Hanging Drop Method: A Technique to Generate 3D Melanoma Spheroids

1. Generation of 3D Melanoma Spheroids Via the Hanging Drop Method

1. Culture melanoma cells (e.g., 451-LU, or any melanoma cell line of interest) according to general protocols, using RPMI containing 10% FCS.
   1. To generate melanoma spheroids of similar size and quality, wash the melanoma cells in PBS, add 5 mL of 1x Trypsin-EDTA in PBS to the cells in a T175 cell culture flask, and incubate for 3 – 5 min at room temperature (RT). Neutralize the Trypsin by adding 5 mL RPMI/10% FCS. Harvest the cells by centrifugation at 200 x g for 5 min. Resuspend the cell pellet in RPMI/FCS at a final concentration of 10,000 cells/mL as determined by counting in a hemocytometer.
2. Spot 40 x 25 µL (= 250 cells) of the cell suspension onto the inner surface of the lid of a sterile non-adhesive cell culture dish (Ø 10 cm) using an electronic multi-pipette. With a fast but smooth movement, turn the lid around and place it on the respective cell culture dish containing 5 mL PBS.
   1. Culture "hanging drop dishes" in a cell incubator at 37 °C and 5% CO₂ for 10-14 days, depending on the cell type used.
      NOTE: Cells from the metastatic melanoma growth phase typically grow faster and form more solid spheroids in the hanging drop compared to melanoma cells derived from the radial growth phase or vertical growth phase. For individual evaluation, observe spheroid growth under a pair of binoculars or under a light microscope (4X magnification). Usually, spheroids become detectable after 48 h under a pair of binoculars or under a light microscope (4X magnification). After 10-14 days, they are visible without any magnification device.
3. Five days after the initial drop spotting, add 10 µL of fresh RPMI/10% FCS medium to each drop. Subsequently, exchange 10 µL of medium every other day. The use of an electronic dispenser is very helpful in this step.
4. Depending on the cell type, harvest the spheroids after 10-15 days. Carefully rinse the spheroids off the lid of the hanging drop cell culture dish with PBS. Collect 10-20 spheroids in a sterile non-adhesive cell culture dish. Carefully remove excessive PBS with a Pasteur pipette.
   NOTE: The cultivation period depends on the tumor growth phase of the melanoma cells when they were initially derived, e.g., spheroids derived from 451-LU cells grow to approximately 500 µm in diameter within 12 days of culturing in the hanging drop.