Formation of Tumor Spheroids of Jurkat T Cell

Culture Conditions

Jurkat T cells ：89%RPMI 1640+10%FBS+1%PS

Cell recovery

1) Retrieve the Jurkat T cells from the liquid nitrogen tank and immediately place the cryovial in a 37℃ water bath. Gently agitate the vial to facilitate thawing.

2) After thawing, transfer the cell suspension to a centrifuge tube containing 3 mL of pre-warmed culture medium. Centrifuge at 1000 rpm for 5 minutes at room temperature to pellet the cells. Carefully discard the supernatant.

3) Resuspend the cell pellet in complete medium and transfer the suspension to a culture dish. Mix gently by pipetting and incubate in a standard humidified incubator at 37℃ with 21% O₂ and 5% CO₂.

Cell Passaging

1) Passage the cells when the density reaches approximately 80%.

2) Collect the cell suspension and centrifuge at 1000 rpm for 3-5 minutes.

3) Discard the supernatant, add 1-2 mL of fresh culture medium, and resuspend the cells by gentle pipetting. Transfer the cell suspension to new culture dishes containing 3-4 mL of fresh medium at a split ratio of 1:2 to 1:4.

4) Incubate the cultures in a standard humidified incubator at 37℃ with 21% O₂ and 5% CO₂ for further expansion.

Cell Seeding

Jurkat cells in the logarithmic growth phase and exhibiting good viability 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 (for one plate in total), with five replicate wells for each density. The peripheral wells of the plate were filled with 100 μL of sterile PBS. The prepared plate was then transferred to the live cell station for culture and time-lapse imaging. Note: The live cell station, installed within a CO₂ incubator, was pre-warmed for 30 minutes and maintained at 37℃ with 21% O₂, 5% CO₂, and saturated humidity throughout the experiment.

Medium Change

During the first three days, due to the low initial cell density and relatively slow metabolic activity, the medium was changed every 2-3 days (i.e., at 24-hour intervals within this period). From day 4 to day 7, as cell proliferation increased the density and accelerated metabolism, the medium was changed every 1-2 days. (Note: Medium changes were performed synchronously for the plate in the live cell station. The medium change frequency for wells seeded at 1000 and 2000 cells/well was lower than that for wells seeded at 4000 and 8000 cells/well). For the medium change process, cell spheroids were carefully transferred using a 200 μL yellow pipette tip into a 35 mm culture dish containing 1 mL of fresh complete medium. All spheroids from the five replicate wells of a given density were transferred simultaneously. The old medium in the wells was then aspirated and discarded. Each well was rinsed 2-3 times with 100 μL 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.