Culture and Phenotyping of Glial Cell Cultures, Gliospheres, and Neurospheres.

Cell cultures constitute an important tool for research as a way to reproduce pathological processes in a controlled system. However, the culture of brain-derived cells in monolayer presents significant challenges that obscure the fidelity of in vitro results. After a few number of passages, glial and neuronal cells begin to lose their morphological characteristics, and most importantly, their specific cellular markers and phenotype. In recent years, the discovery of neural progenitor cells, and the methodology to culture them in suspension maintaining their potentiality while still retaining the ability to differentiate into astrocytes, oligodendrocytes and neurons has been a significant contribution to the fields of neuroscience and neuropathology.In the brain, progenitor cells are located in the Germinal Matrix, the subventricular zone in what later would become the basal ganglia, and play an essential role in the homeostasis of the brain by providing the source to replace differentiated cells that have been lost or damaged by different pathological processes, such as senescence, injury, genetic conditions, or disease. The discovery of these neural stem cells in an organ traditionally thought to have limited or no regenerative capacity has opened the door to the development of novel treatments, which include cell replacement therapy. Here we describe the culture and differentiation of neural progenitor cells into neurospheres, and the phenotyping of the resulting cells using immunocytochemistry . The immunocytological methods outlined are not restricted to the analysis of neurosphere-derived cultures but are also applicable for cell typing of primary glial or cell line-derived samples.