Maintenance of cell line before spheroid generation

After thawing from liquid nitrogen, SW480 cells were maintained in Nunclon Delta T25 cell culture flasks in Gibco DMEM 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:

Media preparation with Collagen I (as extracellular matrix or ECM):

  • Spheroids can be generated with or without extracellular matrix support depending on the nature of the cell line. SW480 cells require ECM adhesion to grow in 3D confined space. Also, it is important to define the concentration of the Collagen I or ECM used to generate spheroid.
  • Some cell lines can require only one concentration of ECM for all different cell densities, whereas SW480 cells require cell number dependent ECM. Here we established the amount of Collagen I required to generate different sizes of spheroids. Use the table below as a reference for the amount of Collagen I required with respect to cell density.
Cell number
to seed
Amount of Collagen I
per ml of media
10,000 cells 30 µg
5,000 cells 15 µg
2,500 cells 8 µg
1,250 cells 4 µg
625 cells 2 µg
312 cells 1 µg
  • Prepare media containing Collagen I according to your cell density before proceeding to the protocol. Store the media mixture at 4°C, and use right before cell seeding. It is always recommended to prepare fresh media mixture every time.


  1. On the day of spheroid generation, bring the designated flask to the biosafety cabinet.
  2. Discard the spent media from the flask, wash the cells with 1x PBS, and dissociate the cells using 1–2 ml of TrypLE reagent. Allow the cells to completely detach from the surface.
  3. Add 4 volumes of fresh complete medium to neutralize the TrypLE reagent.
  4. Spin down the cells, and add fresh complete medium to the pellet.
  5. Perform cell counting using Countess II Cell Counter or hemocytometer.
  6. Make sure the cells are viable above >90%.  It is recommended to proceed with good cell viability.

Spheroid generation:

  1. Different cell densities produce different size of spheroids; hence the required number of cells can be calculated and aliquoted from the main stock. Use the seeding calculator (below) to calculate the number of cells required.
  2. Make sure to aliquot the cells in media containing Collagen I according to their cell density. Refer to the table above to find the right amount of Collagen I for different densities.
  3. Seed 200 µl of cell suspension containing required number of cells in each well of 96-well Nunclon Sphera plate, and label them.
  4. Centrifuge the 96-well plate in a swinging bucket rotor with the recommended plate holder at 1,500 rpm for 10 minutes at room temperature, and incubate the plate at 37°C at 5% CO2.
  5. Note this day as Day 0.

Spheroid maintenance:

  • Spheroids can be maintained up to Day 7 without any media replacement. Observe the spheroid plate at least every 24 hours to monitor morphological changes.
  • SW480 cells start generating spheroid from Day 3 onwards.  Hence, it is recommended to harvest spheroids between Day 3 to Day 7 for high throughput assays.
  • An optimal spheroid diameter should be between 300–500 µm for high-throughput assays. This may vary depending on the seeding density. Please refer to the results below for more information.


  • Centrifugation is an essential step to bring the cells together to form a spheroid. Hence, it is highly recommended to spin the plate after seeding.
  • Avoid shaking or any mechanical stress on the plate after centrifugation. It may disturb the spheroid.
  • Seeding densities between 625–2,500 cells per well is recommended to obtain a spheroid diameter of 300–500 µm.
  • After Day 7, if spheroid growth is to be extended, media replacement must be done. Replace 50% of spent media with 50% of fresh complete media for further observation.
  • Pipetting and aliquoting should be done carefully without disturbing the spheroid. Placing tips on sides of well can avoid this issue.
  • Multi-channel pipettes can be used for seeding when plating large number of spheroids.

Morphology of SW480 Spheroids

Morphology of SW480 spheroids generated in various densities from 625 to 10,000 cells.
Legend: SW480 spheroids generated in Nunclon Sphera Plate in various densities from 625 to 10,000 cells. Images were captured at Day 2 and Day 7 using EVOS 7000 microscope. No ECM bar represents merely cell aggregate and not a spheroid, whereas with Collagen I spheroids were uniform and intact. Scalebar equals to 650 µm.

Size and Sphericity Assessment:

Each cell density tends to generate different spheroid sizes; hence it is important to assess the density of your spheroid before proceeding to high throughput experiments. The graph below projects the different spheroid sizes we obtained with respect to their cell density in SW480 cell line. A good spheroid size for HT assays would be around 300-500 µm of diameter.

Sphericity is another phenomenon that defines the roundness or circular nature of a spheroid. Greater sphericity ensures the spheroid is intact, circular, and hard to disintegrate.

Sphericity of SW480 spheroids generated in various densities from 625 to 10,000 cells