Human Liver Ductal (Differentiation) Organoid Kit

Preparation of Human Liver Ductal Organoid Differentiation Medium I

Prepare Differentiation Medium I according to the table below, mix thoroughly, and keep on ice. This differentiation stage serves as a preconditioning step to enhance the efficiency of subsequent differentiation induction.

Note: Component D contains BMP4. Whether this component should be continuously supplemented during the subsequent differentiation stage should be determined according to the actual differentiation status of the cells.

Preparation of Human Liver Ductal Organoid Differentiation Medium II

Organoid Culture and Passaging Preparation

1. Use the Human Liver Ductal Organoid (Expansion) Kit (MCE Cat. No.: HY-K6117) for expansion culture.

2. When Human Liver Ductal Organoids are >200 μm in diameter or confluence is >80%, aspirate the upper medium and add 1 mL Human Liver Ductal Organoid (Expansion) Basal Medium A. Use a cell scraper or 1 mL pipette tip to detach the contents of the culture wells and transfer them to a 1.5 mL tube.

3. Centrifuge at 250-300 × g for 3 min at room temperature and collect the pellet.

4. Add 1 mL Expansion Basal Medium A and gently pipette until the organoids are dispersed into fragments. If difficult to disperse, use 0.2-0.5 mL Organoid Dissociation Solution (MCE Cat. No.: HY-K6013) at 37°C until clusters containing approximately 10-50 cells are obtained. Limit dissociation to ≤7 min and terminate with 5 mL Expansion Basal Medium A.

5. Centrifuge at 250-300 × g for 3 min at room temperature, discard the supernatant, and wash 1-2 times with Expansion Basal Medium A or PBS.

6. Resuspend the collected cells in MCE Basement Membrane Matrix on ice. Use 100% Basement Membrane Matrix when possible. If dilution is required, maintain a Basement Membrane Matrix-to-organoid-medium ratio > 2 : 1.

7. Using a pre-wetted 200 μL pipette tip, quickly dispense 25-35 μL per well into a 24-well plate while avoiding bubbles. Incubate at 37°C with 5% CO₂ for 15-25 min until gelled.

8. After gelation, slowly add 500 μL pre-warmed Human Liver Ductal Organoid Differentiation Medium I along the edge of each well.

Differentiation Stage I

Culture the human liver ductal organoids in Differentiation Medium I for 5 days. Refresh the medium daily until the organoid diameter reaches approximately 50-150 μm.

Differentiation Stage II

Switch to Differentiation Medium II and culture for an additional 10 days, replacing the medium every 3 days.

Completion of Differentiation

After the 10-day Stage II period, differentiation is complete. The organoids transition into hepatocyte-like organoids expressing liver-specific markers such as Albumin, Ttr, and Cyp3A4.

Note: Prepared Differentiation Medium may be stored at 4°C for 2 weeks or at -20°C for 3 months. Avoid repeated freeze-thaw cycles; freshly prepared differentiation medium is recommended.