Brain organoid containing optic vesicles generated based on H9 induction and eye-brain fusion culture method

An eye-brain fusion culture method for generating a three-dimensional brain organoid containing optic vesicles through the induction of H9 embryonic stem cells, comprising the following steps:

• A) culturing the H9 embryonic stem cells in a feeder-free cell culture and maintenance medium until a cell confluence reaches 70%,
• B) preparing a single cell suspension,
• C) removing the feeder-free cell culture and maintenance medium and adding a neural induction medium containing 10 mM Y27632, inoculating in an orifice plate, changing half of the medium with a neural induction medium without 10 mM Y27632 each day for 0-5 days to form neurospheres,
• wherein the neural induction medium comprises 83% DMEM/F-12, 15% serum replacement supplement, 1% MEM-non-essential amino acids, 1% L-alanyl-L-glutamine dipeptide supplement, 50 μM ß-mercaptoethanol, 10 μM SB431542, and 2 μM XAV939;
• D) removing the neural induction medium and adding a neurosphere medium, and changing half of the medium with the neurosphere medium each day until a 6-7th day,
• wherein the neurosphere medium comprises 48% DMEM/F-12, 48% neuronal cell culture medium, 0.4% N2 supplement, 1% B27 supplement without vitamin A, 0.5% MEM-non-essential amino acids, 1% L-alanyl-L-glutamine dipeptide supplement, 1% penicillin/streptomycin, 50 μM ß-mercaptoethanol, 2.5 M SB431542, 1.5 nM bone morphogenetic protein-4, and 0.1% extracellular matrix;
• E) removing the neurosphere medium and adding an early-stage optic vesicle brain organoid medium on an 8th day,
• wherein the early-stage optic vesicle brain organoid medium comprises 48% DMEM/F-12, 48% neuronal cell culture medium, 0.4% N2 supplement, 1% B27 supplement, 100 nMol retinol acetate, 0.5% MEM-non-essential amino acids, 1% L-alanyl-L-glutamine dipeptide supplement, 0.5 μM Dorsomorphin, 1% penicillin-streptomycin, 50 μM ß-mercaptoethanol, 2.5 μM SB431542, 1.5 nM bone morphogenetic protein-4, and 0.1% extracellular matrix;
• F) removing the early-stage optic vesicle brain organoid medium adding a mature-stage optic vesicle brain organoid medium on a 10th day and culturing under rotational culture, and changing half of the medium with the mature-stage optic vesicle brain organoid medium every week until the three-dimensional brain organoid containing the optic vesicles is formed;
• wherein the mature-stage optic vesicle brain organoid medium comprises 48% DMEM/F-12, 48% neuronal cell culture medium, 0.4% N2 supplement, 1% B27 supplement, 100 nMol retinol acetate, 0.5% MEM-non-essential amino acids, 1% L-alanyl-L-glutamine dipeptide supplement, 0.5 μM Dorsomorphin, 1% penicillin-streptomycin, 50 μM ß-mercaptoethanol, 2.5 μM SB431542, 0.1% ascorbic acid, 10 ng/ml brain-derived neurotrophic factor, and 10 ng/ml ciliary neurotrophic factor;
• G), continuing culture of the brain organoid obtained in step 1 until pigment deposition occurs on one side, and
• H) performing immunostaining and imaging on the brain organoid marked by a forebrain pattern and a visual field specification to verify a structural function and neural development of the brain organoid.