Establishment of a Spheroid Culture Model for Mouse Peripheral Blood Mononuclear Cells(m-PBMCs)

Culture Conditions

mPBMC cells: Blood Cell Culture Medium

Cell recovery

1)  mPBMC cells were retrieved from liquid nitrogen and immediately placed in a 37℃ water bath. The cryovial was gently agitated to thaw the freezing medium.

2)  After thawing, the cell suspension was transferred to a centrifuge tube containing 3 mL of culture medium. The cells were collected by centrifugation at 1000 rpm for 5 minutes at room temperature, and the supernatant was discarded.

3)  The cell pellet was resuspended in complete culture medium, seeded into a culture dish, and gently mixed by pipetting. The cells were then cultured in a standard incubator at 37℃ with 21% O₂ and 5% CO₂.

Cell Passaging

1)  Cells were passaged when they reached 80% confluence.

2)  The cells were collected and centrifuged at 1000 rpm for 3-5 minutes.

3)  The supernatant was discarded. 1-2 mL of fresh culture medium was added to resuspend the cell pellet by gentle pipetting. The cell suspension was then subcultured into new dishes containing 3-4 mL of fresh medium at a split ratio of 1:2 to 1:4.

4)  The cells were subsequently expanded in a standard incubator at 37℃ with 21% O₂ and 5% CO₂.

 Cell Seeding

mPBMC cells in the logarithmic growth phase and in good condition were harvested. They 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 replicate wells per 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 imaging station for culture and imaging. Note: The live-cell imaging station was installed within a CO₂ incubator, pre-warmed for 30 minutes, and maintained at 37℃, 21% O₂, 5% CO₂, and saturated humidity.

Medium Change

For the first 3 days, due to the low initial mPBMC cell count and relatively slow metabolism, the medium was changed every 24 hours. From days 4 to 7, as cell proliferation increased the density and accelerated metabolism, the medium was changed every 1-2 days. (Note: The medium for the plate in the live-cell station was changed simultaneously, following the pattern where the wells with 1000 and 2000 cells/well had a lower change frequency than those with 4000 and 8000 cells/well). During medium change, the 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 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.