Separation of cell types from the developing cerebellum.

The heterogenous population of perikarya (cells) obtained by dissociating cerebellar tissue of developing postnatal rats was separated by sedimentation at unit gravity. Peak fractions, defined by monitoring the distribution of different size classes with a particle analyzer, were enriched in ultrastructurally well-preserved and metabolically competent specific cell types. These fractions included the peak of rapidly sedimenting cells comprising large neurones, such as Purkinje cells, which accounted for about 50% of the cells (vs. 2% in the initial cell suspension) and for a much greater proportion of the total cell mass (over 80%). More slowly sedimenting peak fractions contained 2-5-fold enrichments in replicating cells (assessed in terms of [3H]thymidine incorporation into DNA or by the frequency of mitotic cells), astroglia-like cells and external and differentiating granule cells, respectively. Separated, replicating cells continued synthesizing DNA in vitro; the [3H]thymidine incorporation rate was about 5-fold greater in the fraction enriched in proliferating cells than in the total cell suspension. Besides their structural integrity, the viability of the cells was also indicated by the finding that the proportion of trypan blue-excluding cells in all fractions exceeded 80%. Moreover, protein synthesis, in terms of incorporation of labelled amino acids, continued in the separated cells at a linear rate for a relatively long time. The rate per cell was highest in the large neuronal fraction, and lowest in the astroglia-like fraction.