A Cancer Cell Spheroid Assay to Assess Invasion in a 3D Setting

Remove adherent cancer cells from culture, wash with PBS, treat with trypsin-EDTA for 3 minutes at 37°C, pipet to create single cell suspension, and add culture media. Count cells and dilute to 500-1000 cells per 20 microliters.

Use a multi-channel pipette to create 40 drops of 20 microliters each on the inner surface of a 10 cm dish lid. Add 5 mL of sterile PBS to the bottom of the dish, invert the lid onto it, and incubate at 37°C with 5% CO₂ for up to 72 hours to allow spheroid formation.

Thaw growth factor-reduced basement membrane solution overnight at 4°C. Mix 100 microliters of thawed basement membrane solution with 100 microliters of 2.3 mg/mL cold type 1 collagen solution in a pre-chilled centrifuge tube and keep on ice.

Collect spheroids by tilting the lid to pool droplets and transfer media to a centrifuge tube. Allow spheroids to settle for 10 minutes, then pipette 40 microliters of spheroids and combine with the ECM mixture. Pipette 40 microliter drops into individual wells of a 24-well plate.

Keep the plate level and incubate at 37°C for 30 minutes to allow the 3D matrix to polymerize. Slowly pipette 1 mL of warm culture medium into each well to submerge the cultures, then return to the incubator.

Using an inverted microscope with 20x objective, image the invading cancer cells at predetermined time points. Incubate the dish at 37°C between imaging sessions to monitor cell invasion progression.

Use image analysis software such as ImageJ to determine cell distance from the spheroid edge using the straight line tool and the invasive area using the freehand draw tool. Click Analyze and Measure to display length and area measurements for quantitative analysis.

Analyze invasion variability across different cell lines, noting that spheroid formation and invasion ability vary significantly. Work quickly during matrix preparation to avoid air bubbles, and remember the complete protocol can be performed over 5 days with minimal hands-on time.