Composition for culturing brain organoid based on decellularized brain matrix and method for preparing same
Inventors
Cho, Seung Woo • Cho, Ann Na • Lee, Jung Seung • JIN, Yoonhee
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Assignees
Industry Academic Cooperation Foundation of Yonsei University
Member
Glycos Biomedical LtdWe are UK/US entity with a team based in Maryland and North Carolina comprising an expert team that has linked triggers to disease states including TBI and acute injuries associated with radiation, acute lung injury and infection.
We are UK/US entity with a team based in Maryland and North Carolina comprising an expert team that has linked triggers to disease states including TBI and acute injuries associated with radiation, acute lung injury and infection.
Publication Number
US-12624340-B2
Publication Date
2026-05-12
Expiration Date
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.
Core Innovation
The invention relates to a decellularized brain extracellular matrix (dBEM)-based hydrogel culture platform for brain organoids. It uses a decellularized brain extracellular matrix powder that is re-gelated into a hydrogel composition by mixing with a basement membrane matrix derived from Engelbreth-Holm Swarm (EHS) mouse sarcoma cells. The resulting hydrogel composition contains dBEM in an amount defined for the culture platform and exhibits defined glycosaminoglycan (GAG) content and elastic modulus at 1 Hz.
The hydrogel composition includes defined protein components associated with extracellular matrix constituents. The composition is prepared so that the dBEM hydrogel retains protein components and GAG, while also providing mechanical properties aligned to the hydrogel culture environment. In the disclosed platform, pluripotent stem cell-derived cells are cultured in three-dimensional culture condition to obtain brain organoids.
Brain organoid differentiation and maturation are confirmed by neural marker expression and functional and developmental analyses. The organoids are described as exhibiting neural tube formation, with expression of neuronal markers confirmed, and reactivity to glutamate and gamma amino butyric acid (GABA) assessed. Differentiation confirmation further includes identifying expression of N-Cadherin and analyzing development of cortical layer and forebrain.
Claims Coverage
The partial content provides one independent claim (clm-00001). The claim centers on five inventive aspects: defining a decellularized brain tissue composition with specified cell/DNA removal targets and powder preparation; forming a dBEM hydrogel by mixing with an EHS-derived basement membrane matrix to achieve defined dBEM amount, GAG content, and elastic modulus; specifying a composition having a defined set of protein components; culturing pluripotent stem cell-derived cells in three-dimensional condition to obtain organoids with neural tube formation and confirmed neuronal marker expression; and confirming differentiation using glutamate/GABA reactivity, N-cadherin expression, and cortical layer/forebrain development analysis.
Decellularized brain extracellular matrix powder with defined cell and DNA removal targets
A method that decellularizes brain tissue so that 98% or more of cells are removed and remaining DNA content is 0.01 to 2.0% of that of the brain tissue before decellularization, followed by drying and lyophilizing to obtain a powder.
dBEM hydrogel composition formed with EHS-derived basement membrane matrix and defined properties
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 containing dBEM in an amount of 0.01 to 2.0 mg/mL, having a glycosaminoglycan (GAG) content of 8 to 10 μg/mg, and exhibiting an elastic modulus at 1 Hz of 110 to 130 Pa.
Hydrogel composition containing specified extracellular matrix protein components
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 α1, Laminin α3, Tenascin R, Keratin 1, Brevican, Neurocan, Versican, Heparan sulfate, Prostaglandin, Apolipoprotein E, Apolipoprotein L2, Apolipoprotein O, Galectin-1 and Albumin.
Three-dimensional culturing to obtain brain organoids with neural tube formation and neuronal marker expression
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.
Differentiation confirmation using glutamate/GABA reactivity, N-cadherin expression, and cortical and forebrain development analysis
Confirming differentiation of the brain organoid by identifying reactivity to glutamate and gamma amino butyric acid (GABA) neurotransmitter, identifying expression of N-Cadherin, and analyzing development of cortical layer and forebrain.
Across the single independent claim, the core coverage is a preparation workflow that yields a re-gelated dBEM hydrogel formed with EHS-derived basement membrane matrix to achieve specified dBEM amount, GAG content, and elastic modulus, while also incorporating defined protein components. The claim then specifies culturing pluripotent stem cell-derived cells in that hydrogel to produce brain organoids with neural tube formation and confirmed neuronal marker expression, followed by differentiation confirmation via glutamate/GABA reactivity, N-cadherin expression, and analysis of cortical layer and forebrain development.
Stated Advantages
Better preserves ECM/GAG and laminin/protein components compared to Matrigel®.
Maintains hydrogel mechanics relative to Matrigel®.
Enhances organoid differentiation/maturation compared to Matrigel®, including neural differentiation markers and cortical layer marker development.
Improves neurofunctional readouts, including glutamate/GABA responsiveness and electrophysiological activity.
Drives gene-expression shifts related to nervous system function, synaptic signaling, pluripotency, and ECM/cell adhesion pathways compared to Matrigel®.
Documented Applications
Culturing pluripotent stem cell-derived cells in a three-dimensional hydrogel culture condition to obtain brain organoids for neural tube formation and differentiation confirmation.
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