Maintenance of SKOV-3 cells before spheroid generation

After thawing from liquid nitrogen, cells were maintained in Nunclon Delta T25 cell culture flasks in Gibco McCoy’s 5A medium supplemented with 10% Gibco  FBS and 1% Pen-Strep for 1 passage before seeding for spheroid generation. ATCC protocol was followed for subculturing.

Materials required


  1. On the day of the experiment, medium from the flask was aspirated, the cells were washed once in 1X PBS and dissociated using 1–1.5 ml of TrypLE reagent.
  2. The TrypLE reagent was neutralized using 4 volumes of complete medium, and live-cell count and viability were captured using Countess II cell counting chamber. Cells with >90% viability were taken for spheroid generation.
  3. The stock of cells was diluted 1:10 to 1:20 in complete medium to make calculations for cell seeding density easier.
  4. Seeding cell number was calculated using the cell seeding calculator.
  5. Required number of cells were seeded in respective wells of the Nunclon Sphera plate using a multi-channel pipette. The final volume was maintained at 100 μl.
  6. The plate was centrifuged at 250 g for 5 minutes and placed in the incubator. This is Day 0.
  7. On Day 2, 100 μl of fresh medium was added to each well using a multichannel pipette. The plate was centrifuged at 250 g for 5 minutes after media change and placed back in the incubator.
  8. Spheroids were ready on Day 3.

Cell Seeding Calculator

Number of live cells/mL
(as determined by the Countess Automated Cell Counter)

Number of cells to seed per well
(user-specified number)

... µL

Volume of cell suspension that contains
the specified number of cells


  • Fill the outermost wells of the plate with PBS to prevent evaporation of media during incubation.
  • Seeding densities of <2,500 cells or of >10,000 cells do not result in the formation of good spheroids.


  • There is significant compaction of the cell aggregate on Day 1 compared to Day 0.
  • Beyond Day 3, the compactness and viability of the spheroids are compromised.

Morphology of SKOV-3 spheroids

Microscopic image of multiple SKOV-3 spheroids growing in culture and at different seeding densities

2,500–20,000 SKOV-3 cells were seeded for spheroid generation and brightfield images of spheroids at Day 0, Day 1 and Day 3 were captured using EVOS M7000 microscope under 4x magnification. Scale bar denotes 650 μm.