Formation of Tumor Spheroids of MIA-PACA-2（Pancreatic cancer cell line）

Culture Conditions

MIA-PACA-2 Cells : 89%RPMI 1640 Cell Culture Medium+10% FBS+1% P/S

Cell recovery

1) Retrieve the MIA-PACA-2 cells from the liquid nitrogen tank and quickly place the cryovial into a 37℃ water bath. Gently shake the vial to thaw the freezing medium.

2) After thawing, transfer the cell suspension to a centrifuge tube containing 3 mL of culture medium. Centrifuge to collect the cells at 1000 rpm for 5 minutes at room temperature. Discard the supernatant.

3) Resuspend the cell pellet in complete medium and seed the cells into a culture dish. Mix gently by pipetting. Culture the cells in a normal incubator at 37℃, 21% O₂, and 5% CO₂.

Cell Passaging

1) Passage the cells when they reach approximately 80% confluence.

2) Discard the culture medium and wash the cells once with PBS.

3) Add 1-2 mL of 0.25% trypsin to digest the cells at 37℃ for 3-4 minutes. Observe under a microscope; digestion is complete when the cells detach and become rounded.

4) Quickly discard the trypsin. Add complete medium to neutralize the trypsin, and pipette to create a single-cell suspension. Passage the cells at a split ratio of 1:2 to 1:4. Expand the culture in a normal incubator at 37℃, 21% O₂, and 5% CO₂.

Cell Seeding

MIA-PACA-2 cells in the logarithmic growth phase and in good condition were harvested. Cells were seeded into a 96-well U-bottom ultra-low attachment plate at densities of 1000, 2000, 4000, and 8000 cells per well (one plate total), with five replicates for each density. The peripheral wells of the plate were filled with 100 μL of sterile PBS. The 96-well U-bottom ultra-low attachment plate was then placed in the live cell station for culture and imaging. Note: The live cell station was installed inside a CO₂. constant temperature incubator , pre-warmed for 30 minutes, and maintained at 37℃, 21% O₂, and 5% CO₂., and saturated humidity.

Medium Change

For the first three days, the initial number of MIA-PACA-2 cells was low, and their metabolism was relatively slow. Therefore, the medium was changed every 2-3 days (i.e., every 24 hours within this period). From days 4 to 7, as the cells proliferated and their density gradually increased, metabolism accelerated, necessitating medium changes every 1-2 days. (Note: The medium in the plates within the live cell station was changed synchronously. The medium change frequency for wells seeded at 500, 1000, and 2000 cells/well was lower than that for wells seeded at 4000 and 8000 cells/well). During medium change, cell spheroids were carefully transferred using a 200 μL yellow pipette tip into a 35 mm cell culture dish containing 1 mL of complete medium. All spheroids from the five replicate wells of one density were transferred at once. The old medium in the wells was then aspirated and discarded. The wells were rinsed 2-3 times with 100 μL/well of PBS. Subsequently, the cell spheroids were individually transferred back into their respective wells using a 200 μL yellow pipette tip, maintaining a final medium volume of 100 μL per well.