Establishment of a Spheroid Culture Model for Human Umbilical Vein Endothelial Cells (HUVECs)

Culture Conditions

HUVEC cells: 93% ECM + 5% FBS +1%P/S + 1% ECGS  

Cell recovery

1) Frozen vials of HUVEC cells were retrieved from liquid nitrogen and rapidly thawed in a 37 ℃ water bath with gentle agitation.  

2) The thawed cell suspension was transferred into a centrifuge tube containing 3 mL of culture medium and centrifuged at 1000 rpm for 5 minutes at room temperature. The supernatant was discarded.  

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

Cell Passaging

1) Cells were passaged when reaching 80% confluence.  

2) The culture medium was aspirated, and the cells were washed once with PBS.  

3) 1–2 mL of 0.25% trypsin was added to dissociate the cells. Digestion was performed at room temperature for 1–2 minutes and monitored under a microscope until the cells rounded up and detached.  

4) Trypsin was quickly removed, and complete medium was added to neutralize the enzyme. The cells were gently pipetted to form a single‑cell suspension and subcultured at a split ratio of 1:2 to 1:4. Expanded culture was continued under the same conditions (37 ℃, 21% O₂, 5% CO₂).

Cell Seeding

HUVEC cells in the logarithmic growth phase and in good growth condition 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 was prepared), with five replicate wells per density. The peripheral wells of the 96-well 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 cultivation and imaging. Note: The live-cell imaging station was installed in a CO2 incubator (Thermo, 3111) and pre-warmed for 30 min. The culture conditions were maintained at 37°C, with 21% O₂, 5% CO₂, and saturated humidity.

Medium Change

The culture medium for HUVEC cells was replaced every 24 h (with synchronized replacement for the plate in the live-cell imaging station). The replacement frequency for the 1000 and 2000 cells/well groups was lower than that for the 4000 and 8000 cells/well groups. The replacement was continued for 120 h, totaling five medium changes. During replacement, the old medium was carefully aspirated, with all six replicate wells of one density aspirated at once, followed promptly by replenishment with 100 μL/well of fresh complete medium.