Maintenance of HepG2 cells before spheroid generation

After thawing from liquid nitrogen, cells were maintained in Nunclon Delta T25 cell culture flasks in Gibco MEM medium supplemented with 10% Gibco FBS and 1% Pen-Strep for 2 passages before seeding for spheroid generation. ATCC protocol was followed for subculturing.

Materials required


  1. Once the flask was 60–70% confluent, 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. 

Day 0

  1. Required number of cells was seeded in respective wells of the Nunclon Sphera plate using a multi-channel pipette. The final volume was maintained at 200 μl.
  2. The plate was centrifuged at 1,500 rpm for 10 minutes and placed in the incubator at 37°C and 5% CO2.

Media change

  1. Media was changed 1:1 every alternate day. (aspirate 100 μl of spent medium and add 100 μl of fresh medium). The plate was centrifuged at 1,200 rpm for 5 minutes after media change and placed back in the incubator with above mentioned conditions.

Day 4–8

  1. Spheroids were ready, depending on seeding number.

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. 
  • For HepG2 cells, seeding density of >5,000 cells does not result in the formation of good spheroids.
  • Before seeding the cells for spheroid generation make sure the confluency of the flask is not too high. Always use evenly spaced HepG2 cells for spheroid generation. 
  • Be gentle while pipetting and ensure the tip is not touching the Sphera 96-well plate surface. 

Morphology of HepG2 spheroids

Microscopic image of multiple HepG2 spheroids growing in culture following different seeding densities

312–20,000 HepG2 cells were seeded for spheroid generation and brightfield images of spheroids at Day 2, Day 4, Day 6, and Day 8 were captured using EVOS XL microscope under 4x magnification. Scale bar denotes 1,000 µm.