Matrigel and the expression of liver differentiation markers increased further, the LEM hydrogel concentration was determined as 6 mg/ml LEM and applied to the subsequent liver organoid experiment.

Experimental Example 2-3. Analysis of liver organoids (cholangiocyte-derived liver organoids, CLO) cultured on a hydrogel support derived from decellularized liver tissue

Immunostaining of liver organoids cultured on Matrigel, the most widely used organoid culture, and LEM hydrogel (6 mg/ml) derived from decellularized liver tissue were performed. Liver organoids were produced using cholangiocytes extracted from rat liver tissue, and compared through immunostaining on day 7 of culture.

As shown in Fig. 8, when compared with liver organoids cultured in control Matrigel through immunostaining for liver-specific markers, liver tissue-specific markers were well expressed in CLO type liver organoids cultured in LEM hydrogel scaffolds derived from decellularized liver tissue at a level similar to that in the Matrigel group. * KRT19 (cholangiocyte marker), ECAD (cell-cell junction marker), SOX9 (cholangiocyte progenitor marker), Ki67 (proliferative cell marker), ALB (mature hepatocyte marker)

Experimental Example 2-4. Functional Analysis of Liver Organoids (Choledocholesterol-Derived Liver Organoids, CLO) Cultured on Hydrogel Supports Decellularized Liver Tissue

Liver organoids derived from cholangiocytes were cultured, and liver functionality was evaluated on day 7. Matrigel, a commercialized culture support, was applied as a control group, and LEM hydrogel was applied at a concentration of 6 mg/ml.

As shown in Fig. 9, PAS staining, which analyzes the storage capacity of polysaccharides such as glycogen, showed that liver organoids cultured in LEM hydrogels had a polysaccharide storage capacity similar to that of the control (MAT) group (Fig. 9 (A)). When various liver function analyses (albumin secretion, urea synthesis ability, and cytochrome activity) were performed, it was confirmed that CLO type liver organoids cultured in LEM hydrogels showed similar functionality to liver organoids cultured in MATRIGEL (Fig. 9 (B)).

Experimental Example 2-5. Long-term culture of liver organoids (biliary duct cell-derived liver organoids, CLO) using a hydrogel support derived from decellularized liver tissue

We attempted to culture mouse cholangiocyte-derived liver organoids long-term within previously established decellularized liver tissue-derived LEM hydrogel scaffolds and analyzed liver-specific differentiation markers. The Matrigel group was used as a control group.

As shown in Fig. 10, when examining the morphological image of liver organoids cultured for 1 month while continuing the subculture in LEM hydrogel, it was confirmed that they were cultured at a similar level in shape and size to liver organoids cultured in Matrigel (Fig. 10(A)). Even when liver organoids were cultured long-term for more than 1 month, it was confirmed that the expression of liver differentiation markers increased in liver organoids cultured in LEM hydrogel supports derived from decellularized liver tissue than in Matrigel-based organoids, and it was found that organoid differentiation was further enhanced in LEM hydrogel with longer culture periods (Fig. 10(B)).

Experimental Example 2-6. Culturing and Analysis of Human Liver Organoids (Human Biliary Tissue-Derived Liver Organoids - hCLO) Using Decellularized Liver Tissue-Derived Hydrogel Supports

We investigated whether human liver tissue-derived liver organoids could be cultured using a LEM hydrogel scaffold (6 mg/ml) derived from decellularized liver tissue. Liver cholangiocytes were isolated and human cholangiocyte-derived liver organoids were cultured, and Matrigel was used as a control.

As shown in Fig. 11, similar to Matrigel (MAT), human-derived liver organoids were well formed on the decellularized liver tissue-derived LEM hydrogel support and long-term subculture for more than 50 days was confirmed (Fig. 11(A)). When the expression of liver tissue markers was analyzed using quantitative PCR analysis on the 7th day of culture, the expression of LGR5 gene related to stemness and liver differentiation-related marker genes (HNF4A, SOX9, FOXA2) in liver organoids cultured on LEM hydrogel showed a similar or slightly increased tendency compared to organoids cultured on Matrigel (Fig. 11(B)).

In addition, LEM hydrogel supports derived from decellularized liver tissue were produced at various concentrations and applied to human liver organoid culture, and the differentiation-related gene expression of organoids cultured for 14 days under each concentration condition was compared and analyzed using quantitative PCR (qPCR) method. MAT, a commercialized culture support, was used as a control.

As a result, as confirmed in Fig. 12, when gene expression was compared by LEM concentration condition, both stemness-related genes (LGR5) and liver differentiation-related markers (SOX9, HNF4A, KRT19, FOXA2) showed similar expression levels without significant difference in the LEM hydrogel and MAT groups. Considering that the 6 mg/ml hydrogel among the LEM concentration conditions showed good liver organoid formation efficiency and slightly increased the expression of KRT19 and HNF4A differentiation markers, the LEM hydrogel concentration was determined as 6 mg/ml LEM and applied to the subsequent liver organoid experiment.

Then, immunostaining was performed on liver organoids cultured on MAT and decellularized liver tissue-derived LEM hydrogel (6 mg/ml) supports. Liver organoids were produced using cholangiocytes extracted from human liver tissue, and compared through immunostaining on the 14th day of culture.

Immunostaining for liver-specific markers revealed that liver organoids cultured on decellularized liver tissue-derived LEM hydrogel supports expressed liver tissue-specific markers at levels similar to those in the MAT group, compared to liver organoids cultured on the control MAT (Fig. 13 (A)).

When liver function analysis (urea synthesis ability, albumin secretion) was performed, it was confirmed that human liver organoids cultured in LEM hydrogel showed similar or slightly higher functionality than liver organoids cultured in Matrigel (Figure 13 (B)).

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

Experimental Example 2-7. Culturing of liver organoids (hepatocyte-derived liver organoids; HLO) at different concentrations of decellularized liver tissue-derived hydrogel scaffolds and selection of optimal concentration

Hydrogels were prepared by culturing liver organoids at different concentrations of decellularized liver support, and the formation efficiency and gene expression were compared and analyzed. Matrigel, a commercialized culture support, was used as a control. Liver organoids were prepared using pure hepatocytes extracted by perfusion of mouse liver tissue.

As a result, as shown in Fig. 14, when hepatocyte-derived liver organoids were cultured for 14 days in the decellularized LEM hydrogel scaffold, liver organoids with a shape similar to that of the control group (MAT) were formed (Fig. 14 (A)). The liver organoid formation efficiency according to the decellularized scaffold concentration was compared with the control group (MAT) on the 7th day of culture (Fig. 14 (B)). When the expression of liver tissue markers in organoids cultured in LEM hydrogels manufactured at different concentrations was analyzed using quantitative PCR analysis, it was confirmed that the liver differentiation markers (Krt18, Hnf4a, Cdh1) tended to increase more in the decellularized LEM scaffold group (Fig. 14 (C)). Since the formation efficiency was stable and the expression level of the liver differentiation marker increased at a concentration of 4 mg/ml, it was determined to be an appropriate condition and a concentration of 4 mg/ml was applied for subsequent HLO culture.

Experimental Example 2-8. Comparison of growth of liver organoids (HLO) formed on a decellularized liver tissue-derived hydrogel support (LEM) and a conventional culture support (MAT)

Liver organoids were produced using pure hepatocytes extracted by perfusion of mouse liver tissue, and the growth rates of the organoids were compared by culturing them on decellularized LEM supports (4 mg/ml) and Matrigel, a commercial culture support.

As a result, as shown in Fig. 15, when comparing mouse hepatocyte-derived liver organoids grown on Matrigel, which is the most widely used for organoid culture, and decellularized liver tissue-derived LEM hydrogel (4 mg/ml) supports, it was confirmed that liver organoids formed on each support continued to grow in size and were well tolerated until passage culture.

Experimental Example 2-9. Analysis of hepatocyte-derived liver organoids (HLOs) cultured in a decellularized liver tissue-derived hydrogel scaffold (LEM)

Liver organoids were cultured for 20 days using a decellularized hepatocyte support hydrogel (4 mg/ml), and immunohistochemical staining analysis and liver functional comparative analysis were performed. Matrigel, a commercial culture support, was used as a control. Liver organoids were produced using pure hepatocytes extracted by perfusion of rat liver tissue.

As shown in Figure 16, when comparing the expression of various markers through immunostaining with organoids cultured on the control Matrigel (MAT), it was confirmed that liver tissue-specific markers were well expressed in the decellularized LEM support group as well (Figure 16(A)).

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

Both groups were confirmed to have glycogen storage capacity through PAS (Periodic Acid Schiff) staining, which checks the storage capacity of polysaccharides such as glycogen (Fig. 16 (B)). When the urea synthesis capacity and albumin secretion amount were compared, it was confirmed that the functionality increased in the decellularized LEM support group compared to the control group (MAT) (Fig. 16 (C)).

Experimental Example 3: Confirmation of the usability of a hydrogel scaffold derived from decellularized liver tissue

Experimental Example 3-1. Culturing of Human-induced Pluripotent Stem Cell (hiPSC)-derived Liver Organoids Using Decellularized Liver Tissue-derived Hydrogel Supports

To control the hydrogel properties suitable for liver organoid formation from human-induced pluripotent stem cells, LEM: culture medium = 1:1 (v/v) was mixed to make a hydrogel layer, and cells necessary for liver organoid formation (human-induced pluripotent stem cell-derived hepatic endoderm cells, vascular endothelial cells, and mesenchymal stem cells) were applied on top. Within 72 hours, the cells self-organized to form liver organoids. Protein expression was comparatively analyzed through immunostaining of liver organoids cultured on the decellularized liver support hydrogel (LEM). A commercialized culture support, MATRIGEL (MAT), was used as a control.

As shown in Fig. 17, when iPS cell-derived hepatic endoderm cells + endothelial cells + mesenchymal stem cells were cultured on MAT and decellularized liver tissue-derived hydrogel (LEM) layers, it was confirmed that the cells clumped together over time and both groups formed three-dimensional organoids within 72 hours (Fig. 17 (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 (Fig. 17 (B)).

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

Experimental Example 3-2. Analysis of human induced pluripotent stem cell-derived liver organoids (hiPSC-LO) cultured on a hydrogel scaffold derived from decellularized liver tissue

Human-induced pluripotent stem cell-derived liver organoids were cultured for 7 days using a decellularized intercellular support hydrogel, and their functionality and gene expression were compared and analyzed. The commercialized culture support, Matrigel (MAT), was used as a control. Liver organoids were produced through self-organization of human-induced pluripotent stem cell-derived hepatic endoderm cells, vascular endothelial cells, and mesenchymal stem cells.

As shown in Fig. 18, when PAS staining related to liver functionality was performed, it was confirmed that glycogen storage was performed well in both the Matrigel group and the LEM hydrogel group. In the case of urea synthesis ability, it was confirmed that it was higher in the organoids cultured in the decellularized matrix (Fig. 18(A)). When the expression of liver tissue markers was analyzed using quantitative PCR analysis, the expression of the OCT4 gene, a pluripotency-related marker in liver organoids cultured in LEM hydrogel, slightly decreased, but the expression of liver differentiation-related marker genes (SOX17, HNF4A) and blood vessel-related marker genes (PECAM1, CD34) tended to increase compared to the organoids cultured in Matrigel (Fig. 18(B)).

Experimental Example 3-3. Production of a liver fibrosis model using liver organoids (CLO) cultured on a hydrogel scaffold derived from decellularized liver tissue (LEM)

For disease modeling using liver organoids based on a scaffold derived from decellularized liver tissue, TGF-beta was screened by concentration and its potential to induce liver fibrosis was confirmed. The model was induced using mouse cholangiocyte-derived liver organoids (CLO) cultured for 7 days. To create a liver fibrosis model, TGF-beta was treated by concentration for 48 hours and the organoids were observed.

As shown in Fig. 19, unlike the liver organoids that were not treated with TGF-β, all of the treated liver organoids showed morphological changes and poor growth (Scale bars = 100 μm) (Fig. 19 (A)). To confirm liver fibrosis, SMA (Smooth Muscle Actin) and COL1A1 were stained to confirm the accumulated extracellular matrix (ECM) around them (Scale bars = 100 μm) (Fig. 19 (B)). Quantitative PCR analysis was performed to confirm that the expression of liver fibrosis markers was the highest in the group treated with 4 ng/ml TGF-β (Fig. 19 (C)). Under normal conditions without TGF-β treatment, the expression of the liver fibrosis marker (Col1a1) decreased and the expression of the cholangiocyte marker (Krt19) increased in the decellularized liver tissue-derived scaffold group compared to the control Matrigel group. However, when fibrosis was induced in liver organoids cultured on decellularized liver tissue-derived scaffolds by TGF-β treatment, the expression of fibrosis markers significantly increased, while the expression of cholangiocyte markers was significantly decreased (Fig. 19 (D)). Through this experiment, we confirmed the possibility of creating a fibrosis model using liver organoids cultured on decellularized liver tissue-derived extracellular matrix scaffolds.

Meanwhile, for disease modeling using human cholangiocyte-derived liver organoids (hCLO) cultured on a decellularized liver tissue-derived scaffold, TGF-β was tested at various concentrations, and the most appropriate concentration (80 ng/ml) was used to confirm the possibility of inducing liver fibrosis.

As shown in Fig. 20, to create a liver fibrosis model, TGF-β was treated at various concentrations, and the same organoids were observed after 3 days. It was confirmed that the non-treated liver organoids grew for 3 days, but the treated liver organoids all had changed morphology and did not grow well (Fig. 20(A)). To verify the liver fibrosis model, SMA (Smooth Muscle Actin) was stained to confirm the accumulated extracellular matrix (ECM) around them. Through this experiment, the possibility of creating a human liver fibrosis model using liver organoids cultured on a decellularized liver support was confirmed (Fig. 20(B)).

Experimental Example 3-4. In vivo transplantation of liver organoids using hydrogel scaffolds derived from decellularized liver tissue

Animal experiments and histological analyses were performed to evaluate the use of decellularized liver tissue-derived LEM hydrogel scaffolds as a material for liver organoid transplantation. Liver organoids were transplanted using LEM hydrogel to efficiently deliver and engraft the liver organoids into the liver tissue of a mouse liver toxicity injury model. To adjust the viscosity of LEM hydrogel for easy injection during transplantation, LEM hydrogel was mixed and used in a ratio of 1:10 (v/v) = LEM: culture medium.

As shown in Fig. 21, a liver injury model was induced by intraperitoneal injection of acetaminophen (24 hours), and the injury model was verified through H&E histological analysis (Fig. 21(A)). Liver organoids derived from cholangiocytes (CLO) labeled with DiI fluorescent reagent were transplanted into the mouse liver injury model. Liver organoids were transplanted using Matrigel and decellularized LEM hydrogel as transplantation supports, and engraftment was observed through fluorescence expression one day after transplantation. It was confirmed that the liver organoids transplanted to the injury site survived well and existed in the liver tissue, confirming the potential of LEM hydrogel as a biomaterial for organoid transplantation (Fig. 21(B)).

Experimental Example 3-5. Verification of the Long-Term Storage Possibility of Decellularized Liver Tissue-Derived Hydrogels

Decellularized liver tissue-derived LEM hydrogels were stored for long periods under -80°C freezing conditions and used to culture mouse cholangiocyte-derived liver organoids (CLOs). Matrigel was used as a control.

As a result, as confirmed in Fig. 22, when liver organoids were cultured using the control Matrigel, LEM hydrogel freshly prepared just before organoid culture, and LEM hydrogel stored in a -80 ℃ freezer for a long period of time, it was confirmed that organoids were well formed in all groups (Fig. 22 (A)).

Compared to Matrigel and LEM hydrogels prepared immediately before liver organoid culture, liver organoids were well cultured in hydrogels prepared with LEM solution that had been frozen for 1 or 2 months, and all liver differentiation markers were increased compared to the Matrigel group and showed similar expression levels without significant differences among decellularized LEM hydrogels (Fig. 22(B)).

In addition, the decellularized liver tissue-derived LEM hydrogel was stored for a long period of time under refrigerated conditions at 4°C and then used to culture human cholangiocyte-derived liver organoids (hCLO). Matrigel was used as a control.

As a result, as confirmed in Fig. 23, human liver organoids were cultured well in hydrogels made with LEM solution that had been refrigerated for 2 months compared to Matrigel and LEM hydrogels made immediately before culture, and it was confirmed that both stem cell potential markers and liver differentiation markers showed similar or higher expression levels without significant differences (Fig. 23 (A)).

When liver function analysis (urea synthesis ability, albumin secretion) was performed, it was confirmed that human liver organoids maintained liver functionality well even in long-term stored LEM hydrogels and showed functionality similar to or slightly higher than that of liver organoids cultured in the Matrigel group (Figure 23 (B)).

Through this, it was confirmed that the decellularized liver tissue-derived LEM hydrogel scaffold maintained stability without denaturation even when stored in a solution state at 4°C for more than 2 months, and could be used for liver organoid culture.

Experimental Example 3-6. Culturing of liver cancer organoids using hydrogel scaffolds derived from decellularized liver tissue

The feasibility of culturing human liver cancer organoids using decellularized liver tissue-derived LEM hydrogel scaffolds was investigated. Human liver organoids derived from normal liver tissue and liver organoids derived from hepatocellular carcinoma (HCC) patient tissues were cultured in Matrigel and decellularized liver tissue-derived LEM hydrogel for 20 days, and then analyzed.

As a result, as confirmed in Fig. 24, both normal liver organoids and liver cancer organoids were similarly formed in the Matrigel group and the decellularized liver tissue-derived LEM hydrogel support (Fig. 24 (A)). Immunostaining of liver cancer organoids cultured in Matrigel and liver cancer organoids cultured in LEM hydrogel was performed, and it was confirmed that liver cancer-related marker (SMA, AFP) proteins were stained in both groups (Fig. 24 (B)). When the gene expression of normal liver organoids and liver cancer organoids cultured in decellularized liver tissue-derived LEM hydrogel was compared through qPCR analysis, it was confirmed that the expression of LGR5 related to stemness decreased, while the expression of TNFa and HES1 related to inflammation response and the expression of AFP and PLIN2 markers related to liver cancer increased in liver cancer organoids (Fig. 24 (C)).

Through this, we confirmed that the decellularized liver tissue-derived LEM hydrogel scaffold can be applied to the culture of not only normal liver organoids but also liver cancer organoids, thereby confirming the possibility that it can be utilized as a culture platform for building an in vitro liver cancer model.

Experimental Example 3-7. Verification of the superiority of the decellularized liver tissue-derived scaffold constructed in the present invention through comparison of liver tissue decellularization protocols

The decellularization method developed in this study (Protocol 1) was compared with the decellularization method reported in the literature (Protocol 2). The Protocol 2 method removed DNA using DNase I (3 hours) after treatment with 4% sodium deoxycholate for 4 hours, and the decellularization method developed in this study induced decellularization using only a solution mixed with 1% Triton X-100 and 0.1% ammonium hydroxide, which is a more relaxed condition to reduce protein damage in the tissue.

As a result, as confirmed in Fig. 25, the H&E histological analysis results confirmed that both methods removed cells well, but ECM components were preserved more in the tissue treated with Protocol 1 (Fig. 25 (A)).

After the decellularization process, DNA was significantly reduced in both tissues treated with the two protocols, but GAG quantitative analysis confirmed that the GAG component remaining in the tissue treated with Protocol 2 was significantly less than in the tissue treated with Protocol 1. When the mechanical properties (elastic modulus) of the decellularized liver tissue-derived hydrogels produced with each protocol were measured, it was confirmed that the properties of the hydrogel derived from the decellularized liver tissue through Protocol 2 were lower (Fig. 25 (B)).

These results suggest that Protocol 1 is a more efficient method that can not only induce efficient decellularization but also reduce extracellular matrix (ECM) damage.

In addition, the decellularization method (Protocol 1) constructed in the present invention was compared with a decellularization method (Protocol 3) that is widely used in the past. In Protocol 3, after applying the decellularization method constructed in the present invention, 3% sodium dodecyl sulfate reagent was additionally treated for 24 hours.

As a result, as confirmed in Fig. 26, when H&E histological analysis was performed, it was confirmed that both methods removed cells well, but ECM components were preserved more in the tissue treated with Protocol 1 (Fig. 26 (A)).

After the decellularization process, DNA was significantly reduced in both tissues treated with the two protocols, but GAG quantitative analysis confirmed that the GAG component remaining in the tissue treated with Protocol 3 was significantly less than in the tissue treated with Protocol 1. When the mechanical properties (elastic modulus) of the decellularized liver tissue-derived hydrogels produced with each protocol were measured, it was confirmed that there was no significant difference in the properties of the hydrogels produced with the two protocols (Fig. 26 (B)).

When proteomics analysis of liver tissue-derived LEM scaffolds produced by each decellularization protocol was performed, it was confirmed that the LEM scaffold produced by Protocol 1 contained liver tissue-specific Collagen α1 (XVIII) and Kininogen 1 proteins, and a greater number of extracellular matrix proteins were detected (Fig. 26 (C)).

Therefore, we confirmed that Protocol 1 could induce a more efficient decellularization process that could reduce damage to liver tissue-specific extracellular matrix components compared to Protocol 3.

Experimental Example 3-8. Verification of the superiority of the decellularized liver tissue-derived scaffold constructed in the present invention through comparison of liver tissue decellularization protocols (organoid culture experiment)

Mouse cholangiocyte-derived liver organoids (CLOs) were cultured and compared using hydrogels derived from decellularized liver tissue prepared by the decellularization method constructed in the present invention (Protocol 1), a decellularization method reported in the literature (Protocol 2), and a decellularization method widely used in the past (Protocol 3).

It was confirmed that liver organoids were well formed in all of the decellularized liver tissue-derived LEM hydrogels produced by Protocol 1, 2, and 3 (Day 7) (Fig. 27 (A)). When the gene expression of liver organoids cultured in LEM hydrogels produced by each protocol was compared using the quantitative PCR (qPCR) method on day 7, it was confirmed that the expression of the stem cell potential (Lgr5) marker and the expression of the liver differentiation markers (Afp, Foxa3) were significantly decreased in the liver organoids cultured in the hydrogels produced by Protocol 2 and 3 compared to Protocol 1 (Fig. 27 (B)). When the formation efficiency of liver organoids was compared in the existing culture support, Matrigel (MAT), and the LEM hydrogels produced by each decellularized protocol, it was confirmed that the liver organoid formation efficiency was slightly decreased in the Protocol 1 group, but significantly decreased in the hydrogels produced by Protocols 2 and 3 (Fig. 27 (C)).

These results show that the LEM hydrogel produced by Protocol 1 is more efficient in the formation of liver organoids and can provide a more favorable environment for the growth and differentiation of liver organoids. In other words, it can be seen that the decellularized liver tissue-derived scaffold produced by Protocol 1 constructed in the present invention is a much superior scaffold for liver organoid culture.

Experimental Example 3-9. Cross-species Comparative Analysis of Decellularized Liver Tissue-Derived Hydrogel Scaffolds (Pig vs. Human)

We performed a proteomic analysis of human decellularized liver tissue-derived scaffolds (Human LEM) produced by decellularizing human liver tissue together with Matrigel and porcine LEM. Through a comparison of proteomic analyses of three types of hydrogels, we verified the suitability of the porcine decellularized liver tissue-derived hydrogel developed in the present invention for human liver organoid culture.

The extracellular matrix components of the control Matrigel were mostly composed of ECM glycoproteins, and the rest were ECM regulators and proteoglycans in that order, whereas the porcine and human decellularized liver tissue-derived LEM scaffolds contained the most proteoglycans and had similar levels of collagens and ECM glycoproteins. In addition, when the top 10 ECM proteins were confirmed, most similar components were detected in Porcine LEM and Human LEM, and they were confirmed to be very different from the control Matrigel. Therefore, it can be seen that the porcine decellularized liver tissue-derived hydrogel scaffold can provide a microenvironment similar to human liver tissue, and thus is more suitable for human liver organoid culture than Matrigel. When the extracellular matrix protein components specifically present in each culture matrix were analyzed, the most ECM proteins were detected in the human liver tissue-derived LEM scaffold, and more ECM protein components were detected in the porcine liver tissue-derived LEM scaffold than in the control Matrigel (Fig. 28 (A)).

When Gene Ontology analysis was performed to determine the functions of non-matrisome protein components excluding the extracellular matrix (ECM) included in each scaffold, the non-matrisome components of Matrigel were 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 were mainly involved in roles related to small molecules, various organic acids, and cell metabolism (Fig. 28 (B)). In other words, the non-matrisome protein components excluding the ECM included in the human and porcine LEM scaffolds are expected to play an important role in the formation of functionally mature liver organoids with enhanced organic acid metabolism and various cell metabolism capabilities, which are important functions of hepatocytes.

Experimental Example 3-10. Comparison of human liver organoids (human cholangiocyte-derived liver organoids - hCLO) cultured on hydrogel scaffolds derived from human and porcine decellularized liver tissues

Human liver organoids were cultured on MAT and previously produced human and porcine decellularized liver tissue-derived LEM hydrogel supports. Liver organoids were produced using cholangiocytes extracted from human liver tissue, and gene expression and factor synthesis ability were compared using qPCR on day 14 of culture.

Compared with liver organoids cultured on control Matrigel (MAT), liver organoids cultured on LEM hydrogel supports derived from decellularized liver tissues from two different species were confirmed to form organoids at a level similar to that of the Matrigel group (Fig. 29 (A)).

When the gene expression of liver organoids cultured in each matrix was compared through qPCR analysis, the stemness-related marker (LGR5) was maintained at a similar level, and the expression of differentiation markers (KRT19, KRT7, SOX9) was confirmed to be increased in the decellularized liver tissue LEM hydrogel group. When liver functionality analysis (urea synthesis ability) was performed, it was confirmed that the liver organoids cultured in human and porcine liver tissue-derived LEM hydrogels had a similar or slightly higher level of functionality than the liver organoids cultured in Matrigel (Fig. 29 (B)).

This suggests that porcine tissue-derived LEM scaffolds have equivalent performance to human tissue-derived LEM scaffolds and thus can be applied to liver organoid culture as a replacement for Matrigel and human tissue-derived matrices.

The above description of the present invention is for illustrative purposes only, and those skilled in the art will understand that the present invention can be easily modified into other specific forms without changing the technical idea or essential characteristics of the present invention. Therefore, it should be understood that the embodiments described above are exemplary in all respects and not restrictive.

Claims (4)

(a) a step of producing decellularized liver tissue by chopping the separated liver tissue and stirring it with a decellularization solution;
(b) a step of drying the decellularized liver tissue to produce decellularized liver tissue-derived extracellular matrix (LEM); and
(c) a step of gelling the extracellular matrix derived from the dried decellularized liver tissue; including;
A method for producing a support composition for organoid culture,
A method for producing a support composition for organoid culture, wherein in step (c) above, the concentration of the extracellular matrix derived from decellularized liver tissue is adjusted to 4 to 6 mg/mL.
In the first paragraph,
A method for producing a support composition for organoid culture, wherein the decellularization solution contains 0.1 to 5% Triton X-100 and 0.01 to 0.5% ammonium hydroxide.
In the first paragraph,
A method for producing a support composition for organoid culture, wherein the decellularization in step (a) above removes 95 to 99.9% of liver tissue cells.
A method for culturing liver organoids in a support composition manufactured by the manufacturing method of claim 1.