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 cell