Maintenance of PANC-1 cells before spheroid generation

After thawing from liquid nitrogen, PANC-1 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

Protocol

  1. On the day of spheroid generation, bring the culture flask containing the cells 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 Automated Cell Counter or hemocytometer.
  6. We recommend that you use cells with ≥90% cell viability for spheroid generation.
  7. Spheroids can be generated from wells containing different seeding densities of cells. We recommend that you use the cell seeding calculator (below) to calculate the number of cells you require for each seeding density and then transfer that number of cells from the main stock to the plate.
  8. Seed 200 µl of cell suspension containing required number of cells in each well of 96-well Nunclon Sphera plate and label the wells.
  9. 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 then incubate the plate at 37°C, 5% CO2.
  10. Note this day as Day 0.

Spheroid maintenance

  • PANC-1 cells in Nunclon Sphera plates are stable and do not require ECM for their growth.
  • 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.
  • To obtain a fine spheroid for high-throughput assays, we recommend that you harvest spheroids from Day 5 to Day 7.
  • An optimal spheroid diameter is typically between 300 to 500 µm for high-throughput assays. Depending on the seeding density, this diameter may vary (see the figures below).

Tips

  • Centrifugation is an essential step to bring the cells together to form a spheroid, therefore we strongly recommend that you spin the plate after seeding.
  • Avoid shaking or any mechanical stress on the plate after centrifugation, as it may disturb the spheroids.
  • Seeding densities between 625 and 1,250 cells per well are recommended to obtain spheroid diameters between 300 and 500 µm.
  • After Day 7, if wish to extend the spheroid growth, you must refresh the culture medium. Replace 50% of spent medium with 50% of fresh complete medium for further observation.
  • Perform pipetting and aliquotting very carefully, without disturbing the spheroid. Placing pipette tips on sides of the wells when dispensing and aspirating liquids can help minimize the disturbance of the spheroids.

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

Morphology of PANC-1 spheroids

Microscopic image of multiple PANC-1 spheroids growing in culture following different seeding densities
PANC-1 spheroids generated in a Nunclon Sphera Plate in various densities from 312 to 10,000 cells. Images were captured at Day 3, Day 5 and Day 7 using the EVOS M7000 microscope. Scale bar denotes 650 µm.

Size and sphericity assessment

Spheroid size is typically a function of the starting cell seeding density, therefore it is important to assess this for the cell line you are using before you proceed with high-throughput experiments. The graph below shows the different spheroid sizes we obtained over a range of seeded PANC-1 cell densities. We recommend spheroids in the range of 300–500 µm diameter be used in high-throughput experiments.

Line graph of average spheroid diameter vs days in culture over the range of 10,000 to 312 cells

PANC-1 spheroids were grown in a Nunclon Sphera plate from different cell densities up to 7 days. Spheroid diameter during different days were measured and analyzed using ImageJ.

Sphericity is a measured parameter that indicates the roundness or circular nature of a spheroid. The more spherical the cell is, the more likely it is to remain intact during the course of the experiment, so highly spherical spheroids are preferred. The bar chart below shows the sphericity obtained from spheroids generated from a range of seeding densities.

Bar chart of spheroid sphericity vs number of cells seeded over the range of 10,000 to 312 cells

Sphericity of PANC-1 spheroids at different seeding densities, represented as percentages. 100% sphericity denotes a perfectly rounded spheroid. Error bars represent SEM.