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

- Spheroids are three-dimensional in vitro cell-culture models that mimic in vivo tumor architecture and help study intra-tumoral interactions. To set up the culture, first, prepare a single-cell suspension of melanoma cells in their appropriate medium. Next, spot small volumes of this cell suspension, sufficiently apart onto the inner surface of the lid of a sterile, non-adhesive culture dish. Carefully invert the lid and place it on the base of the culture dish containing PBS to create a hydration chamber. Incubate the plate under appropriate conditions.

Moisture from PBS prevents the evaporation of media from the hanging drops. Refresh the drops by replacing a few microliters of media. Owing to surface tension, the droplets maintain their spherical shape and remain suspended from the plate's inner surface. Gravitational forces facilitate the cells to remain in suspension without spreading but aggregate to form three-dimensional spheroids within the droplets. Finally, rinse the droplets with PBS to harvest the spheroids. Collect them in a non-adhesive culture dish.

In the following protocol, we will demonstrate the generation of three-dimensional spheroids of 451-LU melanoma cells via the hanging drop method.

- To begin, culture melanoma cells 451-LU using RPMI medium containing 10% FCS, according to general cell culture protocols. To generate melanoma spheroids, wash the cultured melanoma cells in PBS. Then, add 5 milliliters of 1x Trypsin-EDTA in PBS. Incubate for 3 to 5 minutes at room temperature. Add 5 milliliters of RPMI containing 10% FCS to neutralize the Trypsin. Then, transfer the cell suspension to a centrifuge tube. Centrifuge at 200 x g for 5 minutes to harvest the cells. Resuspend the cell pellet in RPMI containing 10% FCS.

Next, count the cells and dilute the cell suspension to a final concentration of 10,000 cells per milliliter. Use an electronic multi-pipette to make 40 x 25 microliters spots of cell suspension on the inner surface of the lid of a sterile, non-adhesive 10 centimeter cell culture dish.

Next, turn the lid over with a fast, smooth movement, and place it on the cell culture dish containing 5 milliliters of PBS. Culture the hanging drop dishes at 37 degrees Celsius in 5% carbon dioxide for 10 to 14 days.

Five days after the initial drop spotting, use an electronic dispenser to add 10 microliters of RPMI containing 10% FCS to each drop. After 10 to 15 days, harvest the spheroids by gently rinsing them off the lid with PBS. Collect the spheroids in a fresh, non-adhesive cell culture dish.