Preparation of pure neuronal and non-neuronal cultures from embryonic chick sympathetic ganglia: a new method based on both differential cell adhesiveness and the formation of homotypic neuronal aggregates.
A new method has been developed for the preparation of essentially pure primary cultures of neurons and non-neuronal cells from 11-day embryonic chick sympathetic ganglia. This method utilizes (1) differences in cell-to-substrate adhesiveness between neurons and non-neuronal cells and (2) the capacity of neurons to form homotypic aggragates. The maximum difference in adhesiveness between neuronal and non-neuronal cells occurred when the ganglia were dissociated with trypsin following collection in a salt solution lacking divalent cations. This difference allowed the preparation of highly purified non-neuronal cultures and 85-90% pure neuronal cultures. Intermittent agitation during the period of cell separation markedly increased the purity of the neuronal cultures by (1) inhibiting neuronal but not non-neuronal cell attachment and (2) facilitating the formation of homotypic neuronal aggregates in the supernatant. Neuronal and non-neuronal cultures prepared under these conditions were more than 99% pure on the basis of both morphological and biochemical analyses. Both cell types exhibited attachment efficiencies greater than 95% and have been maintained for several weeks in vitro. Thus, completely isolated neuronal and non-neuronal cultures can be prepared and maintained for prolonged periods in the absence of cells of the other type.
D000818:Animals; D002448:Cell Adhesion; D002449:Cell Aggregation; D002469:Cell Separation / Q000379:methods*; D002478:Cells, Cultured; D002642:Chick Embryo; D002802:Cholinesterases / Q000032:analysis; D004247:DNA / Q000096:biosynthesis; D005725:Ganglia, Autonomic / Q000166:cytology* / Q000201:enzymology / Q000502:physiology; D009474:Neurons / Q000166:cytology / Q000201:enzymology / Q000502:physiology*; D013997:Time Factors
D004247:DNA; D002802:Cholinesterases