Decellularized kidney tissue-derived scaffold for renal organoid culture and transplantation and method for producing the same

Abstract

The present invention relates to a support for renal organoid culture and transplantation using decellularized kidney tissue (Kidney Extracellular Matrix; KEM).

Description

[Technical Field]

[0001] The present invention relates to a decellularized kidney tissue-derived support for renal organoid culture and transplantation, and a method for producing the same. [Background technology]

[0002] Existing 2D cell culture methods have limitations in recreating the actual in vivo microenvironment, resulting in low culture efficiency. Therefore, 2D cell lines are limited as in vitro models. Recently, 3D organoid culture technology has attracted attention as a new approach to address these limitations. Organoids are tissue analogs with a variety of clinical applications, including new drug screening, drug toxicity assessment, disease modeling, cell therapy, and tissue engineering, and are a rapidly growing technology worldwide. Organoids not only comprise various cells that constitute specific organs and tissues in the human body within a 3D structure, but also embody the complex interactions between them. This makes them a far more accurate in vitro model platform than previously used drug evaluation models, such as simple 2D cell line models or animal models.

[0003] Various organ-derived organoid platforms have been established worldwide, and related research is currently underway. To date, Matrigel (Matrigel) has been the most commonly used culture substrate for organoid culture. However, since Matrigel is derived from mouse sarcoma cancer tissue, maintaining consistent product quality is challenging, it is expensive, and there are safety concerns, such as the risk of animal-derived infectious bacteria and viral transfer. Matrigel as an organoid culture system faces many challenges that must be overcome. In particular, as a cancer tissue-derived material, it does not provide the optimal tissue-specific microenvironment required for culturing specific tissue-specific organoids. While some research has been conducted on the development of polymer-based hydrogels to replace Matrigel, no material capable of replacing Matrigel has yet been reported.

[0004] Renal organoids can be produced by culturing adult stem cells extracted from kidney tissue or by culturing pluripotent stem cells, such as human induced pluripotent stem cells or embryonic stem cells. However, because Matrigel, which is used for renal organoid culture, cannot embody the complex kidney tissue-specific microenvironment in vivo, it is necessary to improve the differentiation efficiency and function of renal organoids. Therefore, there is a pressing need to develop a new culture system for producing more mature and functional renal organoids.

[0005] Intractable kidney diseases such as end-stage renal failure and chronic nephritis are serious diseases for which there are no suitable therapeutic agents, forcing patients to undergo hemodialysis for the rest of their lives or kidney transplants, which significantly reduces their quality of life and makes daily life difficult. Even after kidney transplants, patients suffer significant pain from the administration of immunosuppressants and their side effects. Therefore, the development of a system that can efficiently culture kidney organoids as a precise in vitro model for developing kidney disease therapeutics is a very important issue from the perspective of healthcare.

[0006] In the present invention, a hydrogel matrix composed of kidney tissue-specific extracellular matrix components was prepared through a kidney tissue decellularization process and applied to renal organoid culture. Compared to existing organ decellularization methods, the present invention not only greatly simplifies the matrix preparation process by simplifying the decellularization process, but also effectively preserves kidney tissue-specific extracellular matrix components and growth factors, demonstrating its potential as a culture matrix that can replace existing Matrigel. Summary of the Invention [Problem to be solved by the invention]

[0007] The present invention aims to obtain a large amount of decellularized tissue by chemically treating porcine kidney tissue, and then fabricate a hydrogel support from this tissue for use in renal organoid culture.

[0008] However, the technical problems that the present invention aims to solve are not limited to those described 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]

[0009] One aspect of the present invention provides a support for renal organoid culture and transplantation that utilizes kidney tissue-derived extracellular matrix (KEM).

[0010] In one embodiment of the present invention, the kidney tissue-derived extracellular matrix may be prepared using a mixed solution of Triton X-100 and ammonium hydroxide.

[0011] In one embodiment of the present invention, the concentration of the kidney tissue-derived extracellular matrix in the support may be 1 mg / ml to 10 mg / ml.

[0012] Another aspect of the present invention provides a method for producing a support for renal organoid culture and transplantation, comprising: 1) the steps of disrupting isolated kidney tissue; and 2) treating the disrupted kidney tissue with Triton X-100 and ammonium hydroxide to decellularize the tissue and produce a decellularized kidney tissue-derived extracellular matrix (KEM).

[0013] In one embodiment of the present invention, after step 2), the method may further comprise the step of 3) freeze-drying the decellularized kidney tissue-derived extracellular matrix (KEM) to produce a freeze-dried kidney tissue-derived extracellular matrix.

[0014] In one embodiment of the present invention, after step 3), the method may further comprise step 4) of forming the freeze-dried kidney tissue-derived extracellular matrix into a hydrogel-form support for renal organoid culture and transplantation.

[0015] In one specific example of the present invention, step 4) may involve dissolving the freeze-dried kidney tissue-derived extracellular matrix in a pepsin solution to form a solution, and then adjusting the pH to form a hydrogel.

[0016] Another aspect of the present invention provides a method for culturing kidney organoids on the support or a support produced by the production method. [Effects of the Invention]

[0017] The decellularized scaffold developed in this invention enables efficient cultivation of renal organoids, and is expected to replace existing Matrigel, which has various problems, and be widely used in the medical industry for new drug development, drug toxicity and efficacy evaluation, patient-customized drug selection, etc. This is expected to improve the quality of life of the public in health and society, as well as create significant added value in the economy and industry.

[0018] Decellularized kidney tissue-derived artificial matrix scaffolds are expected to be widely applicable in various fields, such as disease modeling research to clarify the mechanisms of various intractable kidney diseases (acute / chronic nephritis, renal failure, etc.) by ex vivo realization and the construction of transplant treatment platforms. As the prevalence of these intractable kidney diseases has recently increased significantly and significant research is required, it is possible to generate revenue by using them as research materials for related basic research.

[0019] Renal organoids have unlimited potential not only as disease models but also as cell therapy agents and tissue regeneration therapeutic agents for regenerative medicine purposes. Patients with end-stage renal failure are unable to lead a normal daily life due to hemodialysis. However, if the underlying kidney disease could be treated through kidney organoid transplantation therapy using the decellularized kidney-derived scaffold developed in the present invention, the quality of life of patients could be greatly improved and related costs could be significantly reduced.

[0020] The artificial scaffold developed in this invention can be applied not only to stem cell-derived kidney organoids but also to the culture of kidney cancer organoids, contributing to the construction of disease models customized for intractable diseases and cancer patients. It can also be used as a precision medicine platform technology, and considering the size of the precision medicine market, which has recently grown rapidly, it is expected to create enormous added value.

[0021] In summary, compared to Matrigel, which is essential for the culture and application of renal organoids, the artificial scaffold developed in the present invention has superior functionality as a culture system, is safer, and has significant cost advantages. Therefore, the replacement effect of Matrigel alone is expected to generate enormous economic benefits. [Brief explanation of the drawings]

[0022] [Figure 1] This shows the fabrication process of a decellularized kidney tissue-derived extracellular matrix (KEM) support for renal organoid culture. [Figure 2] This shows the results of analyzing decellularized kidney tissue-derived KEM scaffolds for renal organoid culture. [Figure 3] 1 shows the results of an analysis of the physical properties of a hydrogel scaffold derived from decellularized kidney tissue as a function of KEM concentration. [Figure 4] This shows the results of analyzing the protein content of decellularized kidney tissue-derived extracellular matrix (Kidney Extracellular Matrix, KEM) for renal organoid culture. [Figure 5] This shows the results of a comparative analysis of the protein bodies of decellularized kidney tissue-derived extracellular matrix (KEM) and Matrigel for renal organoid culture. [Figure 6] This shows the results of an analysis to select the optimal concentration of decellularized kidney tissue-derived KEM hydrogel scaffold for renal organoid culture. [Figure 7] This shows the results of proliferation and differentiation marker expression analysis (cell immunostaining analysis) of kidney organoids cultured on a decellularized kidney tissue-derived KEM hydrogel scaffold. [Figure 8] This shows the growth pattern of kidney organoids formed on a KEM hydrogel scaffold derived from decellularized kidney tissue. [Figure 9] This shows the results of analyzing kidney organoids cultured long-term on a decellularized kidney tissue-derived extracellular matrix support (Kidney Extracellular Matrix, KEM). [Figure 10] This shows the results of verifying the long-term storage potential of decellularized kidney tissue-derived extracellular matrix support (Kidney Extracellular Matrix, KEM). [Figure 11] This shows the results of verifying the long-term storage potential of decellularized kidney tissue-derived extracellular matrix support (Kidney Extracellular Matrix, KEM). [Figure 12] These results confirm the tissue-specific effects of decellularized kidney tissue-derived extracellular matrix support (Kidney Extracellular Matrix, KEM) for renal organoid culture. [Figure 13] This shows the results of an analysis of the functionality of kidney organoids cultured on a decellularized kidney tissue-derived extracellular matrix support (Kidney Extracellular Matrix, KEM). [Figure 14] This is the result of creating a renal fibrosis model using kidney organoids cultured on a decellularized kidney tissue-derived extracellular matrix (KEM). [Figure 15] These are the results of confirming the biocompatibility of the decellularized kidney tissue-derived extracellular matrix (Kidney Extracellular Matrix, KEM) support. [Figure 16]This shows the results of transplanting kidney organoids into the body using an extracellular matrix support (Kidney Extracellular Matrix, KEM) derived from decellularized kidney tissue. [Figure 17] This shows the results of transplanting kidney organoids into the body using an extracellular matrix support (Kidney Extracellular Matrix, KEM) derived from decellularized kidney tissue. BEST MODE FOR CARRYING OUT THE INVENTION

[0023] 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 "comprises" a certain element, this does not mean that it may exclude other elements, but may further include other elements, unless otherwise specified.

[0024] Unless otherwise defined, molecular biology, microbiology, protein purification, protein engineering 、D DNA sequence analysis and recombinant DNA techniques may be performed using conventional techniques well-known to those skilled in the art and described in numerous standard textbooks and references.

[0025] 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.

[0026] Various scientific dictionaries that include the terms contained herein are well known and available in the art. Any methods and materials similar or equivalent to those described herein can be used in the practice or testing of this application, and several methods and materials are described. The present invention is not limited to the specific methodology, protocols, and reagents, as these may vary depending on the context used by those skilled in the art. The present invention is described in more detail below.

[0027] One aspect of the present invention provides a support for renal organoid culture and transplantation that utilizes kidney tissue-derived extracellular matrix (Kidney Extracellular Matrix; KEM).

[0028] 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 may be further processed by dialysis or cross-linking.

[0029] 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.

[0030] The extracellular matrix may comprise approximately 90% collagen in various forms in mammals. Extracellular matrices derived from different biological tissues may differ in overall structure and composition due to the specific roles required by each tissue.

[0031] The terms "derive" and "derived" mean that the component is obtained from the stated source by any useful method.

[0032] In one embodiment of the present invention, the kidney tissue-derived extracellular matrix may be prepared using a mixed solution of Triton X-100 and ammonium hydroxide.

[0033] In one embodiment of the present invention, the concentration of the kidney tissue-derived extracellular matrix in the support may be 1 mg / ml to 10 mg / ml, specifically 1 mg / ml to 7 mg / ml. Examples of the kidney extracellular matrix concentration include 1 mg / ml to 7 mg / ml, 1 mg / ml to 5 mg / ml, 1 mg / ml to 3 mg / ml, 3 mg / ml to 7 mg / ml, 3 mg / ml to 5 mg / ml, or 5 mg / ml to 7 mg / ml, and in one embodiment, 1 mg / ml, 3 mg / ml, 5 mg / ml, or 7 mg / ml. If the kidney extracellular matrix is ​​present at a concentration outside the above range, the intended effect of the present invention will not be achieved.

[0034] The support contains a three-dimensional hydrogel produced from kidney tissue-derived extracellular matrix obtained by decellularization, and can be effectively used for culturing kidney organoids.

[0035] The decellularized kidney tissue contains actual tissue-specific extracellular matrix components, which can provide the physical, mechanical, and biochemical environment of the tissue, and is highly efficient in promoting differentiation into kidney tissue cells and tissue-specific functionality.

[0036] The term "organoid" refers to a micro-organ produced by culturing cells derived from tissues or pluripotent stem cells in a 3D form and fabricating them into an artificial organ-like form.

[0037] 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.

[0038] The organoids may have the innate 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 the adjacent physiological niche). The organoids may have a functional organ-like morphology and tissue-specific functions, with cells and cell functions better arranged through a 3D culture method.

[0039] Another aspect of the present invention provides a method for producing a support for renal organoid culture and transplantation, comprising: 1) the steps of disrupting isolated kidney tissue; and 2) treating the disrupted kidney tissue with Triton X-100 and ammonium hydroxide to decellularize the tissue and produce a decellularized kidney tissue-derived extracellular matrix (KEM).

[0040] Step 1) is a step of disrupting isolated kidney tissue. The kidney tissue may be isolated from a known animal, and specific examples of the animal include cows, pigs, monkeys, and humans. In addition, in the present invention, the isolated kidney tissue is disrupted before being subjected to a decellularization process, resulting in a high efficiency of cell removal. The method for disrupting isolated kidney tissue may be a known method. In the present invention, the kidney tissue is disrupted and subjected to a decellularization process, thereby enabling more efficient and higher level of cell removal.

[0041] Step 2) is a step of treating the disrupted kidney tissue with Triton X-100 and ammonium hydroxide to decellularize the tissue and produce decellularized kidney tissue-derived extracellular matrix (KEM). Unlike existing decellularization methods, the present invention treats only with Triton X-100 and ammonium hydroxide, minimizing tissue damage and thereby preserving a greater variety of proteins in the kidney tissue. As a specific example, the decellularization process may be performed while stirring the disrupted kidney tissue with Triton X-100 and ammonium hydroxide.

[0042] In one embodiment of the present invention, after step 2), the method may further comprise the step of 3) freeze-drying the decellularized kidney tissue-derived extracellular matrix (KEM) to produce a freeze-dried kidney tissue-derived extracellular matrix.

[0043] Step 3) is freeze-drying the decellularized kidney tissue-derived extracellular matrix (KEM) to produce a freeze-dried kidney tissue-derived extracellular matrix. After drying, the freeze-dried kidney tissue-derived extracellular matrix may be exposed to electron beam, gamma radiation, ethylene oxide gas, or supercritical carbon dioxide for sterilization.

[0044] In one embodiment of the present invention, after step 3), the method may further comprise step 4) of forming the freeze-dried kidney tissue-derived extracellular matrix into a hydrogel-form support for renal organoid culture and transplantation.

[0045] Step 4) is a step of forming the freeze-dried kidney tissue-derived extracellular matrix into a hydrogel-type scaffold for renal organoid culture and transplantation. This step may be performed through gelation, specifically, by dissolving the freeze-dried kidney tissue-derived extracellular matrix in a pepsin solution to form a solution, and then adjusting the pH to form a hydrogel. The decellularized kidney tissue-derived extracellular matrix may be crosslinked to form a scaffold in the form of a three-dimensional hydrogel, and the gelled scaffold may be used in various fields related to organoid culture, including experiments and screening.

[0046] The "hydrogel" is a material that forms a porous structure when a liquid containing water as a dispersion medium hardens and loses fluidity through a sol-gel phase transition, and may be formed when a hydrophilic polymer having a three-dimensional network structure and an amorphous structure absorbs water and expands.

[0047] The gelation may be carried out by dissolving the freeze-dried kidney tissue-derived extracellular matrix in an acidic solution with a proteolytic enzyme such as pepsin or trypsin, adjusting the pH, specifically, using 10x PBS and 1 M NaOH to a neutral pH and the electrolyte state of 1x PBS buffer, at 37°C for 30 minutes.

[0048] Another aspect of the present invention provides a method for culturing kidney organoids on the support or a support produced by the production method.

[0049] The existing Matrigel-based culture system is an extract derived from animal cancer tissue, and there is a large difference between batches. kidney In contrast to conventional methods, which are unable to mimic the environment of kidney tissue and therefore result in insufficient efficiency in differentiation and development into kidney organoids, the support can create an environment similar to kidney tissue and is therefore suitable for culturing kidney organoids.

[0050] The term "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, nutrient availability, atmospheric CO2 content, and cell density.

[0051] Suitable 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 organoids can be conditions that facilitate or allow cell differentiation and the formation of multicellular structures. DETAILED DESCRIPTION OF THE INVENTION

[0052] The decellularized kidney tissue-derived extracellular matrix scaffold developed in the present invention can be fabricated using a minimal chemical processing method, which results in less damage to tissue-specific components than scaffolds fabricated using existing decellularization methods, and is more efficient in terms of reducing fabrication time and costs, and is easier to mass-produce. Therefore, it is expected to be more advantageous in terms of commercialization than existing decellularized matrices.

[0053] The decellularized kidney tissue-derived scaffold developed in this invention has been confirmed to have all immunogenic cells removed, while the various extracellular matrix components and growth factors contained in actual kidney tissue are well preserved. Through protein analysis, the extracellular matrix components and related proteins important for kidney tissue were identified. Therefore, the decellularized kidney tissue-derived matrix provides a kidney tissue-specific microenvironment, enabling the efficient cultivation of kidney organoids.

[0054] In fact, it was confirmed that the formation and differentiation of renal organoids was successfully induced in the developed decellularized kidney tissue-derived hydrogel. Decellularized scaffolds with various extracellular matrix concentrations were prepared and applied to renal organoid culture, and the optimal concentration conditions for the most efficient renal organoid culture were selected.

[0055] A comparative analysis of renal organoids cultured on the developed decellularized scaffold with those cultured on a control Matrigel scaffold confirmed that differentiation of the renal organoids cultured on the decellularized scaffold was more enhanced. Based on these results, it was confirmed that renal organoids cultured on the decellularized scaffold can more accurately and precisely reproduce actual kidney tissue than renal organoids cultured using existing methods. As a result, it was confirmed that the decellularized kidney tissue-derived scaffold actually contributes to the efficient differentiation and development of renal organoids, demonstrating its potential as an alternative to Matrigel.

[0056] Preferred examples are presented below to aid in understanding the present invention. However, the following examples are provided only to facilitate understanding of the present invention, and the contents of the present invention are not limited to the following examples.

[0057] Fabrication of decellularized kidney tissue-derived extracellular matrix (KEM) scaffold for renal organoid culture (Figure 1). To prepare a kidney organoid culture scaffold, a kidney extracellular matrix (KEM) was prepared by decellularizing porcine kidney tissue. The decellularization process used in this invention differentiates it from existing decellularization methods by using only a solution containing 1% Triton X-100 and 0.1% ammonium hydroxide, which is less severe than existing methods. This minimizes tissue damage and preserves the various extracellular matrices and growth factor proteins present in the kidney tissue. Furthermore, because the kidney tissue was minced before decellularization, it was possible to more effectively and reliably remove cellular components (DNA) from the tissue.

[0058] (A) Pig kidney tissue was cut into small pieces and decellularized through a series of chemical treatments to remove most of the cells within the tissue, which was then freeze-dried to produce powdered decellularized kidney tissue matrix (Lyophilized KEM).

[0059] (B) 10 mg of powdered decellularized kidney tissue matrix (lyophilized KEM), containing the extracellular matrix of kidney tissue, was treated with a 4 mg / ml pepsin solution (4 mg of porcine gastric mucosa-derived pepsin powder dissolved in 1 ml of 0.02 M HCl) and then left to solubilize at room temperature for 48 hours. 10x PBS and 1 M NaOH were added to the solubilized KEM in appropriate proportions and mixed uniformly to achieve a neutral pH suitable for cell culture and the electrolyte status of 1x PBS buffer. Finally, gelation was induced for 30 minutes at 37°C in an incubator. The KEM hydrogel formed by gelation induction in a glass container did not flow even when the container was turned upside down, confirming the formation of a solidified hydrogel through a temperature-dependent crosslinking reaction.

[0060] Analysis of decellularized kidney tissue-derived KEM supports for kidney organoid culture (Figure 2) A decellularized kidney tissue-derived extracellular matrix (KEM) scaffold was fabricated from porcine kidney tissue and its properties were analyzed.

[0061] (A) H&E tissue staining analysis confirmed that after the decellularization process, most of the cell nuclei of the tissue were removed, but the overall structure was well maintained. Masson's Trichrome staining also confirmed that collagen was well preserved, and Alcian blue staining confirmed that GAG components were well preserved.

[0062] (B) Immunostaining was performed to confirm whether the ECM proteins of kidney tissue were well preserved after the decellularization process. It was confirmed that major ECM proteins, such as fibronectin and laminin, were well preserved after the decellularization process.

[0063] (C) Scanning electron microscopy (SEM) observation of the internal structure of the decellularized kidney tissue-derived KEM hydrogel confirmed that it was composed of a porous microstructure specific to collagen nanofibers. This suggests that KEM hydrogel can provide a structural microenvironment suitable for renal organoid culture.

[0064] (D) DNA and GAG (Glycosaminoglycans) quantitative analyses were performed on kidney tissue before decellularization (Native) and after decellularization (Decell). Comparing the results before and after decellularization, DNA was removed by over 96.8% after the decellularization process, confirming efficient cell removal. Furthermore, GAG was well preserved at a level similar to that of actual kidney tissue, and collagen was also well preserved and present.

[0065] Analysis of the physical properties of decellularized kidney tissue-derived hydrogel scaffolds as a function of KEM concentration (Figure 3) Hydrogel scaffolds were fabricated using decellularized kidney tissue-derived extracellular matrix (KEM) at four concentrations, and the changes in physical properties were measured according to the concentration. Gels at all concentrations were fabricated through a crosslinking reaction in an incubator at 37°C for 30 minutes. The storage modulus (G') was consistently higher than the loss modulus (G") at all concentrations, including the lowest concentration of 1 mg / ml. This confirmed that a stable polymer network was formed through crosslinking within the hydrogel, and that the mechanical properties (modulus) increased with increasing KEM concentration.

[0066] Analysis of protein content of decellularized kidney tissue-derived KEM supports for kidney organoid culture (Figure 4) To identify the extracellular matrix components contained in the decellularized kidney tissue-derived KEM scaffold, proteomics analysis was performed.

[0067] KEM Hydrogel, a decellularized kidney tissue-derived scaffold created through protein analysis, contains various types of extracellular matrix components such as collagens, glycoproteins, and proteoglycans, as well as various secreted factors such as growth factors, coagulation factors, and cytokines, which are secreted by cells within the tissue and exist mainly in a state bound to the extracellular matrix.

[0068] Collagen type VI (COL6), which accounts for a large proportion of the 10 main ECMs that make up KEM hydrogel, is a major component of the renal extracellular matrix and plays an important role in the formation of kidney tissue. Laminin (LAM) glycoprotein is a major component of the basement membrane and is involved in kidney cell and tissue differentiation, homeostasis, and survival. Biglycan (BGN) proteoglycan also plays an important role in regulating the kidney's immune response.

[0069] Furthermore, it was confirmed that the KEM scaffold contains proteins that are more than four times more expressed in kidney tissue than in other tissues (Top 10 kidney-elevated proteins).

[0070] Therefore, it is expected that various proteins present in actual kidney tissue observed within KEM hydrogel can induce the formation and development of kidney organoids.

[0071] Comparative analysis of protein structures between decellularized kidney tissue-derived KEM and Matrigel supports for kidney organoid culture (Figure 5) We compared the protein content of decellularized kidney tissue-derived KEM and Matrigel scaffolds for renal organoid culture. Specifically,

[0072] (A) Comparing the components of decellularized kidney tissue-derived KEM scaffold and Matrigel, it was confirmed that Matrigel is composed almost exclusively of glycoproteins, whereas KEM is composed of various collagens and proteoglycans, including glycoproteins.

[0073] (B) Gene Ontology analysis was used to compare the functions of the top 10 ECM components contained in the decellularized kidney tissue-derived KEM scaffold with the functions of the main components of the existing Matrigel scaffold. It was confirmed that the KEM scaffold contains more proteins involved in the metabolic process of nitrogen compounds in the kidney than Matrigel.

[0074] (C) Heatmap and volcano plot analysis to compare protein expression patterns between Matrigel and KEM confirmed that the protein compositions contained in the two scaffolds are very different. Furthermore, volcano plot analysis also confirmed that the expression trends were clearly different when comparing proteins that were significantly more highly expressed on each scaffold.

[0075] Selection of the optimal concentration of decellularized kidney tissue-derived KEM hydrogel scaffold for kidney organoid culture (Figure 6) Tubular fragments were extracted from mouse kidney tissue. To select the optimal concentration of KEM hydrogel for renal organoid culture, KEM hydrogels were first prepared at various concentrations. The extracted tubular fragments were uniformly mixed with various concentrations of decellularized kidney tissue-derived KEM hydrogel scaffolds, and then 3D culture was performed to induce the formation of renal organoids. Subculture was performed on day 7 of culture, followed by an additional 5 days of culture. On day 12 of total culture, the morphology, formation efficiency, and gene expression of renal organoids formed under each KEM concentration condition were confirmed. Matrigel was used as a control.

[0076] (A) Kidney organoids were formed in all KEM hydrogel concentrations except for the 1 mg / ml concentration condition. They were confirmed to have a spherical morphology similar to organoids cultured in Matrigel, which was used as a control. No organoids were formed in the 1 mg / ml concentration hydrogel.

[0077] (B) The number of organoids was measured on days 0 and 5 based on the time of subculture, and then expressed as a percentage to determine the formation efficiency. When comparing the formation efficiency of kidney organoids at each KEM concentration, the formation efficiency in KEM hydrogel was generally lower than that in Matrigel, but the highest formation efficiency was confirmed in KEM hydrogel at a concentration of 7 mg / ml.

[0078] (C) After culturing kidney organoids in KEM hydrogels prepared at different concentrations for 12 days, gene expression of differentiation markers was compared using quantitative qPCR analysis. Aqp1 (proximal tubule cell), a gene expressed in specific kidney cells, tended to decrease with increasing KEM concentration, but was significantly higher than the Matrigel group at all KEM concentrations. Scl12a1 (loop of Henle cell) showed expression levels similar to those of the Matrigel group, but significantly increased at 7 mg / ml KEM. Furthermore, Aqp2 (collecting duct cell) gene expression was most similar to that of the Matrigel group at 7 mg / ml KEM.

[0079] Through the above analysis, it was confirmed that organoids could be cultured at all concentrations except for the 1 mg / ml KEM hydrogel, but overall, the differentiation potential of kidney organoids was best at the 7 mg / ml KEM concentration condition.

[0080] Proliferation and differentiation marker expression analysis (cell immunostaining analysis) of kidney organoids cultured on decellularized kidney tissue-derived KEM hydrogel scaffolds (Figure 7). When various marker expression analyses were performed using immunostaining on the 12th day of culture, it was confirmed that both AQP3 (collecting duct cell) and CALB1 (distal tubule cell), which are differentiation markers specific to kidney organoids, were expressed at levels similar to those of the control Matrigel group.

[0081] In addition, ZO-1, a marker involved in intercellular tight junctions, was also confirmed to be well expressed in kidney organoids cultured in KEM hydrogel at all concentrations.

[0082] KI67, a marker associated with organoid proliferation, was also confirmed to be highly expressed in kidney organoids cultured in all concentrations of KEM hydrogel.

[0083] Growth pattern of kidney organoids formed on a decellularized kidney tissue-derived KEM hydrogel scaffold (Figure 8). After subculture on day 7 of culture, the growth patterns of kidney organoids cultured in Matrigel and decellularized kidney tissue-derived KEM hydrogel (5 mg / ml, 7 mg / ml) were compared and observed from day 1 to day 4.

[0084] It was confirmed that organoids grew in KEM hydrogel at each concentration, exhibiting a similar appearance to Matrigel, and could be cultured well for 4 days.

[0085] Analysis of mouse kidney organoids cultured long-term on decellularized kidney tissue-derived extracellular matrix (KEM) (Figure 9). (A) When kidney organoids were cultured for up to 52 days on decellularized KEM hydrogel and Matrigel, it was confirmed that the organoids maintained a similar spherical shape and size on both supports, even after nine subcultures.

[0086] (B) Comparison of the mRNA expression levels of Pax8 (renal progenitor cells) and Aqp1 (proximal tubule cells) in kidney organoids cultured 9 times for 52 days on each culture support revealed that Pax8 was expressed at similar levels in both culture supports, while Aqp1 expression was higher in the KEM hydrogel.

[0087] (C) Organoids cultured nine times on each support were immunostained using antibodies against PAX8 (renal progenitor cells), Villin (proximal tubule cells), AQP3 (collecting duct cells), and CALB1 (distal tubule cells). Immunostaining confirmed that the four cell types that make up the kidney tubules were well maintained in culture up to day 52, and that F-actin, a cytoskeleton marker involved in cell-cell interaction, was also well expressed.

[0088] This demonstrated that kidney organoids can be cultured for more than two months in KEM hydrogel, and confirmed that markers for various kidney cell types were expressed at similar or higher levels than in Matrigel, the commonly used organoid culture matrix.

[0089] Verification of the long-term storage potential of decellularized kidney tissue-derived extracellular matrix (Kidney Extracellular Matrix, KEM) (Figures 10 and 11) First, we verified the feasibility of long-term refrigerated storage of decellularized kidney tissue-derived extracellular matrix (Kidney Extracellular Matrix, KEM) (Figure 10).

[0090] (A) Decellularized kidney tissue-derived KEM solution (concentration 7 mg / ml) was refrigerated and stored at 4°C for up to one month, and this was used to culture kidney organoids to confirm the long-term storage potential and stability of the KEM hydrogel.

[0091] (B) It was confirmed that renal organoids were successfully formed and cultured at a similar level to Matrigel on hydrogel scaffolds made with freshly prepared KEM solution immediately before culture, and on hydrogel scaffolds made with KEM solution stored in a refrigerator for one week (Day 7), and one month (Day 31) (image of organoids on day 12 of culture, after one subculture). The number of organoids was measured immediately after subculture and after four additional days of culture, and the percentage was used to measure organoid formation efficiency.

[0092] (C) Quantitative qPCR analysis showed that the stemness-related gene Pax8 was expressed at a similar level in the KEM hydrogel group as in the Matrigel group. The kidney-specific marker Aqp1 (proximal tubule cell) showed generally higher mRNA expression levels in the KEM hydrogel group than in the Matrigel group (analysis was performed on organoids on day 12 of culture, after one subculture).

[0093] (D) Organoid immunostaining analysis confirmed that kidney-specific markers AQP3 (collecting duct cells), CALB1 (distal tubule cells), and PAX8 (renal progenitor cells) were highly expressed in KEM hydrogel at levels similar to those in Matrigel.

[0094] This experiment confirmed that the decellularized kidney tissue-derived KEM hydrogel solution can be stored in a refrigerated liquid state for at least one month without affecting its activity or function. This result proves the long-term storage potential of KEM hydrogel, which is important for product development.

[0095] We then examined the feasibility of long-term frozen storage of decellularized kidney tissue-derived extracellular matrix (Kidney Extracellular Matrix, KEM) (Figure 11).

[0096] (A) Decellularized kidney tissue-derived KEM solution (concentration 7 mg / ml) was frozen and stored at -80°C for up to 3 months, and then re-thawed and used for renal organoid culture to confirm the long-term storage potential and stability of KEM.

[0097] (B) It was confirmed that renal organoids were successfully formed and cultured at a level similar to Matrigel on hydrogel culture scaffolds made with KEM solution freshly prepared immediately before culture (Day 0), and on hydrogel culture scaffolds made with KEM solution stored frozen for one week (Day 7), and three months (Day 90) (image of organoids on Day 12 of culture, after one subculture).

[0098] (C) Quantitative qPCR analysis showed that the stemness-related gene Pax8 was expressed at a similar level in the KEM hydrogel group as in the Matrigel group. Furthermore, the kidney-specific marker Aqp1 (proximal tubule cell) showed higher mRNA expression levels in the KEM hydrogel group than in the Matrigel group (analysis was performed on organoids on day 12 of culture, which had undergone one subculture).

[0099] (D) Immunostaining analysis confirmed that AQP3 (collecting duct cell), a kidney-specific marker, was well expressed in KEM hydrogel at a level similar to that in Matrigel.

[0100] Through this experiment, it was confirmed that the decellularized kidney tissue-derived KEM hydrogel solution can be stored stably for a minimum of three months without affecting its activity or function, even when frozen at -80°C.

[0101] Confirmation of the tissue-specific effect of decellularized kidney tissue-derived extracellular matrix (KEM) for renal organoid culture (Figure 12). To verify the tissue-specific effects of the kidney-specific extracellular matrix components contained in the decellularized kidney tissue-derived KEM scaffold on the formation and development of kidney organoids, we also cultured kidney organoids on decellularized scaffolds derived from other organs and performed comparative analyses.

[0102] (A) Renal organoids were cultured on decellularized hydrogel scaffolds derived from kidney (KEM), intestine (IEM), muscle (MEM), skin (SkEM), and heart (HEM). On day 7, it was confirmed that renal organoids were not successfully formed on skin- and heart-derived scaffolds. Furthermore, after one subculture and an additional 4 days of culture (a total of 11 days of culture), small organoids were observed on all decellularized tissue scaffolds except for the decellularized kidney tissue scaffold. Similarly to day 7, no organoids were formed on the heart-derived scaffold.

[0103] (B) Quantitative analysis of kidney organoid formation efficiency confirmed that the kidney-derived decellularized KEM scaffold had the highest formation efficiency, while decellularized scaffolds derived from other organs had significantly lower formation efficiency. The number of organoids was measured immediately after subculture and after 4 days of additional culture, and then expressed as a percentage to determine formation efficiency.

[0104] (C) To compare the expression levels of marker proteins in kidney organoids cultured for 11 days on each organ-derived decellularized hydrogel scaffold, immunostaining was performed using the KI67 antibody, which is involved in cell proliferation, and the AQP3 antibody, which is associated with collecting duct cells. Kidney organoids cultured on the KEM hydrogel scaffold showed the most pronounced marker expression, while organoids cultured on the remaining tissue-derived scaffolds showed relatively faint expression or poorly formed structures.

[0105] Through this experiment, we can determine the tissue-specific effects of kidney tissue-derived KEM hydrogel on the formation and development of kidney organoids.

[0106] Analysis of the functionality of kidney organoids cultured on decellularized kidney tissue-derived extracellular matrix (KEM) (Figure 13). We conducted an analysis to determine whether renal organoids cultured in decellularized kidney tissue-derived KEM hydrogel can effectively embody the functionality of kidneys in vivo.To confirm the functionality of the P-glycoprotein (P-gp) xenobiotics efflux pump present in proximal tubule cells, one of the many cell types that make up renal organoids, we treated the cells with verapamil, a P-gp inhibitor, and then exposed them to calcein AM, and confirmed the degree of calcein AM accumulation within the cells using fluorescence.

[0107] (A) In the absence of verapamil treatment, P-gp excreted calcein outside the cells, and almost no fluorescent signal was observed within the organoids (No treatment group). As the concentration of verapamil increased, calcein accumulated inside the cells, resulting in relatively higher fluorescence within the organoids.

[0108] (B) The calcein fluorescence intensity in kidney organoids was quantified under conditions where the drug was not treated with verapamil and where it was treated with 100 μM verapamil. As a result, it was confirmed that the fluorescence intensity increased significantly under the condition where verapamil was treated.

[0109] This demonstrates that kidney organoids cultured in KEM hydrogel possess efflux pump functionality that expels external substances.

[0110] Creation of a renal fibrosis model using mouse kidney organoids cultured on decellularized kidney tissue-derived extracellular matrix (KEM) (Figure 14). A renal fibrosis disease model was created using renal organoids cultured in KEM hydrogel. To do this, mouse renal organoids were treated with TGF-β drugs to induce renal fibrosis. Renal organoids were treated for three days at various concentrations on day 9 of culture (subcultured once on day 7, followed by two additional days of culture). Analysis was performed on day 12, and renal organoids not treated with TGF-β (NT, no treatment) served as a control group for comparison.

[0111] (A) To create a renal fibrosis model, kidney organoids were treated with various concentrations of TGF-β. As a result, kidney organoids that were not treated with TGF-β grew well, but kidney organoids that were treated with TGF-β showed morphological changes and did not grow well.

[0112] (B) qPCR analysis of renal fibrosis-related markers Acta2 and Col1a1 confirmed that the expression of fibrosis marker mRNA increased proportionally with increasing TGF-β concentration.

[0113] (C) The expression of renal fibrosis-related markers, vimentin, COL1 (collagen type 1), and α-SMA (alpha smooth muscle actin), was confirmed by immunostaining. When renal organoids cultured in 7 mg / ml KEM were treated with TGF-β, the KEM hydrogel contracted, and the fibrotic organoids consolidated into a single mass. Immunostaining confirmed increased expression of all fibrosis markers in renal organoids treated with TGF-β, confirming that renal organoids grown on Matrigel and KEM scaffolds all showed similar fibrosis marker expression patterns.

[0114] Through this experiment, we will be able to confirm the feasibility of creating a renal fibrosis disease model based on kidney organoids cultured on a KEM hydrogel scaffold.

[0115] Confirmation of the biocompatibility of the decellularized kidney tissue-derived extracellular matrix (KEM) scaffold (Figure 15) To confirm whether the decellularized kidney tissue-derived KEM hydrogel scaffold is suitable as a material for kidney organoid transplantation, 5 mg / ml of decellularized kidney tissue-derived KEM scaffold was implanted into the subcutaneous tissue of mice and subjected to histological analysis. H&E staining was performed to check the degree of inflammatory cell infiltration into the implanted KEM hydrogel scaffold, and Toluidine blue staining was performed to check for the presence of mast cells generated by an immune response. The analysis showed that no inflammation-related cells were found at the KEM hydrogel implantation site, confirming that implantation of KEM hydrogel induces almost no immune or inflammatory responses in vivo.

[0116] Results of in vivo transplantation of kidney organoids using decellularized kidney tissue-derived extracellular matrix (Kidney Extracellular Matrix, KEM) (Figures 16 and 17) To confirm whether decellularized kidney tissue-derived KEM hydrogel can be used as an organoid transplantation material, kidney organoids were transplanted into the kidney capsule of mice using KEM hydrogel. To confirm the survival and presence of transplanted cells, kidney organoids labeled with DiI fluorescent dye were used. To adjust the viscosity of the hydrogel to improve transplantation efficiency into kidney tissue, KEM hydrogel was mixed with the culture medium at a ratio of 1:20 (v / v) and transplanted into the body.

[0117] To observe the degree of engraftment of the transplanted renal organoids in vivo, kidney tissue was harvested on day 4 after transplantation and subjected to histological analysis. As a result, as can be seen in Figure 16, the presence of fluorescent signals was confirmed inside the renal capsule, confirming that the transplanted organoids had successfully engrafted into the tissue.

[0118] To confirm whether decellularized kidney tissue-derived KEM hydrogel scaffolds could be used as a material for kidney organoid transplantation, animal experiments and histological analysis were performed. To induce an acute kidney injury (AKI) model, mice were injected intraperitoneally with 10 mg / kg cisplatin. The following day, 600-700 kidney organoids were transplanted into mouse kidney capsules using 25 μl of 7 mg / ml KEM hydrogel to enhance transplantation efficiency and survival. Kidney tissues from each group were harvested on day 14 after transplantation and analyzed by H&E staining and immunohistochemistry.

[0119] We then confirmed the feasibility of transplanting renal organoids in vivo in an animal model of kidney injury (Figure 17).

[0120] (A) H&E staining revealed widespread acute tubular necrosis (ATN) and hyperemic kidney, typical signs of acute kidney injury, in the damaged kidney tissue without organoid transplantation. In contrast, when kidney organoids were transplanted onto the KEM scaffold, the kidney organoids successfully engrafted into the cortical areas of the damaged kidney tissue, resulting in significantly smaller areas of damage compared to the control kidney tissue. Furthermore, quantitative analysis of the quadrigeminal area was performed to assess the degree of quadrigeminal contraction, a symptom of acute kidney injury. The results showed that kidney tissue transplanted with kidney organoids maintained less damaged quadrigeminal structures than non-transplanted tissue.

[0121] (B) To confirm the degree of proximal tubule cell death, one of the major symptoms of acute kidney injury, immunostaining for Villin, a proximal tubule marker, was performed under each condition. The results confirmed that the proportion of proximal tubules was significantly higher in the organoid-transplanted kidney tissue than in the non-transplanted kidney tissue, confirming that the transplanted organoids had engrafted at the injured site and regenerated the proximal tubules.

[0122] This experiment demonstrated that the KEM hydrogel scaffold can be used as a material for organoid transplantation to treat kidney disease models, and that it can increase the engraftment efficiency of organoids into damaged tissue while also inducing kidney structural recovery.

[0123] 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 spirit or essential features of the present invention. Therefore, it should be understood that the above-described embodiments are illustrative in all respects and are not limiting.

Claims

1. A hydrogel support for renal organoid culture containing kidney tissue-derived extracellular matrix (KEM), the hydrogel support is a freeze-dried product obtained by decellularizing the kidney tissue-derived extracellular matrix, A hydrogel support for renal organoid culture, wherein the concentration of the kidney tissue-derived extracellular matrix in the hydrogel support is 7 mg / ml to 10 mg / ml.

2. 1) disrupting the separated kidney tissue; 2) treating the disrupted kidney tissue with Triton X-100 and ammonium hydroxide to decellularize the tissue, thereby producing a decellularized kidney tissue-derived extracellular matrix (KEM); 3) freeze-drying the decellularized kidney tissue-derived extracellular matrix (KEM) to produce a freeze-dried kidney tissue-derived extracellular matrix; 4) forming a hydrogel support for renal organoid culture in the form of a hydrogel in which the concentration of the freeze-dried kidney tissue-derived extracellular matrix is ​​7 mg / ml to 10 mg / ml; A method for producing a hydrogel support for renal organoid culture, comprising:

3. 3. The method for producing a hydrogel support for renal organoid culture according to claim 2, wherein step 4) comprises dissolving the freeze-dried kidney tissue-derived extracellular matrix in a pepsin solution to form a solution, and then adjusting the pH to form a hydrogel.

4. A method for culturing kidney organoids on a hydrogel support for renal organoid culture according to claim 1 or a hydrogel support for renal organoid culture manufactured by the manufacturing method according to claim 2.

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