Separation and characterization of neuronal and glial cell populations from embryonic chick cerebra in culture

Abstract

A new procedure of separation of glial and neuronal cell population from embryonic chick cerebra is described and their morphology in vitro was examined by SEM. This technique used the differential adhesive properties of the glial and neuronal cells to obtain an initial separation in primary monolayer culture. The neuronal fraction was then further purified by treatment with cytosine arabinoside. The homogeneity of the glial and neuronal cultures produced by this technique was examined by phase contrast and scanning electron microscopy, liquid scintillation counting of incorporation of radioactive precursors into the cultured cells and autoradiographic study of the cultures. The purity of the neuronal culture was estimated to be better than 97 and 98% based on LSC and autoradiography respectively. The purity of glial culture was assessed by phase contrast and SEM and was estimated to have a purity of over 99%. The viability of both cultures was good following initial separation. The glial cells were typically epitheloid and formed a confluent monolayer 7-10 days after the initial separation. These cells have a smooth upper surface and are typically hexagonal in shape. The neuronal cultures formed small aggregates interconnected with compound neuronal processes. It was noted that the neuronal differentiation was closely related to the glial cells. In the presence of a glial carpet, the aggregates became flattened and well differentiated neuronal cells were found. On the contrary, round neuronal aggregates were found. In the case of mixed cultures of glial and neuronal cells neurites were seen growing mainly on the surface of the glial carpet. Only on rare occasions were neurites seen bridging over the bare glass surface.