Long-term effects of brain trypsinization before cell seeding on cell morphology and surface composition.
The relation between the pattern of proteins localized in the surface of astroglial cells and cell differentiation was investigated in primary cultures derived from neonatal rat brains, dissociated either mechanically (MDC) or by 3 (TDC3) and 30 minutes (TDC30) trypsinization. Morphological and ultrastructural studies revealed a bed layer composed of flat, polygonal young and differentiated astrocytes in all types of cultures and a surface layer composed of small, ovoide undifferentiated cells which were more numerous in TDC30 than in TDC3 and MDC. The enrichment in undifferentiated cells, induced by prolonged brain trypsinization prior cell seeding, was observed during two weeks in culture; latter, by day 20, the cell population in all cultures was that of differentiated astrocytes. The presence of structural and enzymatic cell markers indicated that the cell population in MDC and TDC3 as well as in TDC3, including the small cells, was of astroglial origin. Concomitant with the morphological changes, cells in TDC30 were less accessible to surface labeling than those composing MDC. Subsequent electrophoresis of the labeled surface proteins demonstrated that a 140-130 K complex was the most "sensible" to brain trypsinization and that their accessibility to the surface probing was maximal during the differentiation of astrocytes in MDC or of small cells in TDC30. By day 20, these components were not significantly labeled in both, MDC, and TDC30, cultures. The use of two types of astrocytes primary culture which were different in the ratio of differentiated to undifferentiated cells and their surface labeling at different growth stages showed a variation in the composition of surface proteins during the cell maturation. The increased accessibility of some surface proteins to external probing when the cells developed to differentiated astrocytes might suggest their involvement in cell differentiation.