US-12624340-B2 - Composition for culturing brain organoid based on decellularized brain matrix and method for preparing same

Abstract

Provided is a method for preparing a composition for culturing a brain organoid, the method comprising (a) decellularizing brain tissue; (b) drying the brain tissue; and (c) gelating the brain tissue.

Inventors

• Seung Woo Cho
• Ann Na Cho
• Jung Seung Lee
• Yoonhee JIN

Assignees

• INDUSTRY-ACADEMIC COOPERATION FOUNDATION, YONSEI UNIVERSITY

Dates

Publication Date

20260512

Application Date

20230705

Priority Date

20180621

Claims (1)

1. 1 . A method for preparing a brain organoid, comprising: (a) stirring brain tissue for 3 to 24 hours in a decellularizing solution and decellularizing the brain tissue, wherein 98% or more of cells of the brain tissue are removed by the decellularization, and the remaining DNA content is 0.01 to 2.0% of that of the brain tissue before the decellularization; (b) drying the decellularized brain tissue and lyophilizing the decellularized brain tissue to obtain a powder; and (c) gelating the decellularized brain tissue by mixing the powder with a basement membrane matrix derived from Engelbreth-Holm Swarm (EHS) mouse sarcoma cells to obtain a hydrogel composition, wherein the hydrogel composition contains a decellularized brain extracellular matrix(dBEM) in an amount of 0.01 to 2.0 mg/mL, has a glycosaminoglycan (GAG) content of 8 to 10 μg/mg, and exhibits an elastic modulus at 1 Hz of 110 to 130 Pa; wherein the hydrogel composition contains Protein components comprising Collagen type I α2, Collagen type IV α2, Collagen type IV α5, Collagen type VI α1, Collagen type VI α2, Collagen type VI α3, Fibronectin type III, Fibrinogen γ chain, Laminin α5, Laminin β1, Laminin γ1, Tenascin R, Keratin 1, Brevican, Neurocan, Versican, Heparan sulfate, Prostaglandin, Apolipoprotein E, Apolipoprotein L2, Apolipoprotein O, Galectin-1 and Albumin; (d) culturing pluripotent stem cell-derived cells in the hydrogel composition in a three-dimensional culture condition for 45 to 75 days, to obtain a brain organoid exhibiting neural tube formation, wherein expression of neuronal markers selected from Nestin, Tuj1, NeuN, MAP2, Pax6, FoxG1, and Sox2 is confirmed; and (e) confirming differentiation of the brain organoid by (i) identifying of reactivity of the brain organoid to glutamate and gamma amino butyric acid (GABA) neurotransmitter; (ii) identifying expression of N-Cadherin; and (iii) analyzing a development of cortical layer and forebrain.

Description

TECHNICAL FIELD The present invention relates to a composition for culturing a brain organoid using a decellularized brain matrix and a method for preparing the same. BACKGROUND ART Decellularization of tissues and organs has been studied as a promising approach for preparing a functional support or scaffold for cell culture and transplantation. During a decellularization process, cellular components are removed from the tissue, but the extracellular matrix and some growth factor proteins are preserved. Therefore, various extracellular matrix components, which are preserved in the decellularized tissue including collagen, fibronectin, and laminin, provide a three-dimensional microenvironment similar to the inside of intact tissue, and thus can improve survival, proliferation, and differentiation of cultured cells. Additionally, removal of cellular components alters the decellularized matrix into a functional scaffold with minimal immunogenicity for cell transplantation. Recently, studies for functional tissue-engineered scaffold, which are obtained by applying several types of decellularized matrices derived from the brain, heart, blood vessels, heart valve, lungs, and kidneys, have been conducted. In particular, the brain is an organ that serves as a major control tower for regulating an organism, and is an important organ that functions through a close network between neurons and other various cells. However, due to the complicated structure and working principle of the brain, sufficient studies have not been conducted, and studies for causes of neurological diseases and development of new drugs are confronted with limitations. Therefore, there is a growing need for active studies of brain development and brain diseases through the construction of an in vitro model system that recapitulates the brain tissue and functions thereof, and new studies for producing miniature organs using human stem cell-derived organoids are in the spotlight. Nevertheless, the brain organoid produced by the current technique of culturing an organoid has many differences from the actual brain tissue in terms of degree of differentiation and function, and there is a need for the development of an elementary technique for culture of a more mature brain organoid. DISCLOSURE Technical Problem The present invention has been made to solve the above-mentioned problems of the prior art, and an object thereof is to provide a three-dimensional culture platform for improving immature structural development and immature differentiation of a brain organoid according to current culture techniques and for producing a brain organoid similar to the actual brain. Technical Solution According to an aspect of the present invention, there is provided a method for preparing a composition for culturing a brain organoid, comprising (a) decellularizing brain tissue; (b) drying the brain tissue; and (c) gelating the brain tissue. In an embodiment, in the (c), the brain tissue and Matrigel® (basement membrane matrix secreted by Engelbreth-Holm-Swarm (EHS) mouse sarcoma cells) may be mixed to obtain a hydrogel. In an embodiment, the hydrogel may contain a decellularized brain extracellular matrix (dBEM) in an amount of 0.01 to 2.0 mg/mL. In an embodiment, in the (a), the brain tissue may be stirred in a decellularizing solution. In an embodiment, the brain tissue may be stirred for 3 to 24 hours. In an embodiment, 95% or more of cells of the brain tissue may be removed by the decellularization. According to another aspect of the present invention, there is provided a hydrogel composition for culturing a brain organoid, comprising a decellularized brain extracellular matrix (dBEM). In an embodiment, the composition may further comprise Matrigel® (basement membrane matrix secreted by Engelbreth-Holm-Swarm (EHS) mouse sarcoma cells). In an embodiment, the decellularized brain extracellular matrix (dBEM) may have a concentration of 0.01 to 2.0 mg/mL. In an embodiment, the composition may have an elastic modulus at 1 Hz of 100 to 150 Pa. According to still another aspect of the present invention, there is provided a method for culturing a brain organoid in the composition. Advantageous Effects Unlike conventional techniques for culturing a brain organoid, the present invention is highly likely to be applied as an in vitro model for realizing actual brain tissue. The brain matrix-based hydrogel of the present invention can promote differentiation of a brain organoid into neurons and cortical layer neurons, and can be usefully used for the construction of an in vitro model in a brain tissue-like form. It should be understood that the effects of the present invention are not limited to the above-mentioned effects and include all effects which can be deduced from the detailed description of the present invention or the constitution of the invention described in the claims. DESCRIPTION OF DRAWINGS FIGS. 1A and 1B illustrate schematic diagrams for a method of produci