Human iPSC/ESC Whole-Brain Organoid Induction Differentiation Kit

Reagents and Consumables to be Prepared by the User

Trypan Blue Staining Solution (0.4%) (MCE Cat. No.: HY-K2004)

DMEM/F-12 (MCE Cat. No.: HY-K3002)

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

Reduced Growth Factor Basement Membrane Matrix (MCE Cat. No.: HY-K6004)

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

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

Matrix-Selective Gentle Dissociation Solution (MCE Cat. No.: HY-K6022)

Human Pluripotent Stem Cell (PSC) Culture Medium (MCE Cat. No.: HY-K6501)

Human iPSC Brain Organoid Long-Term Culture Medium (MCE Cat. No.: HY-K6502)

Human PSC Embryoid Body (EB) Induction Medium (MCE Cat. No.: HY-K6503)

Equipment/consumables: 6-/96-well plates (low attachment), multichannel pipettes (8-12 channels), etc.

Instructions for Use (Using a 6-Well Plate as an Example)

The Human iPSC/ESC Whole-Brain Organoid Induction and Differentiation Kit (MCE Cat. No.: HY-K6302) can theoretically generate approximately 100 brain organoids. It is recommended to use half of a 6-well plate to initiate the differentiation process for each induction, yielding approximately 100 uniformly sized EBs for subsequent whole-brain organoid induction.

PSC Culture

Depending on the characteristics of the PSC line, thaw one vial of cells from ESC (< passage 60) or iPSC (< passage 50) stock and expand through two passages to reach P3 (refer to the HY-K6501 manual for stem cell culture procedures).

Note: If PSC colonies appear abnormal or show obvious differentiation or floating, the cells should be re-thawed and expanded; continuing induction is not recommended.

EB Induction

1. Timing of EB Induction: Continue culturing P3 PSCs until colony density reaches approximately 80%, colony borders are clear and rounded, and the nuclear-to-cytoplasmic ratio is normal; EB induction can then begin.

2. Reagent Preparation: For 3 wells of a 6-well plate with PSCs at 80% confluence, prepare the following reagents and pre-warm to room temperature:

Human PSC Embryoid Body (EB) Induction Medium (MCE Cat. No.: HY-K6503) 12 mL + CEPT Cocktail Plus (1000×) (MCE Cat. No.: HY-K6021) 12 μL

Matrix-Selective Gentle Dissociation Solution (MCE Cat. No.: HY-K6022) 3 mL + CEPT Cocktail Plus 3 μL

DPBS (MCE Cat. No.: HY-K3008) 50 mL

DMEM/F-12 (MCE Cat. No.: HY-K3002) 10 mL

3. Cell Washing: Wash each of the 3 target wells once with DPBS and aspirate the DPBS.

4. Cell Dissociation: Add 1 mL of Matrix-Selective Gentle Dissociation Solution containing CEPT Cocktail Plus to each well and dissociate at 37°C for 7-10 min.

5. Stopping Dissociation: Monitor cell dissociation under a microscope. Stop dissociation immediately once cell edges become translucent and lift off in sheets; add 1 mL DMEM/F12 to each well to neutralize the dissociation reagent. Pipette gently to fully dissociate cells into a single-cell suspension, and count using Trypan Blue Staining Solution (0.4%) (MCE Cat. No.: HY-K2004). Proceed to the next step only if cell viability is > 90%.

6. EB Induction:

1) Cell Collection: Centrifuge the resulting 6 mL cell suspension at 130 × g for 3 min and discard the supernatant. Resuspend the pellet in 12 mL Human PSC Embryoid Body (EB) Induction Medium containing CEPT Cocktail Plus, mix thoroughly, and recount for viability; the final cell density should ideally be 50,000-80,000 cells/mL.

2) Cell Seeding: Seed 100 μL of the cell suspension per well into a 96-well plate, seal the plate, and centrifuge at 1200 rpm for 3 min in a swinging-bucket centrifuge. Remove the seal and incubate overnight. The following day, add an additional 100 μL of EB Induction Medium (without CEPT Cocktail Plus) to each well.

7. EB Formation: Single cells typically aggregate within 24 h, and smooth, well-rounded EBs form by 48 h, which is the optimal state for proceeding.

Note: If PSC colonies appear abnormal or show obvious differentiation or floating, the cells should be re-thawed and expanded; continuing induction is not recommended.

Neuroectoderm Induction

1. Transfer and Induction: Transfer EBs that have been induced for 48 h into a 6-well plate, 10 EBs per well. Wash once with DPBS to remove residual medium from the previous stage, then aspirate and add 2 mL of Neuroectoderm Induction Medium per well. Culture on an orbital shaker (80 rpm) at 37°C, changing the medium every 2 days, for a total induction period of 4 days.

Note: All culture stages after EB formation must be performed under shaking conditions.

2. Morphological Characteristics: Successfully induced EBs show clear neuroectodermal expansion at the periphery with translucent borders; diameter is typically > 400 μm, with a dense, cavity-free interior resembling primary mouse neurospheres.

Neural Budding

1. Matrix Preparation and Embedding Conditions: Once EBs reach 400-600 μm in diameter, matrix embedding can be performed. Thaw 2 mL of aliquoted Reduced Growth Factor Basement Membrane Matrix (MCE Cat. No.: HY-K6004) at 4°C in advance.

2. Parafilm Preparation: Prepare a 10 × 10 cm sheet of Parafilm and press it against a support (e.g., a 200 μL pipette tip box rack) using the base of a centrifuge tube to create uniform dimples. Place the Parafilm in a 10 cm culture dish, soak in alcohol for 2-5 min, remove and air-dry in a clean dish, then UV-irradiate in a biosafety cabinet for ≥ 30 min before use.

3. EB Embedding: Using a wide-bore 10-100 μL pipette tip, carefully aspirate EBs that have completed neuroectoderm differentiation and transfer them individually into the pre-formed dimples (approximately 10 EBs per batch is recommended). After removing residual medium from each dimple, add approximately 30 μL of Reduced Growth Factor Matrix to each dimple to fully embed the EB. If an EB sits at the edge of the droplet, gently reposition it to the center of the dimple using a 10 μL pipette tip, taking care to avoid introducing air bubbles.

4. Gelation and Transfer: Cover with Parafilm and place at 37°C for 15 min to allow gelation. Using sterile forceps, lift the Parafilm and use 1 mL of Neural Budding Induction Medium to gently flush each row of EBs, close to the dimple base, into a 6-well plate. Place a maximum of 10 organoids per well, with a total medium volume not exceeding 3 mL per well.

5. Alternative Embedding Method (for Large-Batch Processing): If processing a large number of EBs at once, add 10% Reduced Growth Factor Matrix (i.e., 1.2 mL of matrix) to 12 mL of Neural Budding Induction Medium that has been pre-chilled for ≥ 30 min, and vortex thoroughly to mix. Discard the Neuroectoderm Induction Medium from the original plate, add 3 mL of the matrix-containing Neural Budding Induction Medium per well, and culture on an orbital shaker (80 rpm) at 37°C for 48 h. Afterward, discard the medium, wash twice with DPBS, and add 3 mL of matrix-free Neural Budding Medium per well.

6. Neuroepithelial Expansion Culture: Continue culturing the 6-well plate on an orbital shaker (80 rpm) at 37°C for 18 days, changing the medium every 2 days. Early VZ/SVZ-like structures are typically observed around day 4 of neuroepithelial expansion, and neural bud regions gradually fuse and darken from around day 12.

Neural and Brain Maturation

1. When the center of the EBs becomes dense, a loosely organized epithelial structure forms on the outer layer, and the diameter exceeds 1 mm, the organoids have entered the neural maturation stage. Completely remove the Neural Budding Induction Medium, add 3 mL of Neural Maturation Medium per well, and continue culturing until day 38, changing the medium every 2 days.

2. Once organoid diameter exceeds 2 mm, nutrient supply to the interior may become insufficient. Use a 1 mL syringe to carefully trim away excess matrix surrounding the EBs, then add 3 mL of Brain Maturation Medium per well and switch to daily medium changes, while increasing the shaker speed to 120 rpm to improve nutrient circulation. Once mature, brain organoids can be maintained in long-term culture (> 100 days).