Maintenance of MDA-MB-231 cells before 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 1 passage before seeding for spheroid generation. ATCC protocol was followed for subculturing.

Materials required

Protocol

  1. On the day of the experiment, 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.
  5. Required number of cells were seeded in respective wells of the Nunclon Sphera plate using a multi-channel pipette. The final volume was maintained at 100 μl.
  6. The plate was centrifuged at 290 g for 3 minutes and placed in the incubator. This is Day 0.
  7. On Day 1, 100 μl of complete medium containing 6 μg/ml collagen I was added to each well, so that the final concentration of collagen in the medium is 3 μg/ml. The plate was centrifuged at 100 g for 3 minutes and placed back in the incubator.
  8. Spheroids were ready on Day 4 (>2,000 cells) to Day 7 (<2,000 cells) depending on the seeded cell 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

  • Fill the outermost wells of the plate with PBS to prevent evaporation of media during incubation.
  • On Day 1, add the medium carefully through the sides of the wells.
  • On Day 1, the medium should be at room temperature and not at 37 degrees. Also, keep the collagen I on ice until ready to use.

Morphology of MDA-MB-231 spheroids

Microscopic image of multiple MDA-MB-231 spheroids growing in culture following different seeding densities

1,000–10,000 MDA-MB-231 cells were seeded for spheroid generation and brightfield images of spheroids at Day 2, Day 4 and Day 7 were captured using EVOS M5000 microscope under 4x magnification. Scale bar denotes 400 μm.

ECM makes a difference in MDA-MB-231 spheroid formation

Microscopic image of multiple MDA-MB-231 spheroids growing in the presence of collagen and at different seeding densities

1,000–10,000 MDA-MB-231 cells were seeded for spheroid generation with or without collagen according to the above-mentioned protocol. Brightfield images of cell aggregates and spheroids on Day 4 were captured using EVOS M5000 microscope under 4x magnification. Scale bar denotes 400 μm. The addition of collagen was found to dramatically improve the formation of spheroids.