Decellularized spinal cord tissue-derived extracellular matrix and composition containing the same

Abstract

The present invention relates to a decellularized spinal cord tissue-derived extracellular matrix and a composition containing the same. The decellularized spinal cord tissue-derived extracellular matrix of the present invention not only enables the formation of a stable hydrogel but also includes active ingredients derived from spinal cord tissue, allowing for efficient and long-term culture of spinal cord organoids, and has advantages such as low immunogenicity and the potential to induce inflammation.

Description

Technology Field

[0001] This application was filed as a result of the research project [Private / Samsung Future Technology Development Project (Center)] Artificial brain incubator for evaluating the efficacy of Alzheimer's cell therapy (2020-11-1155 / SRFC-TC2003-03 / 2020-09-01 ~ 2023-08-31).

[0002] The present invention relates to a decellularized spinal cord tissue-derived extracellular matrix and a composition containing the same. Background Technology

[0004] The spinal cord is a part of the central nervous system located within the spine and is an important nervous system that acts as an intermediary between the brain and the peripheral nerves. Depending on its location, it is divided into the cervical, dorsal, lumbar, and sacral spinal cords. The spinal cord is surrounded by the meninges and is further enclosed on the outside by a structure called the dura mater, with cerebrospinal fluid flowing in the space between them. A cross-section of the spinal cord is divided into gray matter centered around the middle and white matter on the outside; similar to the brain, the gray matter is where the cell bodies of nerve cells are clustered, while the white matter consists of axon branches.

[0005] The spinal cord is a hub where both motor and sensory nerves converge. Motor nerves descending from the brain are responsible for the motor functions of all muscles in the limbs, while sensory nerves ascending from sensory receptors at the extremities are responsible for sensation throughout the entire body. It also contains nerve bundles responsible for certain autonomic functions, such as bladder control and anal sphincter regulation.

[0006] Meanwhile, organoids, which have recently been gaining prominence, are tissue analogs capable of various clinical applications such as new drug screening, drug toxicity assessment, disease modeling, cell therapy, and tissue engineering; they represent a technology experiencing rapid growth worldwide. Because organoids consist of various cells constituting specific human organs and tissues within a three-dimensional structure and can simulate complex interactions between them, they can be applied as a much more accurate in vitro model platform compared to conventional drug evaluation models, such as simple cell line models or animal models.

[0007] Numerous research teams around the world studying these organoids currently use Matrigel as a culture support. However, because Matrigel is derived from components extracted from mouse sarcoma tissue, it is difficult to maintain consistent product quality. Furthermore, it is expensive and poses safety issues, such as the transmission of animal pathogens and viruses, presenting many challenges that must be addressed as an organoid culture system. In particular, as a material derived from cancer tissue, it fails to provide the optimal tissue-specific microenvironment required for culturing organoids of specific tissues. While some research has been conducted on developing polymer-based hydrogels to replace Matrigel, no material capable of replacing it has been reported to date.

[0008] In particular, when the spinal cord is damaged due to spinal-related diseases such as lordosis, scoliosis, spondylolysis, or spinal stenosis, it can cause significant pain and discomfort due to damage to the motor and sensory nerves contained within the spinal cord. Conventional technologies related to the recovery of damaged spinal cords include Korean Registered Patent Publication No. 10-191458 (a pharmaceutical composition for the prevention, improvement, or treatment of brain or spinal nerve-related diseases containing stem cell-conditioned medium powder) and Korean Published Patent Publication No. 10-2008-0049562 (a treatment for nerve damage diseases containing umbilical cord blood-derived mesenchymal stem cells as an active ingredient), but there is a lack of research or content regarding compositions for treating spinal cord-related diseases or injuries using spinal cord organoids or transplantation treatment methods using such organoids.

[0009] Against this backdrop, the inventors completed the present invention by confirming that efficient culture of spinal cord organoids is possible using an extracellular matrix derived from decellularized spinal cord tissue. The problem to be solved

[0011] The present invention aims to provide a hydrogel composition comprising a decellularized spinal cord tissue-derived extracellular matrix (Spinal cord Extracellular Matrix, ScEM).

[0012] In addition, the present invention aims to provide a spinal cord organoid cultured in three dimensions with the above hydrogel composition.

[0013] In addition, the present invention aims to provide a method for producing the above-mentioned decellularized spinal cord tissue-derived extracellular matrix. means of solving the problem

[0015] One aspect of the present invention relates to a hydrogel composition comprising a decellularized spinal cord tissue-derived extracellular matrix (Spinal cord Extracellular Matrix, ScEM).

[0016] Another aspect of the present invention relates to a spinal cord organoid cultured three-dimensionally with the hydrogel composition.

[0017] Another aspect of the present invention relates to a method for producing the extracellular matrix derived from decellularized spinal cord tissue. Effects of the invention

[0019] The decellularized spinal cord tissue-derived extracellular matrix of the present invention not only enables the formation of a stable hydrogel but also includes active ingredients derived from spinal cord tissue, allowing for efficient and long-term culture of spinal cord organoids, and has advantages such as low immunogenicity and the potential to induce inflammation. Brief explanation of the drawing

[0021] Figure 1 relates to the process of preparing a decellularized spinal cord tissue-derived extracellular matrix (Spinal cord Extracellular Matrix, ScEM) for spinal cord organoid culture. Figures 2 to 4 relate to the component analysis and hydrogel formation of ScEM. Figure 5 shows the results of the analysis of mechanical properties according to the concentration of ScEM. FIGS. 6 to 12 relate to the results of comparative proteomic analysis of ScEM and Matrigel support, etc. Figure 13 shows the results of a comparative analysis of the proteome of ScEM and human spinal cord tissue. Figures 14 and 15 relate to the results of confirming the immunogenicity and biocompatibility of the ScEM hydrogel. Figures 16 and 17 relate to the results of confirming the inflammatory response after in vivo injection of ScEM hydrogel. Figures 18 to 22 relate to the results of selecting the optimal concentration of ScEM hydrogel for spinal cord organoid culture. FIGS. 23 to 25 relate to the results of comparing spinal cord organoids cultured on a ScEM hydrogel with spinal cord organoids cultured on a conventional culture support matrix gel. Figures 26 and 27 relate to the results of analyzing spinal cord-specific marker expression in spinal cord organoids cultured on a ScEM hydrogel compared with spinal cord organoids cultured on a conventional culture support, Matrigel. Figures 28 and 29 relate to the results of comparing and evaluating the spinal cord organoid culture suitability of the ScEM hydrogel with that of spinal cord organoids cultured on a conventional culture support, Matrigel. FIGS. 30 to 32 relate to the results of confirming the long-term culture potential of spinal cord organoids on a ScEM hydrogel compared with spinal cord organoids cultured on a conventional culture support matrix gel. Figures 33 and 34 relate to the results confirming the spinal cord tissue-specific effects of ScEM hydrogel. Figures 35 and 36 relate to the results confirming the long-term storage potential of ScEM hydrogels. Specific details for implementing the invention

[0022] This is explained in detail as follows. Meanwhile, each description and embodiment disclosed in the present invention may be applied to each other description and embodiment. That is, all combinations of the various elements disclosed in the present invention fall within the scope of the present invention. Furthermore, the scope of the present invention should not be considered limited by the specific descriptions provided below.

[0024] One aspect of the present invention provides a decellularized spinal cord tissue-derived extracellular matrix (Spinal cord Extracellular Matrix, ScEM).

[0025] In the present invention, the decellularized spinal cord tissue-derived extracellular matrix may contain a DNA component at a concentration of 1 to 10 ng / mg; a glycosaminoglycan (GAG) at a concentration of 1 to 50, 10 to 40, 15 to 30, 20 to 25, or 20 to 21 μg / mg; and / or a collagen component at a concentration of 1 to 50, 10 to 40, 15 to 30, 20 to 30, 25 to 30, or 25 to 27 μg / mg.

[0026] Another aspect of the present invention provides a hydrogel composition for three-dimensional culture of spinal cord organoids comprising an extracellular matrix derived from decellularized spinal cord tissue.

[0027] In addition, as another aspect of the present invention, the present invention provides a scaffold support for in vivo organoid transplantation comprising the above-mentioned hydrogel composition for three-dimensional culture.

[0028] In addition, as another aspect of the present invention, the present invention provides a composition for coating the surface of a culture vessel comprising the hydrogel composition for three-dimensional culture.

[0029] The above “extracellular matrix” refers to a natural scaffold for cell growth prepared through the decellularization of tissues found in mammals and multicellular organisms. The above extracellular matrix may be further processed through dialysis or crosslinking.

[0030] The above extracellular matrix may be a mixture of structural and non-structural biomolecules, not limited to collagens, elastins, laminins, glycosaminoglycans, proteoglycans, antimicrobials, chemoattractants, cytokines, and growth factors.

[0031] The term “organoid” above refers to a microscopic biological organ produced in the form of an artificial organ by culturing cells derived from tissues or pluripotent stem cells into a 3D form.

[0032] The above organoid is a three-dimensional tissue analog comprising organ-specific cells that originate from stem cells and self-organize (or self-pattern) in a manner similar to in vivo conditions, and can develop into a specific tissue through the patterning of limited factors (e.g., growth factors).

[0033] The organoid described above possesses the original physiological characteristics of cells and may have an anatomical structure that mimics the original state of a cell mixture (including not only limited cell types but also residual stem cells and proximal physiological niches). Through a three-dimensional culture method, the organoid may have better arrangement of cells and cell functions, and may possess a functional organ-like form and tissue-specific functions.

[0034] The above “hydrogel” is a material in which a liquid with water as a dispersion medium solidifies through a sol-gel phase transition, loses fluidity, and forms a porous structure, and can be formed by a hydrophilic polymer having a three-dimensional network structure and a microcrystalline structure expanding by containing water.

[0036] In the present invention, the hydrogel composition comprising the decellularized spinal cord tissue-derived extracellular matrix may have an elastic modulus (G') higher than the viscosity coefficient (G''), specifically, when the concentration of the decellularized spinal cord tissue-derived extracellular matrix is ​​0.8 to 8 mg / mL, it may have an elastic modulus of 3 to 180 Pa, and more specifically, when the concentration is 0.9 to 1.1 mg / mL, the average elastic modulus may be 25 to 30 Pa. Through this, the spinal cord tissue-derived extracellular matrix of the present invention has appropriate elastic modulus and viscosity coefficients, and is characterized by having physical properties that allow a stable polymer network to be formed inside the hydrogel.

[0037] In the present invention, the extracellular matrix derived from decellularized spinal cord tissue has high diversity of constituent protein components and, specifically, may include collagen, ECM glycoproteins, proteoglycans, ECM regulators, ECM-affiliated proteins, and / or secreted factors. The collagen may be COL6A-1, -2, and / or -3.

[0038] In the present invention, the decellularized spinal cord tissue-derived extracellular matrix may further include a central nervous system (CNS) specific protein (a protein expressed four times or more in CNS tissue compared to other tissues) and may further include a CNS tissue-specific protein expressed 18 times or more compared to Matrigel. Specifically, the CNS tissue-specific protein may be one or more proteins selected from the group consisting of ACAN, BCAN, COL4A5, COL9A2, EMID1, HAPLN2, NCAN, and TNR. In addition, the decellularized spinal cord tissue-derived extracellular matrix may include the Matrisome proteins described in FIG. 9. Thus, ScEM containing various proteins that play important roles and functions in central nervous system tissue can serve as an effective culture support for promoting spinal cord organoid growth and differentiation.

[0039] In the present invention, the decellularized spinal cord tissue-derived extracellular matrix may contain or express a protein related to axon induction, and specifically, as a protein related to axon induction described in Fig. 12C, it may contain or express one or more proteins selected from the group consisting of DPYSL2, CRMP1, DPYSL5, SH3GL2, DNM1, AP2A1, AP2M1, CLTC, TLN1, ACTB, and ARPC1A.

[0040] In the present invention, the hydrogel composition comprising the decellularized spinal cord tissue-derived extracellular matrix has low immunogenicity, low potential for inducing inflammation, and excellent biocompatibility. Specifically, the hydrogel composition may be for in vivo implantation, and when the hydrogel composition or an organoid cultured therefrom is implanted in vivo, the ratio of M2 anti-inflammatory macrophages to M1 inflammatory macrophages around the implantation site may increase, and macrophage polarization to the M2 type may be induced.

[0041] In the present invention, the hydrogel composition may be for spinal cord organoid culture. The hydrogel composition may be mixed with human induced pluripotent stem cells to produce a spinal cord organoid, and the hydrogel composition may be applied at a concentration of 0.8 to 7 mg / ml, and more specifically, at a concentration of 0.8 to 3 or 1 mg / ml.

[0042] In the present invention, the hydrogel composition can be stored for a long period, specifically refrigerated or frozen for up to 5 months or 150 days.

[0044] As another aspect of the present invention, a spinal cord organoid cultured three-dimensionally with the hydrogel composition is provided.

[0045] In the present invention, the spinal cord organoid may express one or more genes and / or proteins selected from the group consisting of Nestin, Tuj1, MAP2, Sox2, Nkx6.1, CHAT, PAX6, and OLIG2, and may express them at a level of 80 to 120% compared to a spinal cord organoid differentiated from Matrigel.

[0046] In addition, the spinal cord organoid may have reduced expression of a marker for apoptosis (Caspase-3), and may have reduced expression to 50 to 80% of the level of a spinal cord organoid differentiated from Matrigel. The spinal cord organoid may have expression of a marker related to apoptosis within 1 to 5%.

[0047] In addition, the spinal cord organoid may have enhanced expression of the cell proliferation marker (Ki67), and may have improved expression to a level of 105 to 120% compared to the spinal cord organoid differentiated from Matrigel.

[0048] In the present invention, the spinal cord organoid may be capable of being cultured for 25 to 30 days, and when cultured for 25 to 30 days, the expression of neuronal marker (MAP2), spinal cord ventral motor neuron marker (ISL1), and spinal cord organoid-specific precursor marker (Nkx 6.1) may be at a level of 90 to 200% compared to spinal cord organoids differentiated from Matrigel.

[0050] In another aspect of the present invention, a method for preparing an extracellular matrix derived from decellularized spinal cord tissue is provided, comprising the steps of: separating the spinal cord from the vertebrae to remove the dura mater and pia mater; cutting the spinal cord into pieces of 0.8 to 1.2 cm and storing them for 10 to 14 hours in a solution containing 0.8 to 1.2% Triton X-100 and 0.05 to 0.15% ammonium hydroxide; storing them for 22 to 26 hours in a solution containing 3 to 5% sodium deoxycholate; and treating with DNase I for 2 to 4 hours.

[0051] The process in the present invention has the advantage of significantly reducing tissue damage by using only a solution mixed with 1% Triton X-100 and 0.1% ammonium hydroxide instead of the conventional 3% Triton X-100, while at the same time, by adding 2000 kU Dnase, cells can be completely removed and various proteins within the spinal cord tissue can be preserved more.

[0052] In addition, as another aspect of the present invention, a method for producing a spinal cord organoid is provided, comprising the step of three-dimensionally culturing a hydrogel composition containing the decellularized spinal cord tissue-derived extracellular matrix.

[0054] Another aspect of the present invention provides a composition for implantation into spinal cord tissue comprising the hydrogel composition; and a pharmaceutical composition for the prevention or treatment of spinal cord-related diseases or spinal cord injury.

[0055] Another aspect of the present invention provides a method for transplanting the spinal cord organoid to an individual with spinal cord injury or an individual having a spinal cord-related disease.

[0056] Another aspect of the present invention provides a method for improving or treating spinal cord injury in a spinal cord injury subject by implanting the spinal cord organoid.

[0057] Another aspect of the present invention provides a method for preventing or treating a spinal cord-related disease in an individual having a spinal cord-related disease by implanting the spinal cord organoid.

[0058] Another aspect of the present invention provides the use of the spinal cord organoid for the prevention or treatment of spinal cord-related diseases.

[0059] Another aspect of the present invention provides a use for manufacturing a drug for the prevention or treatment of spinal cord-related diseases of the spinal cord organoid.

[0060] In the present invention, the spinal cord-related disease may be a cerebral infarction, stroke, inflammatory brain disease, intracranial injury, traumatic brain injury, Alzheimer's disease, Parkinson's disease, depression, epilepsy, multiple sclerosis, proximal spinal cord injury, spinal stenosis, spinal injury, neuromyelitis optica spectrum disorder (NMOSD), amyotrophic lateral sclerosis (ALS), or mania.

[0061] In the present invention, an individual refers to an animal including humans.

[0062] Spinal cord organoids can be cultured and produced using the hydrogel composition for three-dimensional culture of spinal cord organoids containing the decellularized spinal cord tissue-derived extracellular matrix of the present invention, and such spinal cord organoids have high similarity to spinal cord tissue in vivo in various aspects, so they can be utilized for the aforementioned purposes.

[0064] The above composition of the present invention can be administered in a pharmaceutically effective amount.

[0065] In the present invention, the term "pharmaceuticalally effective amount" means an amount sufficient to treat a disease with a reasonable benefit / risk ratio applicable to medical treatment, and the effective dose level may be determined based on factors including individual type and severity, age, sex, type of disease, drug activity, sensitivity to the drug, time of administration, route of administration and elimination rate, duration of treatment, concurrently used drugs, and other factors well known in the medical field. The preferred dosage of the composition of the present invention depends on the patient's condition and weight, degree of disease, drug form, route of administration, and duration, but for a desirable effect, the pharmaceutical composition of the present invention may be administered at a dose of 0.0001 to 100 mg / kg per day. Administration may be performed once a day or divided into several doses. The method of administration includes, without limitation, any method conventional in the art, and may be administered, for example, orally, rectally or intravenously, intramuscularly, or by subcutaneous injection.

[0066] The pharmaceutical composition of the present invention may be prepared in a unit volume form or contained in a multi-volume container by formulating it using a pharmaceutically acceptable carrier and / or excipient according to a method that can be easily carried out by a person skilled in the art to which the invention belongs.

[0067] In the present invention, the term "prevention and improvement" refers to any act of inhibiting the progression of spinal cord-related diseases or delaying their onset by administering or implanting the above composition, and the term "treatment" refers to any act of improving or beneficially altering the symptoms of spinal cord-related diseases by administering or implanting the above composition.

[0070] The structure and effects of the present invention will be explained in more detail below through examples. These examples are solely for the purpose of illustrating the present invention, and the scope of the present invention is not limited by these examples.

[0072] Example 1: Preparation of decellularized spinal cord tissue-derived extracellular matrix (Spinal cord Extracellular Matrix, ScEM) for spinal cord organoid culture

[0074] To prepare a spinal cord tissue-derived extracellular matrix (ScEM) to be used as a spinal cord organoid culture scaffold, a decellularization process was performed on porcine spinal cords. The spinal cord was isolated from porcine vertebrae, and the dura mater and pia mater were removed. The trimmed spinal cord was cut into approximately 1 cm lengths and subjected to a decellularization process involving treatment in a mixed solution of 1% Triton X-100 and 0.1% ammonium hydroxide for 12 hours, in a 4% sodium deoxycholate solution for 24 hours, and in DNase I for 3 hours to produce the ScEM, which was then freeze-dried and stored (Fig. 1).

[0076] Example 2: Component analysis of ScEM and hydrogel formation

[0078] To confirm whether the decellularization process was successfully carried out, the amount of residual dsDNA was measured (Fig. 2).

[0079] As a result, 3.15 ng / mg of dsDNA was detected in the decellularized spinal cord tissue-derived extracellular matrix (ScEM), which was confirmed to be lower than the clinically applicable standard of 50 ng / mg in decellularized tissue. Furthermore, it was confirmed that the decellularization process was successful, with 99.38% of DNA components removed compared to the original spinal cord tissue (513.48 ng / mg). Thus, the decellularized spinal cord tissue-derived extracellular matrix can contain DNA components at a level of 1 to 10 ng / mg.

[0080] Additionally, quantitative analysis was performed to confirm whether major extracellular matrix components, namely glycosaminoglycans (GAGs) and collagen, were well preserved even after the decellularization process. As a result, while 18.12 μg / mg and 16.11 μg / mg, respectively, were detected in the original spinal cord tissue, it was confirmed that GAGs and collagen were present at 20.19 μg / mg and 26.10 μg / mg, respectively, in the ScEM. Consequently, it was confirmed that the concentrations of the two extracellular matrix components were measured to be higher in the ScEM because all cellular components were removed during the decellularization process, leaving only extracellular matrix components. This indicates that these extracellular matrix components were well preserved without damage during the decellularization process.

[0082] Next, hematoxylin and eosin (H&E) staining was performed to confirm that all cellular components were removed from the prepared decellularized spinal cord tissue scaffold (ScEM), and Masson's trichrome staining was performed to confirm that collagen components were well preserved and maintained even after the decellularization process. Additionally, toluidine blue staining confirmed that GAG components were also well preserved in the ScEM (Fig. 3).

[0083] In addition, immunohistochemical staining was performed to confirm the preservation of major extracellular matrix components such as fibronectin and laminin within the ScEM (Fig. 3).

[0084] Through these results, it can be seen that cellular components within ScEM are effectively all removed through the spinal cord tissue decellularization process of the present invention, while major extracellular matrix components are well preserved.

[0086] Meanwhile, lyophilized decellularized spinal cord tissue-derived extracellular matrix (ScEM) was dissolved by treating it with a mixed solution of 4 mg / ml pepsin and 0.02 M hydrochloric acid for 48 hours at 240 rpm. Subsequently, salinity and pH were adjusted using 10X PBS and 0.5 M sodium hydroxide, and ScEM hydrogel formation was induced by crosslinking at 37°C for 30 minutes (Fig. 4). The ScEM hydrogel thus prepared was applied as a spinal cord organoid culture support.

[0087] Field Emission Scanning Electron Microscope (FE-SEM) analysis was performed to verify whether a three-dimensional microenvironment could be provided. As a result, it was confirmed that the decellularized spinal cord tissue-derived hydrogel scaffold (ScEM) had a porous internal structure in the form of nanofiber bundles and could provide a three-dimensional microenvironment suitable for the culture of spinal cord organoids (Fig. 4).

[0089] Example 3: Analysis of physical properties according to ScEM concentration

[0091] To analyze the changes in physical properties according to the concentration of ScEM, hydrogels were prepared at different concentrations of ScEM (0.8, 1, 3, 5, 7 mg / ml), and mechanical properties were measured through rheological analysis. It was confirmed that the storage modulus (G') was higher than the loss modulus (G'') under all five concentration conditions, and that polymer networks were stably formed within the hydrogels at all concentrations (Fig. 5).

[0092] In addition, it was confirmed that the average elastic modulus also increased as the ScEM concentration increased. The average elastic modulus was measured to be 3.81 Pa, 28.57 Pa, 41.56 Pa, 118.50 Pa, and 173.50 Pa at 0.8 mg / ml, 1 mg / ml, 3 mg / ml, 5 mg / ml, and 7 mg / ml, respectively. Meanwhile, the average elastic modulus of Matrigel was 140.93 Pa, which was found to be similar to that of the 5 mg / ml ScEM hydrogel (Fig. 5).

[0094] Example 4: Comparative proteomic analysis of ScEM and Matrigel supports

[0096] To analyze the proteome of decellularized spinal cord tissue-derived extracellular matrix (ScEM), high-resolution liquid chromatography tandem mass spectrometry (LC-MS / MS) was used to detect matrisome proteins, which are extracellular matrix proteins. In addition, the proteome of Matrigel (MAT), a commonly used organoid culture support, was also analyzed and compared with the matrisome proteins detected in ScEM.

[0097] First, quantitative analysis was performed to compare the protein components of ScEM and Matrigel (MAT). It was confirmed that ScEM consists of 21.51% matrisome proteins and 78.49% non-matrisome proteins, whereas Matrigel, derived from mouse sarcoma, consists of 80.00% matrisome proteins and 20.00% non-matrisome proteins, indicating that the overall protein composition of the two scaffolds is different (Fig. 6A).

[0098] As a result of comparing the composition of matrisome proteins in ScEM and Matrigel by type, it was confirmed that while ScEM is composed of more than 50% collagen, it is also made up of various types of matrisome proteins, including ECM glycoproteins, proteoglycans, ECM regulators, ECM-affiliated proteins, and secreted factors. However, Matrigel is composed almost entirely of ECM glycoproteins, indicating that it has lower diversity of constituent protein components compared to ScEM (Fig. 6B). Therefore, it can be seen that a ScEM support composed of more diverse protein components would serve as a more suitable support for organoid culture than a Matrigel composed mainly of ECM glycoproteins.

[0099] When comparing the 10 most expressed matrisome proteins in ScEM and Matrigel, as mentioned above, 4 out of the 10 in ScEM consist of collagen, and among them, the most expressed components COL6A-1, -2, and -3 play an important role in protecting neurons. On the other hand, it was confirmed that Matrigel is composed of Lamc1, Lamb1, and Lama1, which are representative ECM glycoproteins (Fig. 7C).

[0101] Next, a comparative analysis of the matrisome proteins in decellularized spinal cord tissue-derived extracellular matrix (ScEM) and Matrigel (MAT) revealed that ScEM consisted of an average of 101 matrisome proteins, while Matrigel consisted of an average of 41 matrisome proteins, confirming that ScEM contains a greater variety of matrisome proteins (Fig. 8A).

[0102] As a result of comparing the number of each type of matrisome protein contained in ScEM and Matrigel, an average of 23 and 3 collagens were detected, respectively; 46 and 21 ECM glycoproteins were detected, respectively; 14 and 1 proteoglycan were detected, respectively; 8.3 and 13 ECM regulators were detected, respectively; 6 and 2 ECM-affiliated proteins were detected, respectively; and finally, 3.6 and 1 secreted factor were detected, respectively. It was confirmed that ScEM contained a greater variety of proteins in all matrisome types, except for ECM regulators (Fig. 8B).

[0103] In addition, central nervous system (CNS) specific proteins (referring to proteins expressed four times more than in other tissues) in ScEM and Matrigel were compared. As a result, ScEM contained an average of 117 CNS specific proteins, whereas Matrigel contained an average of 6.3 proteins; therefore, ScEM contained approximately 18 times more CNS tissue-specific proteins compared to Matrigel and was predicted to have a better effect on spinal cord organoid culture and differentiation (Fig. 8C).

[0105] It was confirmed that the decellularized spinal cord tissue-derived extracellular matrix (ScEM) contained central nervous system-specific proteins (referring to proteins expressed four times more than in other tissues) such as ACAN, BCAN, COL4A5, COL9A2, EMID1, HAPLN2, NCAN, and TNR among the matrisome proteins. On the other hand, central nervous system-specific proteins were not detected in the Matrigel (MAT) matrisome proteins (Fig. 9).

[0106] Meanwhile, TNC and TNR, ECM glycoprotein components contained in ScEM, are known to be involved in regulating neurite growth and axon function, while LAMA1 and LAMC1 are known to play a role in promoting neurite growth and neuronal differentiation. Additionally, ACAN, BCAN, NCAN, and VCAN, which are major proteoglycans of the central nervous system, play important roles in regulating neural stem cell differentiation and synaptic maturation and stabilization, and LGALS1 and LGALS3 are neuroprotective proteins that regulate inflammation in the central nervous system or remodel damaged tissues.

[0107] Therefore, it can be seen that ScEM containing various proteins that play important roles and functions in central nervous system tissues will serve as an effective culture support for promoting spinal cord organoid growth and differentiation.

[0109] Next, the total proteins constituting the decellularized spinal cord tissue-derived extracellular matrix (ScEM) and Matrigel were compared. As a result, it was confirmed that 143 proteins were commonly expressed when comparing the total proteins. (Fig. 10A)

[0110] Here, when comparing the expression levels of 143 proteins commonly expressed in ScEM and Matrigel, it was confirmed that 83 of the 143 proteins showed different expression patterns. Among them, 32 proteins were overexpressed in ScEM and 51 proteins were expressed at low levels in ScEM, as confirmed through a volcano plot (Fig. 10B) and heatmap analysis (Fig. 10C).

[0111] Through this, it can be seen that ScEM contains protein components that are commonly expressed as Matrigel, a conventional organoid culture support, but differs significantly in protein content and expression patterns, thus showing superior effects in spinal cord organoid culture.

[0113] Next, the expression patterns of proteins related to neuron development (Fig. 11A), axon development (Fig. 11B), and central nervous system development (Fig. 11C) were compared in decellularized spinal cord tissue-derived extracellular matrix (ScEM) and Matrigel (MAT).

[0114] As a result, it was confirmed that proteins related to the three biological processes were hardly expressed in Matrigel, whereas both the number and amount of expressed proteins were higher in ScEM, suggesting that ScEM culture supports may be more effective than Matrigel supports for spinal cord organoid formation and development.

[0116] Next, Gene Ontology (GO) analysis was performed on central nervous system-specific proteins (referring to proteins expressed at a level four times higher in CNS tissue compared to other tissues) expressed in decellularized spinal cord tissue-derived extracellular matrix (ScEM). As a result, it was confirmed that proteins expressed in ScEM play roles related to biological processes associated with nervous system development, such as neuronal development, projection, formation, and differentiation, and also play roles related to axonal development. Therefore, it was expected that the ScEM scaffold would have a significant effect on neuronal and axonal development in spinal cord organoids during culture (Fig. 12A).

[0117] In addition, Gene Ontology (GO) analysis was performed on the metrisome proteins included in ScEM, and they were identified as proteins related to biological processes such as extracellular matrix structure and composition, cell adhesion, collagen structure, and tissue development; thus, it was confirmed that the ScEM scaffold is composed of proteins that can play an important role in spinal cord organoid culture (Fig. 12B).

[0118] In addition, after analyzing the inter-protein interactions of all proteins expressed in ScEM using the STRING database, reactome pathway analysis was performed to investigate the involved pathways, and a pathway involved in axon induction was identified. Based on this, it was expected that ScEM would effectively contribute to axon formation in spinal cord organoids (Fig. 12C).

[0119] Through these results, it can be seen that ScEM can be utilized as a highly effective scaffold for spinal cord organoid formation by serving as a support that induces structure formation during organoid culture, while simultaneously promoting the development and function of nerves and axons.

[0121] Example 5: Comparative analysis of proteomes between ScEM and human spinal cord tissue

[0123] Proteomic comparative analysis was performed to evaluate the similarity of decellularized spinal cord tissue-derived extracellular matrix (ScEM) to actual human spinal cord tissue. As a result, it was confirmed that ScEM is composed of various types of metrisome proteins, such as collagen, ECM glycoproteins, proteoglycans, ECM regulators, ECM-affiliated proteins, and secreted factors, just like actual human spinal cord tissue (Fig. 13A).

[0124] Furthermore, when comparing the metrisome proteins of ScEM with those of actual human spinal cord tissue, it was found that the majority of the metrisomes (93 out of 111) in the decellularized spinal cord tissue-derived scaffold (ScEM) consisted of metrisome proteins commonly expressed in human spinal cord tissue. Additionally, when comparing ScEM with actual human spinal cord tissue for central nervous system (CNS) specific proteins (referring to proteins expressed four times more frequently in CNS tissue compared to other tissues), it was confirmed that 123 out of 127 proteins were commonly expressed in human spinal cord tissue (Fig. 13B).

[0125] Principal component analysis was performed on all proteins expressed in ScEM, actual human spinal cord tissue, and Matrigel, and it was confirmed that ScEM has high similarity to human spinal cord tissue, and that the protein components and expression differ when compared to Matrigel (Fig. 13C).

[0126] Through this, it can be seen that ScEM contains extracellular matrix protein components that are very similar to spinal cord tissue in vivo, and that it will contribute to better replicating the microenvironment within actual spinal cord tissue in spinal cord organoid culture.

[0128] Example 6: Confirmation of Immunogenicity and Biocompatibility of ScEM Hydrogel

[0130] To confirm the immunogenicity of decellularized spinal cord tissue-derived extracellular matrix (ScEM) hydrogel, macrophages were co-cultured with 5 mg / ml ScEM hydrogel, and the secretion of the pro-inflammatory cytokines tumor necrosis factor alpha (TNF-α) and Interleukin 6 (IL-6) from the macrophages was confirmed. To determine the secretion amounts of TNF-α and IL-6 from the macrophages, the culture medium was obtained after co-culture with the ScEM hydrogel for 6 hours and 12 hours, respectively, and an enzyme-linked immunosorbent assay (ELISA) was performed to quantify the secretion amounts of each cytokine.

[0131] In the case of TNF-α cytokine, 30.33 pg / ml was detected in the negative control (No treatment) and 31.12 pg / ml in the macrophage group co-cultured with ScEM hydrogel, confirming that there was almost no difference between the two groups. On the other hand, the positive control was the macrophage group treated with 1 μg / ml lipopolysaccharides (LPS), an immune-inducing substance, with a secretion of 17,749.94 pg / ml detected; thus, it was confirmed that there was a significant difference in the secretion amount of macrophages co-cultured with ScEM hydrogel (Fig. 14A).

[0132] IL-6 cytokine was detected at 2.44 pg / ml in the negative control (No treatment) and 4.92 pg / ml in the macrophage group co-cultured with ScEM hydrogel, confirming that there was almost no difference in cytokine secretion between the two groups. On the other hand, 1929.30 pg / ml of secretion was detected in the positive control group of macrophages treated with 1 μg / mg lipopolysaccharides (LPS), confirming a significant difference from the secretion of macrophages co-cultured with ScEM hydrogel (Fig. 14B).

[0133] Through the above results, it was confirmed that the ScEM hydrogel has very low immunogenicity and will not induce an immune response when used for implantation in the body, and is a material with excellent biocompatibility.

[0135] Next, to evaluate the biocompatibility of the ScEM hydrogel after in vivo injection, 200 μl of 5 mg / ml ScEM hydrogel was subcutaneously injected into the dorsal area of ​​mice. Samples were collected after 1, 4, and 7 days and subjected to histological analysis using H&E staining and Toluidine blue (TB) staining. As a result, H&E staining confirmed that the ScEM hydrogel adhered well to the tissue on days 1, 4, and 7 of injection, and no tissue damage, such as necrosis, was observed.

[0136] In addition, TB staining was performed to analyze whether mast cells, which are immune cells, were observed around the injected ScEM hydrogel. As a result, no mast cells were observed around the ScEM hydrogel on days 1, 4, and 7. Therefore, it was confirmed that the ScEM hydrogel does not cause an immune response after injection into the body and has excellent biocompatibility (Fig. 15).

[0138] Example 7: Confirmation of inflammatory response after in vivo injection of ScEM hydrogel

[0140] To determine whether decellularized spinal cord tissue-derived extracellular matrix (ScEM) hydrogel induces an inflammatory response upon in vivo injection, 200 μl of 5 mg / ml ScEM hydrogel was injected subcutaneously into the dorsal region of mice. Skin tissues were collected on days 1, 4, and 7, and macrophage staining was performed using immunohistochemical staining. F4 / 80 is a marker that stains all general types of macrophages, while CD206 stains M2 macrophages, which exhibit anti-inflammatory traits.

[0141] As a result, on day 1 of ScEM hydrogel transplantation, inflammatory M1 macrophages (F4 / 80+CD206-) increased at the site where the hydrogel was injected due to injection and material transplantation, but it was confirmed that they gradually decreased on days 4 and 7 after injection. On the other hand, the expression of anti-inflammatory M2 macrophages (F4 / 80+CD206+) was low on day 1 but increased on days 4 and 7, confirming that the proportion of anti-inflammatory M2 macrophages gradually increased (Fig. 16A and Fig. 17B).

[0142] In addition, the M1 macrophage / M2 macrophage ratio increased compared to normal tissue on day 1 immediately after injection, and many inflammatory macrophages were observed, but as the ratio between the two cell types progressed to days 4 and 7, it was found that the macrophage type around the ScEM hydrogel changed from inflammatory to anti-inflammatory (Fig. 17C).

[0143] These results indicate that ScEM hydrogel is highly unlikely to cause severe inflammatory responses in the body and, furthermore, can induce macrophage polarization into the M2 type, which aids in tissue regeneration.

[0145] Example 8: Selection of Optimal Concentration of ScEM Hydrogel for Spinal Cord Organoid Culture

[0147] Spinal cord organoids were prepared through the differentiation of human induced pluripotent stem cells (hiPSCs), and 10-day-old spinal cord organoids that had completed initial formation were placed in a ScEM hydrogel for three-dimensional culture. To select optimal culture conditions, ScEM hydrogels with various concentrations were prepared and applied to the spinal cord organoid culture. Matrigel was used as a scaffold for a positive control for comparison.

[0148] As a result, when decellularized spinal cord tissue-derived ScEM hydrogel was applied for human spinal cord organoid culture under various concentration conditions, it was confirmed that spinal cord organoids similar in form to those in the control Matrigel (MAT) were formed at all concentration conditions (0.8, 1, 3, 5, 7 mg / ml) (Fig. 18A).

[0149] In addition, when culture was performed on ScEM hydrogels of various concentrations, it was confirmed that there was no significant difference in the rate of increase in spinal cord organoid area at days 8 and 15 after encapsulation compared to Matrigel under all concentration conditions (Fig. 19 B).

[0150] Therefore, it was confirmed that ScEM hydrogel can induce the development of human spinal cord organoids to a level similar to Matrigel.

[0152] Next, decellularized spinal cord tissue-derived ScEM hydrogel scaffolds were prepared at various concentrations and applied to the culture of human induced pluripotent stem cell-derived spinal cord organoids. Gene expression in spinal cord organoids cultured in three dimensions for 8 days under each concentration condition was compared using quantitative PCR (qPCR). A commercially available culture scaffold, Matrigel (MAT), was used as a positive control.

[0153] As a result, when gene expression was compared according to ScEM concentration conditions, the gene (Nkx6.1) associated with spinal cord organoid-specific precursor cells showed a tendency to decrease slightly at concentrations of 5 mg / ml and 7 mg / ml compared to the control group (MAT), while the expression of MAP2, a marker related to neuronal differentiation, showed a similar expression pattern to the MAT group except for the 5 mg / ml and 7 mg / ml concentration conditions. In addition, ISL1, a marker specific to motor neurons expressed in the ventral side of the spinal cord, showed expression levels similar to the MAT group at 0.8 and 1 mg / ml conditions, whereas expression tended to decrease as the concentration increased; and in the case of BAX, a marker related to cell necrosis, there was no significant difference compared to the control group (MAT), but a slight increase was observed at high concentrations (Fig. 20).

[0154] Through these results, it was confirmed that ScEM hydrogel may not be relatively effective for the differentiation and development of spinal cord organoids under high concentration conditions of 5 mg / ml or higher, and that the expression of neuronal differentiation markers was highest under concentration conditions of 0.8 to 3, or 1 mg / ml.

[0156] Next, immunohistochemistry was performed on spinal cord organoids cultured on decellularized spinal cord tissue-derived ScEM hydrogels and control Matrigel. Spinal cord organoids were prepared from human induced pluripotent stem cells and, on day 10 of culture, placed in ScEM hydrogel scaffolds of different concentrations and control Matrigel for an additional 8 days of three-dimensional culture.

[0157] Comparative analysis of the expression of neuron-specific markers (Tuj1), neuron precursor markers (Sox2), spinal motor neuron precursor markers (Nkx6.1), and choline receptor neuron-specific markers (CHAT) in spinal cord organoids cultured in decellularized spinal cord tissue-derived ScEM hydrogels and control Matrigel at various concentrations revealed that while the expression of spinal cord-related markers was observed under all concentration conditions, it was found that the expression decreased slightly under high concentration conditions of 5 mg / ml and 7 mg / ml (Fig. 21).

[0158] In addition, the expression ratio of major motor neuron progenitor cells located in the ventral spinal cord was compared for each group using images obtained through immunohistochemistry. It was confirmed that compared to the control group Matrigel, expression decreased slightly under 5 mg / ml and 7 mg / ml conditions, but showed a similar expression pattern to the Matrigel group under other concentration conditions (Fig. 22).

[0159] In summary, among the ScEM hydrogel concentration conditions tested, the ScEM hydrogel group at a concentration of 1 mg / ml showed no significant difference in the rate of increase in spinal cord organoid area compared to the Matrigel group, similar expression of spinal cord and neuronal-specific markers was observed, apoptosis was at a low level, and the expression pattern of spinal cord-specific progenitor cells also showed no significant difference. Therefore, the optimal ScEM hydrogel concentration was determined to be 1 mg / ml and subsequently applied to spinal cord organoid culture.

[0161] Example 9: Analysis of spinal cord organoids cultured in ScEM hydrogel

[0163] Spinal cord organoids were cultured three-dimensionally for 14 days on a decellularized spinal cord tissue-derived ScEM hydrogel (1 mg / ml) scaffold and compared with spinal cord organoids cultured on Matrigel (MAT), which is the most widely used organoid culture medium.

[0164] When comparing the morphological images of spinal cord organoids cultured in the control group Matrigel and ScEM hydrogel for 2, 10, and 16 days, it was observed that some interfaces became smoother compared to the Matrigel group (Fig. 23A).

[0165] Next, when the change in spinal cord organoid area was graphed after culturing in the control Matrigel and ScEM hydrogel for 2, 10, and 16 days, the spinal cord organoid cultured in the ScEM hydrogel showed a slightly smaller area but overall similar rate of area increase (Fig. 24 B).

[0166] In addition, quantitative PCR (qPCR) analysis was performed after culturing for 15 days (Day 25) in decellularized spinal cord tissue-derived ScEM hydrogel and a control Matrigel. Among the neuronal differentiation markers (Nestin, Tuj1, MAP2), Nestin, an early neuronal differentiation marker, was slightly reduced compared to the Matrigel group, while mature neuronal markers (Tuj1, MAP2) showed similar expression patterns. Furthermore, no significant difference was found between the two groups for spinal cord organoid differentiation-related markers (PAX6, OLIG2, Nkx6.1) (Top of Fig. 25). Additionally, no significant difference was observed between the two groups in the expression of the ventral motor neuron marker (ISL1) and the cholinergic neuron marker (CHAT), which represents major neuronal types in the central nervous system that utilize acetylcholine as a neurotransmitter (Bottom of Fig. 25).

[0167] Through the above results, it was confirmed that ScEM hydrogel has substantially equivalent performance to Matrigel in spinal cord organoid culture.

[0169] Nestin, Tuj1, MAP2: neuronal markers

[0170] PAX6: dorsal spinal cord progenitor

[0171] Olig2: hindbrain & spinal cord progenitor

[0172] Nkx6.1: ventral spinal cord progenitor

[0174] Example 10: Analysis of Spinal Cord-Specific Marker Expression in Spinal Cord Organoids Cultured on ScEM Hydrogel

[0176] After inducing the formation of early spinal cord organoids from human-derived induced pluripotent stem cells, on day 10 of culture, immunostaining was performed on spinal cord organoids cultured for 15 days in decellularized spinal cord tissue-derived ScEM hydrogel (1 mg / ml) and a control Matrigel.

[0177] As a result, immunohistochemistry confirmed that the expression of neuronal (Tuj1), neural progenitor cell (Sox2), and spinal cord-associated progenitor cell (Olig2, Nkx6.1) markers in spinal cord organoids cultured in ScEM hydrogel was at a similar level compared to the control Matrigel group (Fig. 26).

[0178] In addition, quantitative analysis of images obtained by immunohistochemistry compared the expression of neural progenitor cells (Sox2), hindbrain and spinal cord progenitor cells (Olig2), and ventral spinal cord progenitor cells (Nkx6.1), and confirmed that spinal cord organoids cultured in ScEM hydrogel contained these cells at levels similar to those of the control Matrigel group (Fig. 27).

[0180] Example 11: Evaluation of ScEM Hydrogel's Suitability for Spinal Cord Organoid Culture

[0182] To evaluate the suitability of spinal cord organoid culture in decellularized spinal cord tissue-derived ScEM hydrogels, a comparative analysis was performed with spinal cord organoids cultured in a control Matrigel through immunohistochemistry of markers related to apoptosis and proliferation.

[0183] When the expression of markers for apoptosis (Caspase-3) and cell proliferation (Ki67) was compared by immunohistochemistry of spinal cord organoids cultured for 15 days on ScEM (1 mg / ml) hydrogel and Matrigel, apoptosis was detected at negligible levels in both scaffold groups, whereas the expression of markers for cell proliferation was observed at high levels (Fig. 28).

[0184] In addition, the expression of markers related to apoptosis in spinal cord organoids cultured on each culture support was low at around 5%, and there was no significant difference between the two groups, and in the case of Ki67, a marker related to cell proliferation, similar expression levels were observed in both groups (Fig. 29).

[0185] As a result, since ScEM hydrogel can maintain the survival of spinal cord organoids at a level similar to that of the positive control Matrigel, it can be seen that it has the role and function as a suitable scaffold for spinal cord organoid culture.

[0187] Example 12: Confirmation of Long-term Culture Potential of Spinal Cord Organoids on ScEM Hydrogel

[0189] After forming initial spinal cord organoids from human induced pluripotent stem cells, on the 10th day of culture, the organoids were placed in a decellularized spinal cord tissue-derived ScEM hydrogel (1 mg / ml) and a control Matrigel, and the expression of markers related to differentiation and development in spinal cord organoids cultured for 8 days (Day 18) and 27 days (Day 37) was compared and analyzed.

[0190] First, when comparing the morphology of spinal cord organoids cultured for 8 and 27 days in decellularized spinal cord tissue-derived ScEM hydrogel (1 mg / ml) and a control Matrigel, it was confirmed that the spinal cord organoids cultured in the ScEM hydrogel were cultured to a level similar in shape and size to the spinal cord organoids cultured in the Matrigel (Fig. 30).

[0191] In addition, quantitative qPCR analysis of spinal cord organoids cultured for 8 and 27 days in decellularized spinal cord tissue-derived ScEM hydrogel (1 mg / ml) and control Matrigel confirmed that there was no significant difference between the two groups in the expression of neuronal marker (MAP2), spinal cord ventral motor neuron marker (ISL1), and spinal cord organoid-specific precursor marker (Nkx 6.1) (Fig. 31).

[0192] In addition, immunohistochemical analysis of spinal cord organoids cultured for 27 days in decellularized spinal cord tissue-derived ScEM hydrogel (1 mg / ml) and control Matrigel confirmed that neuronal markers (MAP2) and ventral motor neuron markers (ISL1) were well expressed in both groups, and that excitatory neuron markers (vGLUT2) and inhibitory neuron markers (GAD67), which act as interneurons within the spinal cord, were also expressed in both groups (Fig. 32).

[0193] Based on the above results, it can be seen that long-term culture of spinal cord organoids is sufficiently possible in decellularized spinal cord tissue-derived ScEM hydrogels, similar to the control Matrigel.

[0195] Example 13: Confirmation of Spinal Cord Tissue-Specific Effects of ScEM Hydrogel

[0197] To verify whether decellularized spinal cord tissue-derived ScEM hydrogel can serve as the most suitable scaffold for spinal cord organoid culture by replacing the existing culture scaffold Matrigel and providing a spinal cord-specific microenvironment, the differentiation ability of spinal cord organoids cultured in a control Matrigel and extracellular matrix hydrogels derived from other organs besides ScEM (1 mg / ml) hydrogel—namely liver (LEM, 1 mg / ml), small intestine (IEM, 1 mg / ml), stomach (SEM, 1 mg / ml), and skeletal muscle (MEM, 1 mg / ml) hydrogel—was compared.

[0199] As a result of culturing on each scaffold for 2 and 15 days, spinal cord organoids were formed on all culture scaffolds; however, spinal cord organoids cultured on a decellularized spinal cord tissue-derived ScEM hydrogel showed the most similar size increase to organoids cultured on a control Matrigel, whereas organoids cultured on hydrogel scaffolds derived from decellularized tissue of other organs were found to be relatively small in size (Fig. 33).

[0200] In addition, quantitative PCR analysis of spinal cord-related markers in spinal cord organoids cultured for 15 days on each scaffold showed that spinal cord organoids cultured on a decellularized spinal cord tissue-derived ScEM hydrogel exhibited similar levels of marker expression compared to spinal cord organoids cultured on a control Matrigel, whereas spinal cord organoids cultured on hydrogel scaffolds derived from decellularized tissues of other organs showed significantly reduced expression (Fig. 34).

[0201] Through the above results, the tissue-specific effects of the decellularized spinal cord tissue-derived extracellular matrix (ScEM) hydrogel were confirmed, and it was confirmed that it is the most suitable culture support compared to extracellular matrix hydrogels derived from other organs for spinal cord organoid culture.

[0203] Example 14: Confirmation of Long-term Storage Possibility of ScEM Hydrogel

[0205] The long-term storage and stability of the ScEM hydrogel were confirmed by storing a decellularized spinal cord tissue-derived ScEM pre-gel solution (10 mg / ml) for a long period under refrigeration (4 ℃) and freezing (-80 ℃) conditions, then adjusting the solution concentration to 1 mg / ml at room temperature for reuse as a ScEM hydrogel, and using it for spinal cord organoid culture.

[0207] As a result, when spinal cord organoids were cultured for 15 days on a ScEM 1 mg / ml hydrogel prepared immediately before spinal cord organoid culture (Fresh ScEM), a ScEM 1 mg / ml hydrogel reused after storage at 4°C for 1 month and up to 5 months (1-month ScEM, 5-month ScEM), and a control Matrigel, it was confirmed that spinal cord organoids formed and grew on all scaffolds at a level similar to that of the control Matrigel (Fig. 35A). In addition, when quantitative qPCR analysis was performed on the spinal cord organoids cultured on each scaffold, it was confirmed that there were no significant differences in the expression of spinal cord tissue-specific markers (Fig. 35B).

[0208] Next, when spinal cord organoids were cultured for 15 days on a ScEM 1 mg / ml hydrogel prepared immediately before spinal cord organoid culture (Fresh ScEM), a ScEM 1 mg / ml hydrogel reused after being stored at -80℃ for 5 months (5-month ScEM), and a control Matrigel, it was confirmed that spinal cord organoids could be cultured on all scaffolds at a level similar to that of the control Matrigel (Fig. 36C). Furthermore, when the expression of spinal cord tissue-specific markers was compared via quantitative qPCR analysis, it was confirmed that all scaffold groups showed similar expression patterns without significant differences (Fig. 36D).

[0209] Therefore, it can be seen that ScEM hydrogel can be reused for spinal cord organoid culture even after long-term refrigeration and freezing, and can be developed as an organoid culture support with excellent storage properties.

[0211] From the foregoing description, those skilled in the art to which the present invention pertains will understand that the present invention may be implemented in other specific forms without altering its technical concept or essential features. In this regard, the embodiments described above should be understood as illustrative in all respects and not restrictive. The scope of the present invention should be interpreted as including all modifications or variations derived from the meaning and scope of the claims set forth below and their equivalents, rather than from the detailed description above.

Claims

Claim 1 A hydrogel composition comprising a decellularized spinal cord tissue-derived extracellular matrix (ScEM), wherein the concentration of the decellularized spinal cord tissue-derived extracellular matrix is ​​0.8 to 7 mg / mL, the hydrogel composition has an elastic modulus of 3 to 180 Pa, and the hydrogel composition is for spinal cord organoid culture and is applied at a concentration of 0.8 to 7 mg / ml. Claim 2 A hydrogel composition according to claim 1, wherein the hydrogel composition is for three-dimensional culture of spinal cord organoids. Claim 3 A hydrogel composition according to claim 1, wherein the decellularized spinal cord tissue-derived extracellular matrix comprises a DNA component at a concentration of 1 to 10 ng / mg; a glycosaminoglycan (GAG) at a concentration of 1 to 50 μg / mg; or a collagen component at a concentration of 1 to 50 μg / mg. Claim 4 delete Claim 5 A hydrogel composition according to claim 1, wherein the decellularized spinal cord tissue-derived extracellular matrix comprises or expresses one or more proteins selected from the group consisting of ACAN, BCAN, COL4A5, COL9A2, EMID1, HAPLN2, NCAN, and TNR as central nervous system-specific proteins. Claim 6 The hydrogel composition of claim 1, wherein the hydrogel composition is for in vivo implantation and, when implanted in vivo, increases the ratio of M2 anti-inflammatory macrophages to M1 inflammatory macrophages around the implantation site. Claim 7 A hydrogel composition according to claim 1, wherein the decellularized spinal cord tissue-derived extracellular matrix comprises or expresses one or more proteins selected from the group consisting of DPYSL2, CRMP1, DPYSL5, SH3GL2, DNM1, AP2A1, AP2M1, CLTC, TLN1, ACTB, and ARPC1A as proteins associated with axon induction. Claim 8 delete Claim 9 Spinal cord organoid cultured three-dimensionally with the hydrogel composition of claim 1. Claim 10 In claim 9, the spinal cord organoid is a spinal cord organoid that expresses one or more genes or proteins selected from the group consisting of Nestin, Tuj1, MAP2, Sox2, Nkx6.1, CHAT, PAX6, and OLIG2. Claim 11 In claim 9, the spinal cord organoid is a spinal cord organoid in which the expression of a marker (Ki67) regarding cell proliferation is enhanced to a level of 105 to 120% compared to a spinal cord organoid differentiated from Matrigel. Claim 12 A method for preparing a decellularized spinal cord tissue-derived extracellular matrix according to claim 1, comprising the steps of: separating a spinal cord from the spine of a non-human animal and removing the dura mater and pia mater; cutting the spinal cord into pieces of 0.8 to 1.2 cm and storing them for 10 to 14 hours in a solution containing 0.8 to 1.2% Triton X-100 and 0.05 to 0.15% ammonium hydroxide; storing them for 22 to 26 hours in a solution containing 3 to 5% sodium deoxycholate; and treating with DNase I for 2 to 4 hours.

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