Formation of Tumor Spheroids of A549 (Human Non-Small Cell Lung Cancer Cells)

Culture Conditions

A549 Cells: 89% DMEM/F12 medium, 10% FBS and 1% P/S

Cell recovery

• RetrieveA549 cells from liquid nitrogen and quickly place them in a 37℃ water bath, gently shaking the cryovial to thaw the freezing medium.
• After thawing, transfer the cells to a centrifuge tube containing 3 mL of medium, centrifuge to pellet the cells (1000 rpm, 5 min, room temperature), and discard the supernatant.
• Resuspend the cells in complete medium, seed them into a culture dish, gently pipet to mix, and culture in a normal incubator at 37℃, 21% O₂, 5% CO₂.

Cell Passaging

• When cell density reaches 80%, passage the cells.
• Discard the medium and wash once with PBS.
• Add 1-2 mL of 0.25% trypsin to digest the cells at 37℃for 2-5 min. Observe under a microscope; digestion is complete when cells separate and become rounded.
• Quickly discard the trypsin, add complete medium, pipet to create a single-cell suspension, and passage at a ratio of 1:2 to 1:4. Expand the culture in a normal incubator at 37℃, 21% O₂, 5% CO₂.

Cell Seeding

Take A549 cells in the logarithmic growth phase with good growth status. Seed them into a 96-well U-bottom ultra-low attachment plate at densities of 500, 1000, 2000, 4000, and 8000 cells/well (one plate total), with 5 replicate wells per density. Add 100 μL of sterile PBS to the peripheral wells of the plate. Place the 96-well U-bottom ultra-low attachment plate in the live cell station for culture and imaging. Note: The live cell station was installed in the CO₂ incubator (Thermo, 3111) and prewarmed for 30 min, maintaining conditions at 37℃, 21% O₂, 5% CO₂, and saturated humidity.

Medium Change

For the first 3 days, the initial number of A549 cells is low and metabolism is relatively slow; change the medium every 24-48 hours. From days 4-7, as cell proliferation increases and density gradually rises, metabolism accelerates, requiring medium changes every 1-2 days (Note: Medium changes are also performed synchronously for the plate in the live cell station. The medium change frequency for the 500, 1000, and 2000 cells/well densities is lower than that for the 4000 and 8000 cells/well densities). During medium change, carefully transfer the cell spheroids gently using a 200 μL yellow pipette tip to a 35 mm cell culture dish containing 1 mL of complete medium. Transfer the spheroids from all 6 replicate wells of one density at once. Then, aspirate and discard the old medium from the wells, rinse the wells 2-3 times with 100 μL/well of PBS. Subsequently, use the 200 μL yellow pipette tip to transfer the cell spheroids back into the wells one by one, maintaining a medium volume of 100 μL per well.