Materials and Reagents

75 cm² cell culture flasks (Thermo Fisher, catalog number: 156499 )
10 cm dishes (Thermo Fisher, catalog number: 150464 )
15 ml conical tubes (Corning, catalog number: 352196 )
Falcon^TM permeable support for 24-well plate with 0.4 µm transparent PET membrane (Corning, catalog number: 353095 )
Falcon^TM 24-well TC-treated cell polystyrene permeable support companion plate (Corning, catalog number: 353504 )
Mus musculus B16F10 skin melanoma cells (ATCC, catalog number: CRL-6475 )
PBS, pH 7.4 (Gibco, catalog number: 10010023 )
0.25% Trypsin-EDTA, phenol red (Gibco, catalog number: 25200072 )
Trypan Blue solution (0.4%) (Thermo Fisher, catalog number: 15250061 )
RPMI-1640 medium with L-glutamine and sodium bicarbonate, liquid, sterile-filtered (Sigma-Aldrich, catalog number: R8758 )
Heat inactivated Fetal Bovine Serum (Japan Bioserum, catalog number: S1820-500 ) (Testing of different batches is recommended)
MEM Non-essential Amino Acid solution (100×) (Sigma-Aldrich, catalog number: M7145 )
Sodium Pyruvate (100 mM) (Gibco, catalog number: 11360070 )
HEPES solution (1 M) (Sigma-Aldrich, catalog number: H3537 )
Penicillin-Streptomycin (10,000 U/ml) (Gibco, catalog number: 15140122 )
2-Mercaptoethanol (Gibco, catalog number: 21985023 )
L-Glutamine (200 mM) (Lonza, catalog number: 17-605E )
Complete cell culture medium (see Recipes)

Equipment

Pipettes
Hemocytometer
Incubator (37 °C, 5% CO₂)
Water bath (37 °C)
Refrigerated benchtop centrifuge for spinning conical tubes
Inverted microscope

Procedure

Single Cell Suspension Preparation
1. Plate early (3-4) passage of B16F10 cells in a 75 cm² cell culture flask in complete cell culture medium. Incubate at 37 °C until cells reach 80-90% confluency.
2. Remove and discard cell culture medium.
3. Briefly rinse the monolayer with PBS to remove all traces of serum since it contains the trypsin inhibitor.
4. Detach adherent cells from the surface of a cell culture flask by adding 1.0 to 2.0 ml of 0.25% Trypsin-EDTA solution to the flask. Incubate at 37 °C until the monolayer is dispersed (usually within 5-10 min). Monitor cells under an inverted microscope.
5. As soon as cells have detached, neutralize the Trypsin-EDTA solution by adding 8.0 to 9.0 ml of complete cell culture medium (shown below). Gently pipette up and down the mixture until cells are in suspension.
6. After spinning down, count cells using a hemocytometer and perform a dilution to allow for the seeding of 10,000 cells per 25 μl of complete cell culture medium.
Generation of Spheroids
1. Remove the lid from the 10 cm dish and carefully deposit 25 μl drops on the inner side of the lid.
2. Fill the bottom of the dish with 10 ml PBS to humidify the culture chamber after distribution of the drops.
3. Flip the lid in a confident, yet controlled manner. Cells under the force of gravity will be aggregated at the bottom of the hanging drop. Make sure to place the drops sufficiently apart from each other and not too close to the dish edges.
4. Carefully place the dish in the incubator and preferably avoid touching or moving for the first 2 days.
5. Change cell culture medium on the 3^rd and 6^th day in each drop. To this end, carefully remove the lid from the dish and replace approximately 10 μl of used media with 10-15 μl of fresh media in each drop, in order to maintain constant osmolality and nutrient supply. Replace the medium from the edge of the drop. Avoid touching spheroids. Flip the lid in a confident, yet controlled manner.
6. Monitor the drops under an inverted microscope. Pay attention not to disturb the droplets while moving the dish.
  
  Figure 2. Representative photographs of skin melanoma spheroids. B16F10 melanoma cells (10,000 cells per 25 ul drop of cell culture medium) placed on the lid of a PBS containing dish and incubated for 3 and 7 days. Objective magnification: 3.2x (lower panel).
Co-cultures with Immune Cells
1. Collect spheroids by the wide end of the tip (100 or 1,000 μl pipette tip cut widely 5-6 mm from the end) and transfer to an appropriate cell culture plate.
2. Co-culture spheroids with immune cells in complete cell culture medium by either layering two components one on top of the other in the same well of the cell culture plate or by employing cell culture inserts with porous membranes, to keep immune cells separated from spheroids.

Notes

Antibiotics (and antimycotics) may be added to PBS in order to minimize bacterial (and fungal) contamination during the period of spheroid generation.
Always prepare a surplus number of spheroids in case some of the drops detach from the lid or slide on the surface of the lid during the change of the medium process.
The longer time of incubation is possible, however the cell viability should be assessed. We have observed around 90% cell viability following 7 days of incubation.
For the use of other types of cancer cells, the number of cells and the volume of the drops should be adjusted.

Recipes

Complete cell culture medium
RPMI-1640 medium
10% Fetal Calf Serum
100 μM MEM Non-essential Amino Acid
10 mM HEPES
100 U/ml Penicillin-Streptomycin
1 mM Sodium Pyruvate
50 μM 2-mercaptoethanol
400 μM L-Glutamine

Acknowledgments

This work was supported by the FRIPRO Mobility Grant Fellowship from the Research Council of Norway (302241) to M.W., and by a Grant-in-Aid for Scientific Research (A) (20H00511) from the Japan Society for the Promotion of Science, to S.K. This protocol was adapted from the original research paper published in Wagner et al. (2020).

Competing interests

The authors declare no competing interests.

References

Berens, E. B., Holy, J. M., Riegel, A. T. and Wellstein, A. (2015). A cancer cell spheroid assay to assess invasion in a 3D setting. J Vis Exp (105): 53409.
Foty, R. (2011). A simple hanging drop cell culture protocol for generation of 3D spheroids. J Vis Exp (51): 2720.
Moro, K., Yamada, T., Tanabe, M., Takeuchi, T., Ikawa, T., Kawamoto, H., Furusawa, J., Ohtani, M., Fujii, H. and Koyasu, S. (2010). Innate production of T_H2 cytokines by adipose tissue-associated c-Kit⁺Sca-1⁺ lymphoid cells. Nature 463(7280): 540-544.
Moro, K., Ealey, K. N., Kabata, H. and Koyasu, S. (2015). Isolation and analysis of group 2 innate lymphoid cells in mice. Nat Protoc 10(5): 792-806.
Sant, S. and Johnston, P. A. (2017). The production of 3D tumor spheroids for cancer drug discovery. Drug Discov Today Technol 23: 27-36.
Wagner, M., Ealey, K. N., Tetsu, H., Kiniwa, T., Motomura, Y., Moro, K. and Koyasu, S. (2020). Tumor-derived lactic acid contributes to the paucity of intratumoral ILC2s. Cell Rep 30(8): 2743-2757 e2745.
Wagner, M. and Koyasu, S. (2019). Cancer immunoediting by innate lymphoid cells. Trends Immunol 40(5): 415-430.