Rhabdomyosarcoma Organoid Kit

1. Preparation of Complete Culture Medium for Rhabdomyosarcoma

Prepare the complete organoid medium according to the table below, mix thoroughly, and keep on ice. Perform all steps under sterile conditions.

2. Extraction of Tumor Cells from Primary Tissues

a. Soak freshly extracted tumor tissue in pre-cooled primary tissue storage solution and keep temporarily at 4°C.

b. Under sterile conditions, wash the tissue with Rhabdomyosarcoma Organoid Basal Medium A or PBS and remove non-target components such as fat, muscle, and necrotic tissue.

c. Cut the tissue into pieces approximately 1-2 mm in diameter and transfer them to a 15 mL conical tube.

d. Add an appropriate amount of tumor tissue dissociation solution without exceeding two-thirds of the tube volume. Incubate on a horizontal shaker at 37°C for approximately 0.5-1 h until most tissue fragments can be aspirated with a 1 mL pipette tip.

e. Add FBS to a final concentration of 2% to terminate digestion and filter through a 100 μm cell strainer on ice.

f. Centrifuge at 250 × g for 3 min at 4°C and collect the pellet. If the pellet appears red, remove the supernatant, gently resuspend in 1-2 mL red blood cell lysis buffer, lyse for approximately 1 min at room temperature, and centrifuge again at 250 × g for 3 min at 4°C.

g. Resuspend the pellet in an appropriate amount of Rhabdomyosarcoma Organoid Basal Medium A, centrifuge at 250 × g for 3 min at 4°C, and repeat the wash 1-2 times.

3. Construction of Organoid

a. Keep the entire procedure on ice. Resuspend the collected tumor cells in MCE Basement Membrane Matrix at a recommended density of approximately 4 × 10⁴ cells/100 μL. Use 100% Basement Membrane Matrix whenever possible. If dilution is required, maintain a Basement Membrane Matrix-to-organoid-medium volume ratio > 2 : 1.

b. Using a pre-wetted 200 μL pipette tip, quickly dispense 25-35 μL matrix/cell suspension per well into a 24-well plate while avoiding bubbles. Incubate at 37°C with 5% CO₂ for 15-30 min until gelled.

c. After gelation, slowly add 500 μL Rhabdomyosarcoma Organoid Complete Medium along the edge of each well without disrupting the gel and continue incubation at 37°C with 5% CO₂.

d. Replace 500 μL pre-warmed complete medium every 3-4 days. Organoids are generally visible after approximately 7-10 days of culture.

4. Organoid Passages

a. When organoids reach >200 μm, become darkened, or reach high culture density, aspirate the upper medium and add approximately 500 μL Basal Medium A. Detach the well contents using a cell scraper or 1 mL pipette tip and transfer to a 1.5 mL tube.

b. Gently pipette to separate organoids from Basement Membrane Matrix and centrifuge at 250-300 × g for 3 min at room temperature.

c. Add 1 mL Basal Medium A and gently pipette to disperse organoids into smaller fragments. If dissociation is difficult, add 0.2-0.5 mL organoid dissociation solution and incubate at 37°C until clusters of approximately 10-50 cells are obtained. Limit dissociation to approximately 5-7 min and terminate with approximately 5 mL Basal Medium A.

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

e. Re-embed the cells in Basement Membrane Matrix on ice, seed 25-35 μL per well as described above, and incubate at 37°C with 5% CO₂ until gelled.

f. Slowly add 500 μL complete medium per well and continue culture, replacing the medium every 3-4 days.