Human iPSC/ESC Cortical Brain Organoid Maturation Medium

1. DPBS (MCE Cat. No.: HY-K3008)

2. Basement Membrane Matrix IPSC-qualified (MCE Cat. No.: HY-K6006)

3. iPSC/ESC Dissociation Solution (Enzyme-Free) (MCE Cat. No.: HY-K6020)

4. CEPT Cocktail Plus (1000×) (MCE Cat. No.: HY-K6021)

5. ECM Gentle Dissociation Solution (MCE Cat. No.: HY-K6022)

6. Human iPSC/ESC Cortical Brain Organoid Induction Differentiation Kit (MCE Cat. No.: HY-K6301)

7. Human PSC Maintenance Medium (MCE Cat. No.: HY-K6501)

8. Equipment and Consumables: 6/96-well low-attachment plates, and multichannel pipettes (8-12 channels), among other related equipment and consumables.

Human iPSC/ESC Cortical Brain Organoid Induction Differentiation Kit (MCE Cat. No.: HY-K6301) can theoretically generate no fewer than 100 brain organoids. For the initial induction, it is recommended to start with one 6-well plate, which typically yields approximately 60-100 uniformly sized neuroepithelial spheres (ideal formation rate ~60%).

1. (Refer to the HY-K6301 Manual) Neuroepithelial Sphere Induction and Neural Rosette Expansion

Using the Human iPSC/ESC Cortical Brain Organoid Induction Differentiation Kit (MCE Cat. No.: HY-K6301), PSCs are induced to form neuroepithelial spheres and initiate neural rosette expansion. When black punctate structures appear in the center and periphery of the spheres, indicating well-developed neural rosettes, discard the expansion medium completely.

2. Transition to the Maturation Stage

Add 2 mL of Human iPSC/ESC Cortical Brain Organoid Maturation Medium to each well and transfer the plate to a cross-shaker/rotary shaker at 80 rpm for 18-26 d, refreshing the medium every 2-3 d. Mature brain organoids at this stage are suitable for characterization and downstream applications. When cultured long-term on a shaker using the maturation medium, organoids can be maintained up to 120 d without central apoptosis.

Note: Mature organoids exhibit layered VZ- and SVZ-like structures composed of TUJ1⁺ neurons and SOX2⁺ radial glia, as confirmed by immunofluorescence (IF). qPCR analysis shows a 3-5-fold increase in glutamatergic markers such as SLC17A6/VGLUT2.

3. (Refer to the HY-K6301 Manual) Organoid Sectioning

When cortical organoids exceed 1 mm in diameter, perform mechanical sectioning to alleviate inner-core apoptosis. After cutting, change the medium daily to remove apoptosis-related debris and continue shaking culture at 80 rpm for 4 d.

4. Long-Term Maintenance

After 4 d, switch to the Cortical Brain Organoid Maturation Medium and maintain the organoids at 37°C, 120 rpm. This regimen supports extended culture and organoid viability for up to 180 d.

Note: During culture, organoid quality can be monitored by TUNEL assay for central necrosis and by evaluating markers such as Synaptophysin to assess maturation and functional status.