Establishment of a Spheroid Culture Model for Human Umbilical Cord Mesenchymal Stem Cells(HUC-MSCs)

Culture Conditions

HUC-MSC cells: 93% MSCM + 5% FBS + 1% P/S + 1% MSCGS

Cell recovery

1)  HUC-MSC 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 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 proceeded at 37℃ for 1-2 minutes and was monitored under a microscope until cell detachment and rounding were observed, indicating completion.

4)  The trypsin was quickly aspirated, and complete culture medium was added. The cells were gently pipetted to generate a single-cell suspension. The cells were subcultured at a split ratio of 1:2 to 1:4 and expanded in a standard incubator at 37℃ with 21% O₂ and 5% CO₂.

Cell Seeding

HUC-MSC 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

The culture medium for HUC-MSC cells was changed every 24 hours (the plate within the live-cell station was changed simultaneously). The medium change frequency for the 1000 and 2000 cells/well densities was lower than that for the 4000 and 8000 cells/well densities. This process continued for 120 hours, totaling 5 medium changes. During medium change, the old medium was carefully aspirated, with all five replicate wells of one density aspirated at once, and then quickly replenished with 100 μL/well of fresh complete culture medium.