Decellularized cardiac tissue-derived support for cardiac organoid culture and transplantation, and method for producing the same.

Abstract

The present invention relates to a support for cardiac organoid culture and transplantation using decellularized cardiac tissue (Heart Extracellular Matrix; HEM).

Description

[Technical Field]

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

[0002] Organoids are three-dimensional cell structures composed of various cells constituting tissues, and have recently attracted attention because they can mimic the environment of living tissues. For this reason, they are widely used from fundamental biological research to various applied research fields including new drug development, disease modeling, and regenerative therapy.

[0003] In the case of disease models for new drug development, most animal models are currently widely used. However, since animals differ from humans in genetic and physiological aspects, there are limitations to actually reproducing human diseases in animals, and ethical issues regarding animal experimentation have continuously emerged. The use of organoid models produced from patient-derived cells has gained much attention because it enables the study of disease mechanisms as well as patient-specific diagnosis and treatment.

[0004] Currently, various types of organoid models derived from stem cells of many in vivo organs have been established. When culturing these various types of organoids, most researchers worldwide almost exclusively use Matrigel as a culture support. However, Matrigel is a component derived from mouse sarcoma, and concerns over attempts to transplant organoids cultured in Matrigel into humans have been raised due to the risk of infection and immune rejection. In addition, since Matrigel is not a component of the extracellular matrix contained in actual tissues, it cannot reproduce the microenvironment of tissue-specific extracellular matrix that is essential for cell growth and differentiation. Therefore, there is an urgent need for the development of a biocompatible and tissue-specific organoid culture support that can solve the problems of such Matrigel.

[0005] This invention presents a novel culture platform for culturing cardiac organoids by producing a matrix containing only extracellular matrix components from cardiac tissue through a decellularization process. Since this decellularized cardiac tissue-derived support contains various extracellular matrix components and growth factors present in cardiac tissue, it is expected to promote the formation, growth, and differentiation of cardiac organoids by providing a cardiac tissue-specific microenvironment. The mature cardiac organoids thus produced are expected to be useful not only as a platform for cardiac disease modeling and drug toxicity evaluation, but also in the field of regenerative medicine. [Overview of the Initiative] [Problems that the invention aims to solve]

[0006] This invention provides a method for producing a large quantity of decellularized cardiac tissue-derived support by subjecting porcine cardiac tissue to a series of chemical treatments, and for applying this support to cardiac organoid culture.

[0007] However, the technical problems that this invention aims to solve are not limited to those described above, and other problems not mentioned should be clearly understood by those skilled in the art from the following description. [Means for solving the problem]

[0008] The present invention will be described below with reference to the attached drawings. However, the present invention can be embodied in various different forms and is therefore not limited to the embodiments described herein. When a part is said to "include" a certain component, this means that, unless otherwise stated, it may include other components rather than excluding them.

[0009] Unless otherwise defined, this may be carried out by conventional techniques commonly used in molecular biology, microbiology, protein purification, protein engineering, and DNA sequencing analysis and, to the extent of the skills of those skilled in the art, in the field of DNA recombination. These techniques are known to those skilled in the art and are described in many standardized textbooks and reference books.

[0010] Unless otherwise defined herein, all technical and scientific terms used have the meanings that are ordinarily understood by an ordinary person in the art.

[0011] Various scientific dictionaries containing the terminology used herein are well known and available in the art. Any methods and substances similar or equivalent to those described herein have been used in the practice or testing of this invention, and several methods and substances are described. The invention is not limited to any particular methodology, protocol, and reagent, as they can be used in various ways depending on the context in which those skilled in the art use them. The invention will be described in more detail below.

[0012] One aspect of the present invention provides a support composition comprising a decellularized cardiac tissue-derived extracellular matrix (HEM).

[0013] The term "extracellular matrix" above refers to a natural support for cell growth produced through the decellularization of tissues found in mammals and multicellular organisms. The extracellular matrix may be further treated by dialysis or cross-linking.

[0014] The extracellular matrix may be a mixture of structural and unstructured biomolecules, not limited to collagen, elastins, laminins, glycosaminoglycans, proteoglycans, antimicrobials, chemoattractants, cytokines, and growth factors.

[0015] The extracellular matrix described above may contain approximately 90% collagen in various forms in mammals. Extracellular matrix derived from various biological tissues may differ in overall structure and composition due to the specific roles required by each tissue.

[0016] The terms "derive" and "derived" above refer to components obtained from the source mentioned by a useful method.

[0017] Furthermore, in one specific example of the present invention, the extracellular matrix derived from decellularized cardiac tissue may contain 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, or 6 mg / mL, and in an optimized specific example, 2 mg / mL. If it is included at a concentration outside the above range, the effects intended by the present invention may not be obtained, or it may be unsuitable for manufacture or use.

[0018] In one specific example of the present invention, the composition may have an elastic modulus of 1 to 150 Pa based on 0.1 to 10 Hz, and by having an elastic modulus within this range, the composition can form a stable polymer network.

[0019] The above support composition includes a three-dimensional culture hydrogel manufactured based on a cardiac tissue matrix composition obtained by decellularization, and can be effectively utilized for culturing cardiac organoids or single cardiac cells.

[0020] Since the decellularized cardiac tissue described above contains actual tissue-specific extracellular matrix components, it can provide the physical, mechanical, and biochemical environment of the tissue, making it highly efficient in promoting differentiation into cardiac tissue cells and enhancing tissue-specific functionality.

[0021] The term "organoid" above refers to a miniature biological organ created by culturing cells derived from tissue or pluripotent stem cells in a 3D form and fabricating it into a shape similar to an artificial organ.

[0022] The above organoids are three-dimensional tissue analogs containing organ-specific cells that originate from stem cells and self-organize (or self-pattern) in a manner similar to that in vivo, and can develop into specific tissues through patterning of restricted elements (e.g., growth factors).

[0023] The above organoids may possess the original physiological characteristics of cells and have anatomical structures that mimic the original state of the cell mixture (including not only limited cell types but also residual stem cells and the surrounding physiological niche). The above organoids may have a more efficient arrangement of cells and cell functions through a three-dimensional culture method, and may possess organ-like morphology and tissue-specific functions that result in functional organs.

[0024] The above composition may be for encapsulating cardiac organoids or single cardiomyocytes. Specifically, a single cardiomyocyte itself may be encapsulated and cultured to produce cardiac organoids, or cultured cardiac organoids may be encapsulated. When encapsulating a single cardiomyocyte, other cells may be further included to reflect the microenvironment in the body, specifically, vascular cells and cardiac fibroblasts may be further included. Cardiac organoids or single cardiomyocytes may be encapsulated as the above composition to promote cardiomyocyte differentiation.

[0025] Another aspect of the present invention provides a method for producing a support composition, comprising the steps of (a) producing decellularized cardiac tissue by decellularizing isolated cardiac tissue, and (b) producing decellularized cardiac tissue-derived extracellular matrix (Heart Extracellular Matrix, HEM) by drying the decellularized cardiac tissue.

[0026] Said step (a) is a step of producing decellularized heart tissue by decellularizing isolated heart tissue.

[0027] In one embodiment of the present invention, said step (a) comprises treating said isolated heart tissue with 0.1 to 2% sodium dodecyl sulfate (SDS) at 1 to 10°C for 12 to 36 hours, specifically 0.5 to 1.5% SDS at 2 to 9°C for 15 to 30 hours, more specifically 1% sodium dodecyl sulfate (SDS) at 4°C for 24 hours, followed by decellularization by stirring in a decellularization solution. By adding such a treatment before stirring with the decellularization solution, the overall decellularization efficiency can be increased.

[0028] Said decellularization solution may contain various components for removing cells from heart tissue, for example, it may contain components of hypertonic saline, peracetic acid, Triton X-100, SDS or other detergents. In one embodiment of the present invention, said decellularization solution may comprise 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 decellularization solution as described above, decellularization proceeds under milder conditions compared with existing processes, whereby DNA in the produced scaffold can be effectively removed, and at the same time, more various proteins in the heart tissue can be preserved.

[0029] The above stirring may be carried out for 3 to 24 hours, more specifically 4 to 12 hours, most specifically 5 to 8 hours, or as an example, 6 hours, and the stirring may be carried out at 1 to 10°C, more specifically 2 to 9°C, or most specifically 4°C. Through such stirring (decellularization) process, 95 to 99.9%, more specifically 96 to 98%, of the cardiac tissue cells may be removed. If decellularization is carried out for a time outside the above range or at a cardiac tissue cell removal level, problems such as a decrease in the quality of the manufactured support composition or a decrease in process economics may occur.

[0030] Step (b) above is a step of producing decellularized cardiac tissue-derived extracellular matrix (Heart Extracellular Matrix, HEM) by drying the decellularized cardiac tissue.

[0031] The method for drying the decellularized cardiac tissue described above may be carried out by known methods, including natural drying or freeze-drying. For sterilization purposes, the tissue may be exposed to ethylene oxide gas or supercritical carbon dioxide using an electron beam or gamma radiation after drying.

[0032] In one specific example of the present invention, after step (b) above, the extracellular matrix derived from decellularized cardiac tissue may be included at a concentration of 0.01 to 10 mg / mL, specifically 0.5 to 9 mg / mL, more specifically 1 mg / mL to 8 mg / mL, most specifically 2, 4, or 6 mg / mL, and in an optimized specific example, 2 mg / mL. If it is included at a concentration outside the above range, the effects intended by the present invention may not be obtained, or it may be unsuitable for manufacture or use.

[0033] The above-mentioned dried extracellular matrix may be subdivided by a method including tearing, milling, cutting, grinding, and shearing steps. The above-mentioned subdivided extracellular matrix may be processed into a powder by methods such as grinding or milling while frozen or lyophilized.

[0034] Furthermore, the method for producing the support composition may further include the additional step of (c) gelating the dried decellularized cardiac tissue-derived extracellular matrix.

[0035] Step (c) above is a step of gelling the dried decellularized cardiac tissue-derived extracellular matrix.

[0036] Through the gelation process described above, a three-dimensional hydrogel support can be fabricated by crosslinking the extracellular matrix derived from decellularized cardiac tissue. This gelled support can be used in a variety of applications, including experiments, screening, and organoid culture.

[0037] The above-mentioned "hydrogel" is a substance in which a liquid with water as the dispersion medium solidifies and loses its fluidity through a sol-gel phase transition, forming a porous structure. It may also be formed by a hydrophilic polymer having a three-dimensional network structure and an uncrystalline structure expanding when it contains water.

[0038] The gelation described above may also be carried out by dissolving the extracellular matrix derived from decellularized cardiac tissue in an acidic solution with a proteolytic enzyme such as pepsin or trypsin, adjusting the pH to neutral and the electrolyte state of the 1x PBS buffer using 10×PBS and 1M NaOH, and then performing the gelation at a temperature of 37°C for 30 minutes.

[0039] The above gelation process may be used to encapsulate cardiac organoids or single cardiomyocytes. Specifically, the above gelation process may be used to encapsulate a single cardiomyocyte itself and then culture it to produce cardiac organoids, or cultured cardiac organoids may be encapsulated. When encapsulating a single cardiomyocyte, other cells may be included to reflect the microenvironment within the body; specifically, vascular cells and cardiac fibroblasts may be included. Cardiac organoids or single cardiomyocytes may be encapsulated in the above composition to promote cardiomyocyte differentiation.

[0040] Another aspect of the present invention provides a method for culturing cardiac organoids with the above-described support composition or a support composition produced by the above-described manufacturing method.

[0041] Existing Matrigel-based culture systems use extracts derived from animal cancer tissue, and the significant differences in placement prevent them from accurately simulating the actual environment, resulting in insufficient differentiation and development into cardiac organoids. In contrast, the above-mentioned support composition can create a cardiac tissue-like environment and is therefore suitable for cardiac organoid culture.

[0042] Specifically, the support composition described above or the support composition produced by the above manufacturing method may be used to encapsulate cardiac organoids or single cardiomyocytes and culture cardiac organoids. More specifically, single cardiomyocytes themselves may be encapsulated and cultured to culture cardiac organoids, or cultured cardiac organoids may be encapsulated and cultured. When encapsulating single cardiomyocytes, other cells may be further included to reflect the microenvironment in the body, specifically, vascular cells and cardiac fibroblasts may be further included. Cardiac organoids or single cardiomyocytes may be encapsulated as the above composition to promote myocardial differentiation.

[0043] The above culture refers to the process of maintaining and growing cells under suitable conditions, which may include, for example, the temperature at which the cells are maintained, nutrient availability, atmospheric CO2 content, and cell density.

[0044] Appropriate culture conditions for maintaining, proliferating, expanding, and differentiating different types of cells are publicly known and documented in the art. Conditions suitable for the formation of the above organoids may also be conditions that facilitate or allow cell differentiation and the formation of multicellular structures. [Effects of the Invention]

[0045] By utilizing the decellularized cardiac tissue-derived support produced in this invention, it becomes possible to realize the cardiac microenvironment with cardiac tissue-specific extracellular matrix components abundantly present within the support, enabling the production of even more advanced organoids that mimic actual cardiac tissue. This can replace Matrigel, a representative organoid culture support that has been commonly used, and create industrially high added value.

[0046] Furthermore, highly functional human cardiac organoids possess high potential for use in the development of next-generation drugs and the evaluation of drug toxicity. Accurately predicting the cardiotoxicity of a drug is a crucial success factor in the drug development process. Existing cell lines or animal models often fail to accurately predict drug cardiotoxicity, resulting in significant wasted time and resources and posing a major obstacle to drug development. Human cardiac organoids are expected to contribute greatly to the pharmaceutical industry in drug development because they offer greater accuracy in judging drug efficacy and safety compared to existing models, and are more economical in terms of time and cost.

[0047] The human cardiac organoid established in this invention can precisely simulate cardiac tissue structurally and functionally, and can therefore be used to create various cardiac disease models that are currently impossible to realize. Thus, a cardiac disease modeling platform applying organoid culture technology based on decellularized cardiac tissue-derived supports can be applied to mechanistic research on intractable cardiac diseases and contribute to the development of the medical industry. If organoids are produced from a patient's stem cells, it becomes possible to develop a patient-specific precision medical platform, and ultimately, it is expected to be applicable to regenerative medicine uses such as reconstructing damaged cardiac tissue, including that caused by myocardial infarction. [Brief explanation of the drawing]

[0048] [Figure 1] This study describes the fabrication and analysis of a decellularized cardiac tissue-derived extracellular matrix support (Heart Extracellular Matrix, HEM) for culturing cardiac organoids. [Figure 2] This study describes the fabrication and analysis of a decellularized cardiac tissue-derived extracellular matrix support (Heart Extracellular Matrix, HEM) for culturing cardiac organoids. [Figure 3] This study analyzed the proteins in a decellularized cardiac tissue-derived extracellular matrix support (HEM) used for culturing cardiac organoids. [Figure 4] This shows the types and quantitative analysis results of matrixsome proteins in a decellularized cardiac tissue-derived extracellular matrix support (Heart Extracellular Matrix, HEM). [Figure 5] This is the result of non-matrisome protein analysis of extracellular matrix support (Heart Extracellular Matrix, HEM) derived from decellularized cardiac tissue. [Figure 6] This document presents a comparative analysis and selection of the optimal method for decellularization of cardiac tissue. [Figure 7] This describes the process of fabricating cardiac organoids using a decellularized cardiac tissue-derived extracellular matrix support (Heart Extracellular Matrix, HEM). [Figure 8] This paper presents the results of producing human cardiac organoids based on a decellularized cardiac tissue-derived extracellular matrix (HEM) support using human dedifferentiated stem cell-derived cardiomyocytes and organoid encapsulation methods. [Figure 9] This study analyzed gene expression in human cardiac organoids based on a decellularized cardiac tissue-derived extracellular matrix support (Heart Extracellular Matrix, HEM) using an organoid encapsulation method. [Figure 10] This study evaluated and analyzed the degree of myocardial differentiation of human cardiac organoids based on a decellularized cardiac tissue-derived extracellular matrix support (Heart Extracellular Matrix, HEM) using an organoid encapsulation method. [Figure 11]This study evaluated and analyzed the degree of differentiation of human cardiac organoids based on a decellularized cardiac tissue-derived extracellular matrix support (Heart Extracellular Matrix, HEM) using an organoid encapsulation method. [Figure 12] This report presents the results of an analysis of the drug responsiveness of human cardiac organoids based on a decellularized cardiac tissue-derived extracellular matrix (HEM) support, utilizing an organoid encapsulation method. [Figure 13] This paper describes a method for producing human cardiac organoids based on a decellularized cardiac tissue-derived extracellular matrix (HEM) support through the encapsulation of a single cardiomyocyte, and presents the results of gene expression analysis. [Figure 14] This shows the results of vascular cell culture using a decellularized cardiac tissue-derived extracellular matrix support (Heart Extracellular Matrix, HEM). [Figure 15] This paper demonstrates the production of human cardiac organoids based on a decellularized cardiac tissue-derived extracellular matrix (HEM) support through a method of encapsulating cardiomyocytes and two types of non-myocytes, and the selection of optimized culture conditions. [Figure 16] This paper describes the production of an advanced human cardiac organoid with a vascular structure using a method for encapsulating cardiomyocytes and two types of non-myocytes, as well as the concentration selection process and results of the decellularized cardiac tissue-derived extracellular matrix support (HEM) used for this purpose. [Figure 17] This paper describes the production of an advanced human cardiac organoid with a vascular structure using a method for encapsulating cardiomyocytes and two types of non-myocytes, as well as the concentration selection process and results of the decellularized cardiac tissue-derived extracellular matrix support (HEM) used for this purpose. [Figure 18]This paper describes the production of an advanced human cardiac organoid with a vascular structure using a method for encapsulating cardiomyocytes and two types of non-myocytes, and the process for selecting the concentration of the decellularized cardiac tissue-derived extracellular matrix (HEM) support for this purpose. [Figure 19] This paper presents a method for encapsulating cardiomyocytes and two types of non-myocytes using HEM (Heat-Enhanced Microscopy), as well as a method and results for culturing human cardiac organoids using a microfluidic chip. [Figure 20] This paper describes a method for encapsulating cardiomyocytes and two types of non-myocytes, as well as the process and results of fabricating cardiac organoids from LQT patients using a decellularized cardiac tissue-derived extracellular matrix support (Heart Extracellular Matrix, HEM). [Figure 21] This paper presents a method for HEM encapsulation of cardiomyocytes and two types of non-myocytes, as well as the results of culturing and analyzing cardiac organoids from LQT patients using a microfluidic chip. [Modes for carrying out the invention]

[0049] In this invention, a large amount of decellularized cardiac tissue-derived support was produced from porcine cardiac tissue through a series of chemical treatments, and this was applied to the culture of cardiac organoids. To culture cardiac organoids, the decellularized support was produced using an optimal protocol that allowed for sufficient retention of extracellular matrix components, and the experiment proceeded accordingly. The decellularization process confirmed that cells within the cardiac tissue were effectively removed while simultaneously preserving cardiac tissue-specific extracellular matrix components. It was confirmed that the produced decellularized cardiac tissue-derived extracellular matrix components could be used to form a hydrogel for three-dimensional organoid culture.

[0050] In this invention, cardiac organoids were produced by differentiating human dedifferentiated stem cells into cardiomyocytes and then culturing these cells in a hydrogel made from decellularized cardiac tissue-derived extracellular matrix in a three-dimensional manner. Gene expression analysis confirmed that cardiac organoids cultured on a decellularized cardiac tissue-derived hydrogel support showed enhanced differentiation into cardiac tissue compared to commonly used supports, Matrigel and collagen hydrogel.

[0051] Furthermore, cardiac organoids cultured on a hydrogel support derived from decellularized cardiac tissue exhibited the most vigorous beating, superior expression of cardiac muscle-specific proteins, and the clearest observation of cardiomyocyte-specific sarcomere structures compared to organoids cultured on Matrigel and collagen hydrogel. This confirms that the decellularized cardiac tissue-derived support has the potential to be developed as a novel functional culture platform that can replace existing culture supports for cardiac organoid culture.

[0052] The newly developed decellularized cardiac tissue-derived support culture platform has the advantages of being easier and more economical to manufacture compared to existing expensive culture matrices and polymer supports, making it highly advantageous for commercialization.

[0053] The following are preferred embodiments to aid in understanding the present invention. However, the following embodiments are provided solely to facilitate understanding of the present invention, and the scope of the present invention is not limited by these embodiments.

[0054] Example 1: Fabrication and analysis of decellularized cardiac tissue-derived extracellular matrix (HEM) for cardiac organoid culture. Example 1-1. Preparation and analysis of extracellular matrix derived from decellularized cardiac tissue. As shown in Figure 1, a decellularized heart tissue-derived extracellular matrix (HEM) support was fabricated from porcine heart tissue through a decellularization process, and its properties were analyzed.

[0055] The cardiac decellularization process was carried out by treating the tissue with 1% sodium dodecyl sulfate (SDS) under 24-hour refrigeration conditions, followed by additional treatment with 1% Triton-X100 and 0.1% NH4OH under 6-hour refrigeration conditions. The extracellular matrix components produced through the decellularization process were prepared as a powder by lyophilization. Undecellularized cardiac tissue was used as the control group.

[0056] In the fabricated decellularized cardiac tissue-derived HEM supports, DNA and GAG (Glycosaminoglycans) were analyzed to confirm whether cells were sufficiently removed and whether extracellular matrix components were well preserved. The results showed that in all fabricated decellularized cardiac tissue supports, DNA was sufficiently removed after the decellularization process, and GAG was present at levels similar to those found in actual cardiac tissue (Figure 1(A)).

[0057] Examples 1-2. Preparation and analysis of support compositions 10 mg of lyophilized HEM (HEM), a powdered decellularized cardiac tissue-derived support prepared by freeze-drying, was treated with a 4 mg / ml pepsin solution (a solution of 4 mg of porcine gastric mucosa-derived pepsin powder dissolved in 1 ml of 0.02 M HCl), and the solution was allowed to dissolve at room temperature for 48 hours. Subsequently, the solution was adjusted to a neutral pH and electrolyte state using 10 × PBS and 1 M NaOH for use in cell culture. Finally, hydrogelation was induced for 30 minutes at a temperature of 37°C in an incubator.

[0058] Fluid dynamics analysis was performed to measure the physical properties of the hydrogel, confirming that the fabricated hydrogel support derived from decellularized cardiac tissue was composed of a stable polymer network structure and possessed mechanical properties suitable for organoid culture (Figure 1(B)).

[0059] On the other hand, a decellularized support (Heart Extracellular Matrix, HEM) was fabricated from porcine heart tissue and its properties were analyzed.

[0060] As a result, as can be seen in Figure 2, when cardiac tissue was observed before and after the decellularization process through H&E histological analysis, it was confirmed that although the cell nuclei were almost completely removed, the overall structure of the tissue was maintained (Figure 2(A)). Furthermore, in order to confirm the internal structure of the hydrogel formed from the decellularized cardiac tissue-derived support, scanning electron microscopy (SEM) analysis was performed. The results showed that the interior of the HEM hydrogel was composed of extracellular matrix components in the form of nanofibers, and it was confirmed that the hydrogel produced in this way can provide a microenvironment suitable for the growth of cardiac organoids (Figure 2(B)).

[0061] Experimental Example 1: Analysis of extracellular matrix derived from decellularized cardiac tissue Experimental Example 1-1. Analysis of Heart Extracellular Matrix (HEM) proteins derived from decellularized cardiac tissue for cardiac organoid culture. To identify the extracellular matrix components contained in the decellularized cardiac tissue-derived support, protein analysis was performed using a mass spectrometer.

[0062] As a result, as can be seen in Figure 3, it was confirmed that many extracellular matrix components and glycoproteins, such as various collagens, proteoglycans, and glycoproteins, were contained in the decellularized cardiac tissue-derived support. Therefore, it is expected that these cardiac-specific components present in actual cardiac tissue can promote the differentiation, structural development, and functional enhancement of cardiac organoids.

[0063] Experimental Example 1-2. Analysis of matrixsome protein types and quantitative analysis of extracellular matrix supports (Heart Extracellular Matrix, HEM) derived from decellularized cardiac tissue. Matrisome proteins contained in HEM were detected through protein analysis using a mass spectrometer, classified by protein type, and then relative quantitative analysis was performed.

[0064] We confirmed that various types of cardiac tissue proteins are present in HEM, and we were able to predict that these components have a positive effect on the culture of cardiac organoids (Figure 4(a, b)).

[0065] Furthermore, through relative quantitative analysis of proteins, the 10 most abundant matrixome proteins in HEM were identified. High concentrations of collagen and fibrinogen subtypes were detected, along with large amounts of lumican, perlecan, and laminin (gamma1). Additionally, various matrixome proteins known to be specifically abundant in actual cardiac tissue (such as laminin (alpha 2), thrombospondin 4, collagen type 6 (alpha 6), and fibulin 2) were detected within HEM (Figure 4(c)).

[0066] Therefore, compared to existing culture systems based on single-component proteins or cardiac nonspecific proteins such as Matrigel, collagen, and fibrin, HEM hydrogel, which contains matrixomyces from various cardiac tissues, provides a cardiac tissue-specific microenvironment that is useful for the development of cardiac organoids, thereby helping to produce more advanced and mature cardiac organoids.

[0067] Experimental Examples 1-3. Analysis of non-matrisome proteins in decellularized cardiac tissue-derived extracellular matrix supports (Heart Extracellular Matrix, HEM). The non-matrisome proteins of the decellularized cardiac tissue-derived extracellular matrix support (Heart Extracellular Matrix, HEM) of the present invention were analyzed.

[0068] As a result, it was confirmed that HEM contains not only matrixomyces but also a large amount of non-matrisome proteins (Figure 5(a)). Furthermore, it was confirmed that various non-matrisome proteins that are abundant in actual cardiac tissue are contained within HEM (Figure 5(b)).

[0069] On the other hand, non-matrisome proteins were analyzed using the GOBP (gene ontology biological process) method to determine which biological processes the proteins involved were present in large quantities. As a result, it was confirmed that the proteins contained particularly large amounts of proteins involved in tissue formation, such as cytoskeleton organization, cellular component organization, cellular component assembly, and organelle organization (Figure 5(c)).

[0070] Therefore, it is evident that mature cardiac organoids can be cultured using HEM supports containing such non-matrisome proteins.

[0071] Experimental Examples 1-4. Comparative Analysis and Optimization of Decellularization Protocols for Cardiac Tissue Experiments were conducted to compare the cell decellation method of the present invention (Protocol 1) with other cell decellation methods (Protocol 2). Unlike Protocol 1, Protocol 2 did not involve treatment with 1% sodium dodecyl sulfate (SDS), but instead involved treatment with 1% Triton-×100 and 0.1% NH4OH for only 6 hours.

[0072] As a result, when comparing the amount of DNA remaining after the decellularization process of the heart, it was confirmed that almost all of the DNA was removed from the decellularized cardiac matrix produced by Protocol 1, while a considerable amount remained in the tissue produced by Protocol 2. Furthermore, when comparing the remaining extracellular matrix components through GAG quantitative analysis, it was confirmed that the GAG ​​components were well preserved after processing with Protocol 1, while the remaining GAG components were significantly reduced after processing with Protocol 2 (Figure 6(a)).

[0073] Then, after preparing hydrogels using the decellularized matrix, the mechanical properties (modulus) were measured, and it was confirmed that the hydrogels prepared using Protocol 1 had significantly higher properties (Figure 6(b)).

[0074] Furthermore, H&E histological analysis revealed that the matrix produced using Protocol 2 retained a large number of cells and still maintained a morphology similar to that of actual cardiac tissue (Figure 6(c)).

[0075] On the other hand, proteomics analysis of the matrices produced by each decellularization method revealed that protocol 1 preserved a greater number of extracellular matrix proteins compared to protocol 2 (Figure 6(d)).

[0076] Therefore, the decellularization method established in this study is more effective than other decellularization methods in preserving extracellular matrix proteins in cardiac tissue, and enables the creation of a support more suitable for culturing cardiac organoids.

[0077] Example 2: Fabrication of cardiac organoids using decellularized cardiac tissue-derived extracellular matrix support (Heart Extracellular Matrix, HEM) Two methods for producing cardiac organoids using decellularized cardiac tissue-derived HEM hydrogel and cardiomyocytes were performed, as shown in Figure 7.

[0078] Specifically, the first method involves fabricating a single cardiomyocyte into a three-dimensional organoid using a microwell, and then encapsulating the fabricated cardiac organoid in HEM hydrogel and proceeding with the culture. The second method involves encapsulating a single cardiomyocyte in HEM hydrogel, inducing organoid formation, and then fabricating cardiac organoids in which the HEM hydrogel is mixed between the cells.

[0079] Below, we will verify that cardiac organoids with enhanced myocardial differentiation can be produced using HEM hydrogel by each method.

[0080] Experimental Example 2: Verification of encapsulation methods for organoids Experimental Example 2-1. Production of human cardiac organoids based on decellularized cardiac tissue-derived extracellular matrix (HEM) using human dedifferentiated stem cell-derived cardiomyocytes and organoid encapsulation methods. Human induced pluripotent stem cells were differentiated into cardiomyocytes, and the differentiated cardiomyocytes were fabricated into a three-dimensional organoid morphology using microwells. Subsequently, cardiac organoids with enhanced differentiation and maturation were produced by three-dimensional culture in HEM hydrogel (4 mg / ml).

[0081] When human induced pluripotent stem cells were differentiated into cardiomyocytes on a standard plate, organic cell linkage and pulsation were confirmed on day 15 of differentiation. Furthermore, on day 17, the expression of α-actin, a heart-specific protein, and sarcomere structure were confirmed through immunohistochemistry (Figure 8(A)).

[0082] Differentiated cardiomyocytes were fabricated into three-dimensional organoid morphologies using microwells. The formed organoids were then cultured in three dimensions within HEM hydrogel. Two groups were used as comparison groups: one cultured using collagen hydrogel (Col I) and Matrigel (Mat), and the other cultured in suspension without matrix (No ECM). The results showed that cardiac organoids cultured in HEM hydrogel pulsated more actively than organoids cultured under the comparison conditions (Figure 8(b)).

[0083] Experimental Example 2-2. Gene expression analysis of human cardiac organoids based on decellularized cardiac tissue-derived extracellular matrix (HEM) support using organoid encapsulation method. To confirm the degree of differentiation enhancement in human cardiac organoids cultured in three dimensions within HEM hydrogel (4 mg / ml), qPCR analysis was performed on day 7 of culture. Two groups were used as comparison groups: one cultured using collagen hydrogel (Col I) and Matrigel (Mat), and the other cultured in suspension without matrix (No ECM).

[0084] As a result, as can be seen in Figure 9, it was confirmed that the expression of the cardiomyocyte-specific genes TNNT2, NPPA, and SCN5A was more enhanced in human cardiac organoids fabricated using HEM hydrogel compared to the control group.

[0085] Therefore, it was confirmed that HEM hydrogel further promotes myocardial differentiation of human cardiac organoids compared to existing organoid culture supports.

[0086] Experimental Example 2-3. Evaluation and Analysis of Myocardial Differentiation of Human Cardiac Organoids Based on Decellularized Cardiac Tissue-Derived Extracellular Matrix (HEM) Support Using Organoid Encapsulation Method To investigate the effects of HEM hydrogel on the myocardial differentiation of human cardiac organoids, immunohistochemical staining for Troponin I (TNNI), a myocardial-specific marker, was performed on day 7 of culture. Groups cultured using collagen hydrogel (Col I) and Matrigel (Mat) were used as comparison groups, while groups cultured in suspension without matrix (No ECM) were also used.

[0087] As a result, as can be seen in Figure 10, in human cardiac organoids prepared using HEM hydrogel (4 mg / ml), the cardiomyocytes were found to be in a more mature morphology, and TNNI expression was confirmed to be very high. In addition, sarcomere-specific structures were clearly and distinctly observed compared to the control group.

[0088] Experimental Example 2-4. Evaluation and analysis of the degree of differentiation of human cardiac organoids based on decellularized cardiac tissue-derived extracellular matrix (HEM) support using organoid encapsulation method. To investigate the effects of HEM hydrogel on cardiac organoid myocardial differentiation, immunohistochemical staining for the cardiomyocyte-specific markers α-actinin and cTnT was performed on day 7 of culture, and the protein expression levels of these markers were compared. Groups cultured using collagen hydrogel (Col I) and Matrigel (Mat) were used as comparison groups.

[0089] As a result, as shown in Figure 11, cardiomyocytes were found in a more mature morphology in human cardiac organoids fabricated using HEM hydrogel (4 mg / ml), and cardiomyocyte-specific sarcomere structures were observed more clearly and distinctly compared to the control group.

[0090] Experimental Example 2-5. Analysis of drug responsiveness of human cardiac organoids based on decellularized cardiac tissue-derived extracellular matrix (HEM) support using organoid encapsulation method. To confirm the drug responsiveness of human cardiac organoids cultured in HEM hydrogel (4 mg / ml), two representative drugs that affect myocardial beat rate (isoproterenol and propranolol) were treated on day 7 of culture, and the organoid response was confirmed by calcium imaging.

[0091] As a result, as can be seen in Figure 12, it was confirmed that the heart rate of cardiac organoids increased when treated with isoproterenol at a concentration of 1 μM, and that the heart rate slowed down when treated with propranolol at a concentration of 10 μM.

[0092] This confirmed that human cardiac organoids cultured in HEM hydrogel can exhibit appropriate responsiveness to drugs, thus verifying their potential for use in evaluating cardiac responses to various drugs.

[0093] Experimental Example 3: Verification of a method for encapsulating single cardiomyocytes Experimental Example 3-1. Method for producing human cardiac organoids based on decellularized cardiac tissue-derived extracellular matrix (HEM) support via single cardiomyocyte encapsulation and analysis of gene expression. Unlike methods that encapsulate formed organoids within HEM hydrogel, we attempted a method to produce human cardiac organoids by encapsulating human-induced pluripotent stem cell-derived cardiomyocytes in a single-cell state within HEM hydrogel.

[0094] As a result, the hydrogel contracted as the cells were cultured, forming a firm, solid cardiac organoid. Cardiac organoids were fabricated using various concentrations of HEM hydrogel, and it was confirmed that relatively larger cardiac organoids could be produced compared to the method of encapsulating the organoids (Figure 13(A)).

[0095] Then, to confirm the myocardial differentiation-promoting effect of HEM hydrogel, qPCR analysis was performed on day 7 of culture, and cardiac organoids cultured in Matrigel (Mat) were used as a control group for comparison.

[0096] As a result, it was confirmed that the expression of the cardiomyocyte-specific genes TNNT2 and NPPA was more enhanced in human cardiac organoids fabricated using HEM hydrogel compared to cardiac organoids cultured in the control group (Figure 13(B)).

[0097] Therefore, it was confirmed that HEM hydrogel further promotes the formation of human cardiac organoids and myocardial differentiation, even through a method of encapsulating single cardiomyocytes.

[0098] Experimental Example 3-2. Vascular cell culture based on decellularized cardiac tissue-derived extracellular matrix (HEM) support. Since the formation of vascular structures within organoids is a crucial element in organoid growth, development, and function, we investigated whether vascular cells can be cultured in HEM hydrogel for the vascularization of cardiac organoids.

[0099] Specifically, to determine whether HEM hydrogel is suitable for culturing vascular endothelial cells, human umbilical vein endothelial cell (HUVEC) cells were encapsulated in single cells in various concentrations of HEM hydrogel and cultured.

[0100] Analysis of the cells on day 7 of culture by immunohistochemical staining for the vascular markers CD31 and VE-Cadherin, as shown in Figure 14, revealed that while culture was possible at all concentrations, the expression of vascular cell markers was highest and cell spreading was best observed under 2 mg / ml HEM hydrogel conditions, confirming that this concentration was optimal for culture.

[0101] Experimental Example 3-3. Production of human cardiac organoids based on decellularized cardiac tissue-derived extracellular matrix (HEM) support through encapsulation of cardiomyocytes and two types of non-myocytes, and selection of optimized culture conditions. To create a cardiac model with a structurally and functionally more advanced vascular structure, cardiac organoids were produced by co-culturing three types of cells: endothelial cells and cardiac fibroblasts, which are non-myocytes present in the actual heart, together in HEM hydrogel using a single-cell encapsulation method. HUVEC cells were used for the endothelial cells, and the cardiac fibroblasts were differentiated from human induced pluripotent stem cells (Figure 15(A)).

[0102] Specifically, in order to select co-culture medium conditions, cardiomyocyte culture medium and vascular cell culture medium were mixed in various proportions, and experiments were conducted to select culture conditions.

[0103] On day 7 of culture, immunohistochemical staining was used to confirm the expression of cardiomyocyte markers (cTnT) and vascular cell markers (CD31). It was found that angiogenesis was best achieved under CM (cardiomyocyte culture medium) + 50% EGM (vascular cell culture medium) conditions, and that there were no problems with the expression of cardiomyocyte-specific proteins (Figure 15(B)).

[0104] Therefore, the subsequent production of co-cultured cardiac organoids proceeded under CM + 50% EGM culture medium conditions.

[0105] Experimental Example 3-4. Production of advanced human cardiac organoids with vascular structures using encapsulation methods for cardiomyocytes and two types of non-myocytes, and concentration selection of decellularized cardiac tissue-derived extracellular matrix (HEM) for this purpose. We fabricated advanced human cardiac organoids with vascular structures by applying encapsulation methods for cardiomyocytes and two types of non-myocytes, and selected the concentration of decellularized cardiac tissue-derived extracellular matrix (HEM) support for this purpose.

[0106] Specifically, in HEM hydrogels at concentrations of 2, 4, and 6 mg / ml, three types of cells (cardiomyocytes, vascular cells, and cardiac fibroblasts) were encapsulated in single cells, totaling 2 × 10⁶ cells. 5 Cardiac organoids were created by culturing cells in a ratio of 2:1:1.

[0107] As a result, microscopic analysis on day 7 of culture confirmed the successful fabrication of spherical cardiac organoids (Figure 16(A)). Furthermore, a comparison of the sizes of cardiac organoids formed from HEM hydrogel at different concentrations on day 7 of culture revealed that the lower the HEM concentration, the more condensed the organoids became, resulting in the formation of smaller organoids with a denser structure (Figure 16(B)).

[0108] Then, on day 7 of culture, qPCR analysis was performed to compare mRNA expression levels at different HEM hydrogel concentrations. The results showed that the expression of myocardial-specific markers TNNT2 and NPPA, and the expression of vascular-specific markers PECAM1 and vWF, were confirmed, and myocardial differentiation and vascular maturation were further enhanced under lower HEM hydrogel concentrations (Figure 16(C)).

[0109] Therefore, it was confirmed that cardiac organoids co-cultured with three types of cells in HEM hydrogel at a concentration of 2 mg / ml were cultured into the most condensed form with a dense structure, further enhancing vascularization and myocardial differentiation.

[0110] Then, on day 7 of culture, the distribution of cardiomyocytes (cTnT), vascular cells (CD31), and cardiac fibroblasts (VIM, DDR2) was confirmed through immunohistochemical staining.

[0111] As a result, as can be seen in Figure 17, it was confirmed that the lower the concentration, the more condensed the cardiac organoids were, the better the cell-to-cell connections were, and the better the expression of each marker.

[0112] Therefore, it was confirmed that cardiac organoids co-cultured with three types of cells in a 2 mg / ml HEM hydrogel cultured to the most condensed form, while vascularization and myocardial differentiation were further enhanced.

[0113] Experimental Example 3-5. Production of advanced human cardiac organoids with vascular structures using encapsulation methods for cardiomyocytes and two types of non-myocytes, and concentration selection of decellularized cardiac tissue-derived extracellular matrix (HEM) support for this purpose. To confirm the internal structure of cardiac organoids co-cultured for 14 days in HEM hydrogels of various concentrations using three cell types, immunohistochemical staining was performed using histological analysis.

[0114] As can be seen in Figure 18, the results of examining the cardiomyocyte marker cTnT, the vascular cell marker CD31, and the cardiac fibroblast marker VIM confirmed that cells were tightly linked and formed dense structures in HEM hydrogels at concentrations of 2 mg / ml and 4 mg / ml. In the case of the 6 mg / ml HEM group and the control group (Matrigel), cells remained in a single-cell morphology and did not form organic structures within the organoids.

[0115] Experimental Example 3-6. Method for HEM encapsulation of cardiomyocytes and two types of non-myocytes, and cultivation of human cardiac organoids using microfluidic chips. To create a more advanced human heart model, we utilized a microfluidic chip capable of providing a fine flow of culture medium that simulates human blood flow, and cultured cardiac organoids within the chip using HEM hydrogel. Matrigel (Mat) was used as the control group.

[0116] On day 14 of culture, immunohistochemical staining was used to compare the expression of cardiomyocyte (cTnT), vascular cell (CD31), and cardiac fibroblast (VIM) markers. As can be seen in Figure 19, cardiac organoids in 2 mg / ml HEM hydrogel showed the best expression of all cellular markers, and were able to form highly developed organoids with organic structures.

[0117] Therefore, this experiment confirmed that by combining HEM hydrogel and a microfluidic chip, it is possible to produce a more advanced human cardiac organoid in which three types of cells are co-cultured.

[0118] Experimental Example 4: Confirmation of the potential use of the support composition of the present invention Experimental Example 4-1. Method for encapsulating cardiomyocytes and two types of non-myocytes, and the production of cardiac organoids from LQT patients using decellularized cardiac tissue-derived extracellular matrix (HEM). Cardiac organoids were produced using cardiomyocytes differentiated from induced pluripotent stem cells of patients with long QT syndrome (LQT) through a single-cell encapsulation method. Specifically, the process was carried out using HEM hydrogel at a concentration of 2 mg / ml. In addition to cardiomyocytes, two types of non-cardiomyocytes (vascular cells and cardiac fibroblasts) were encapsulated together, and cardiac organoids were produced using cardiomyocytes differentiated from stem cells of different LQT patients (LQT2 and LQT3).

[0119] As a result, as can be seen in Figure 20, on day 14 of culture, LQT cardiac organoids were formed within the HEM hydrogel, confirming that the culture was successful, as demonstrated by optical microscopy analysis.

[0120] This confirms that it is possible to create human cardiac disease organoid models using HEM hydrogel, demonstrating that HEM hydrogel can be applied as an effective organoid culture matrix for in vitro disease modeling.

[0121] Experimental Example 4-2. Method for HEM encapsulation of cardiomyocytes and two types of non-myocytes, and culture and analysis of cardiac organoids from LQT patients using a microfluidic chip. As in Experimental Example 4-1, cardiomyocytes, vascular cells, and cardiac fibroblasts derived from induced pluripotent stem cells of LQT patients were co-cultured in 2 mg / ml HEM hydrogel, and cardiac organoids of LQT disease were fabricated through dynamic culture by applying this to a microfluidic chip to provide a fine flow of culture medium.

[0122] As a result, as can be seen in Figure 21, pulsation analysis of cardiac organoids on day 14 of culture confirmed that normal cardiac organoids exhibited a regular pulsation, while organoids from LQT disease exhibited an irregular pulsation.

[0123] This experiment confirmed that organoids of LQT disease fabricated using HEM hydrogel possessed the characteristics of the disease.

[0124] The above description of the present invention is illustrative, and a person with ordinary skill in the art to which the present invention pertains should understand that it can be easily modified into other specific forms without altering the technical idea or essential features of the present invention. Therefore, the above-described embodiments should be understood to be illustrative in all respects and not limiting.

Claims

1. (a) A step of producing decellularized cardiac tissue by decellularizing the isolated cardiac tissue, (b) A step of producing a decellularized cardiac tissue-derived extracellular matrix (Heart Extracellular Matrix, HEM) by drying the decellularized cardiac tissue, (c) The step of adjusting the amount of the decellularized cardiac tissue-derived extracellular matrix to be 0.01 to 2 mg / mL, The aforementioned heart tissue is of pig origin, Step (a) involves treating the separated cardiac tissue with 0.1-2% SDS (sodium dodecyl sulfate) for 12-36 hours under conditions of 1-10°C, and then decellularizing it by stirring with a decellularization solution. The method for producing a culture support composition, wherein the decellularization solution contains 0.1 to 5% Triton X-100 and 0.01 to 0.5% ammonium hydroxide.

2. The method for producing a culture support composition according to claim 1, wherein the decellularization in step (a) is performed in which 95 to 99.9% of cardiac tissue cells are removed.

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