Preparation of a tissue-like cortical primary culture from embryonic rats using Matrigel and serum free Start V Medium.

To address the scientific quest for unravelling signalling pathways crucial in CNS development and function, cell culture systems have to be developed that are mimicking the physiological state of brain cells more efficiently. Here, we describe a method for cultivation of a virtual three-dimensional structure consisting of neural stem cell-derived cell types by using Matrigel as surface substrate and Start V as a serum free medium. We demonstrate that free floating dissociated cells form attached neurospheres from which cells start migration to surrounding areas and develop a virtual three-dimensional cell structure composed of neurons, glia and neural stem cells. Neuronal precursor cells differentiate into cholinergic and GABAergic cells and express vesicle proteins. Further, neuronal cells are interwoven with Nestin positive stem cells and GFAP positive astrocytes. Additionally, oligodendrocytes and microglia can also be detected in this neural tissue-like structure. As an example for studying cell migration we added externally microglial cells (BV2) and performed a confocal time lapse study. It revealed, that co-cultivated microglial cells migrated towards neurospheres within 14 h. Thus, the described method provides a serum free, tissue-like primary cell culture system of neural cells useful for the investigations of basic cell-cell interactions under in vitro conditions.