Spheroid confrontation assay: a simple method to monitor the three-dimensional migration of different cell types in vitro.

Cell migration and tissue invasion is an important issue in tumour and developmental research. Here, we describe the spheroid invasion assay in detail, a method for monitoring migration of different cell types in a three-dimensional model in vitro. Different (or same) cell types are fluorescently labelled with two different dyes, e.g. Vybrant® CFDA SE Kit ([5(6)carboxyfluorescein diacetate] succidimidylester, green) or SNARF-1® (red), and spheroids of these cells are formed in a medium with 0.24% methylcellulose. After this, spheroids are detached, picked and confronted with each other. After different periods of time, cell invasion can be easily followed microscopically. As an example, the method was applied here to visualize the migration and invasion of microglial and glial precursor cells into spheroids of tumour cells driven by chemokines or chemotactic growth factors. Antibodies to chemokines or chemotactic growth factors/receptors or inhibitors of signal transduction/proteases generating soluble factors can be used to demonstrate the specificity of the chemotactic agents. Thus, this method provides an easy in vitro-method to monitor chemotaxis and three-dimensional cell migration.