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

Protocol for spheroid generation

  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. 
  • We have observed that centrifuging the plate after seeding cells facilitates cell aggregation leading to uniform spheroid formation consistently. However, this step is optional.

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 = 1,000 µm.

Characterization of HepG2 spheroids

Assessing cell viability using PrestoBlue-HS cell viability reagent

  1. On Day 6, cell viability assay was performed using the PrestoBlue HS cell viability reagent
  2. The PrestoBlue HS reagent was warmed up to room temperature. Then, 20 μl was added to each well containing 200 μl of medium using a multichannel pipette, and gently mixed by pipetting 2-3 times. Wells containing only fresh medium and the reagent were used as normalization control (blank).
  3. The plate was incubated at 37°C for 4 hours.
  4. Following this, fluorescence was read using the Varioskan LUX Multimode Microplate Reader using the following settings:
  • Excitation: 560 nm; Emission: 590 nm
  • 12 nm excitation bandwidth
  • Measurement time: 100 ms
  • Bottom optics reading
  • Instrument temperature: 37°C

The data was exported to Microsoft Excel for analysis. Individual fluorescence values were normalized to Blank and mean of at least 6 replicates per cell seeding number were plotted using graphing and statistics software. The experiment was repeated 3 times.

bar chart showing relative fluorescence units as a function of cell number

Graph showing relative viabiltiy of HepG2 spheroids of Day 6 post-cell seeding. Error bar represents SEM.

Visualizing live and dead cells using LIVE/DEAD Viability/Cytotoxicity kit

  1. The population of live and dead cells on Day 6 HepG2 spheroids were visualized by staining them using the LIVE/DEAD Viability/Cytotoxicity kit.
  2. Working solution was prepared by adding Calcein AM and EthD-1 at a final concentration of 1 μM and 3 μM respectively  respectively in fresh medium (See our application note for more information on fluorescence staining of spheroids).
  3. NucBlue Live ReadyProbes Reagent was added (2 drops per ml) to the working solution for nuclear staining of the spheroids. Spent medium of spheroids was changed 1:1 with working solution and incubated at 37°C for 3 hours.
  4. Following this, they were washed three times with PBS + 5% FBS (1:1 change), and finally resuspended in PBS + 0.5% FBS (1:1 change), each time centrifuging the plate at 500 rpm  for 5 minutes at room temperature to settle the spheroids and minimize background during fluorescence imaging.
  5. The spheroids were then imaged using CellInsight CX7 High-Content Screening Platform under 4X objective.
Note: With increasing spheroid diameter, there is reduced penetration of dyes and an increase in the dead cell population (red staining).
microscopic views of fluorescently stained spheroids

Representative images of Day 6 HepG2 spheroids showing the live (green staining) and dead (red staining) cell population (top row). The bottom row shows nuclear staining. Scale bar = 100 µm. 

Bar chart of percent dead cells as a function of number of cells seeded

Graph showing percent dead cells in HepG2 spheroids on Day 6. Fluorescence reading was captured using Varioskan LUX Multimode Microplate Reader. Values were plotted using GraphPad Prism. Spheroids treated with 70% methanol overnight were used as positive control for dead cells for respective seeding densities.