Culturing Human Melanoma Spheroids

Human wild-type MV3 melanoma cells, kindly provided by G. van Muijen (Department of Pathology, Radboud University Medical Center, Nijmegen; van Muijen et al., 1991 (link)), were cultured (humidified atmosphere, 10% CO₂, 37°C) in Dulbecco's modified Eagle's serum medium (DMEM, Invitrogen) supplemented with 10% fetal calf serum (Sigma), penicillin (100 U/ml, PAA), streptomycin (100 µg/ml, Invitrogen), L-glutamine (2 mM, Lonza) and sodium pyruvate (1 mM, Gibco). Identity of the MV3 cells was verified by short tandem repeat (STR) DNA profiling (IDEXX BioResearch), and no mammalian interspecies contamination was detected. Lack of contamination with mycoplasma was routinely verified using the MycoAlert Mycoplasma Detection Kit (Lonza). Cells were detached with EDTA (2 mM, Invitrogen), and multicellular spheroids were generated according to the hanging drop method (Korff and Augustin, 1998 (link)). Briefly, hanging droplets of a 30 µl volume containing 5000 cells, methylcellulose (40% dissolved in DMEM; Sigma) and bovine collagen (10 µg/ml; Advanced Biomatrix) were incubated for 24 h to ensure multicellular aggregation. Spheroids were washed and resuspended in medium.