Maintenance of cells before PC-3 spheroid generation

After thawing from liquid nitrogen, 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. Once the flask was 70-80% 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 and cell suspension was diluted in ice cold DMEM medium.
  5. Geltrex matrix was then thawed completely on ice and added at a final concentration of 3% to the above cell suspension. (for lower cell densities reduce the concentration of Geltrex matrix)
  6. A volume of 200 μl of the cell suspension was added to each well of 96-well Sphera plates.
  7. Plates were then centrifuged at 1,500 rpm for 10 min at 4°C and placed in the incubator at 37°C and 5% CO2.

Day 5–9

  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

Tips

  • Be very careful about the temperature conditions of Geltrex matrix. Keep it ice-cold at all times.
  • While preparing the cell suspension make sure the media is cold. This cold media doesn’t harm the cells.
  • Make sure after adding Geltrex matrix into the suspension it is mixed properly without forming bubbles.

Morphology of PC-3 spheroids cultured in the presence of Geltrex matrix

Microscopic image of multiple PC-3 spheroids growing in the presence of Geltrex matrix and at different seeding densities

150–5,000 PC-3 cells were seeded using Geltrex matrix for spheroid generation and brightfield images of spheroids at Day 3, Day 5, Day 7 and day 9 were captured using EVOS M7000 microscope under 4x magnification. Scale bar denotes 650 μm.

Morphology of PC-3 spheroids cultured in the absence of Geltrex matrix

Microscopic image of multiple PC-3 spheroids growing in the absence of Geltrex matrix and at different seeding densities

150–5,000 PC-3 cells were seeded without using Geltrex matrix for spheroid generation and brightfield images of spheroids at Day 3, Day 5, Day 7 and day 9 were captured using EVOS M7000 microscope under 4x magnification. Scale bar denotes 650 μm.