Embryonic micromass limb bud and midbrain cultures: Different cell cycle kinetics during differentiation in vitro

Rat embryo micromass limb bud (LB) and midbrain (CNS) cultures have been proposed as a screening system for teratogenic potential as well as a model system for differentiation. To further characterize these in vitro differentiating cultures, their population kinetics have been examined. Cellular proliferation, cell cycle kinetics and cell differentiation were monitored at days 1, 2 and 5 in culture. For cell cycle analysis, cellular DNA was stained with 4,6-diamidino-2-phenyl indole and nuclei were analysed by flow cytometry. Cell viability was assessed using trypan blue and differentiation was monitored using haematoxylin (CNS) or alcian blue (LB) stain. CNS cultures underwent 2-3 cell doublings during the 5 days of culture. There was a gradual accumulation of cells in the G(0)/G(1) compartment (54% +/- 10%, day 1 to 78% +/- 2%, day 5) (P < 0.05, chi(2) test). This accumulation appeared to occur at the expense of the S and G(2) + M compartments since both decreased over this same period of time. LB cultures underwent 1-2 cell doublings during the same culture period. In contrast to the CNS cultures, the limb bud cultures exhibited only minimal changes in the size and distribution of the cell cycle compartments during the 5 days in culture. Characterization of the cellular kinetics of these two widely used embryo cell culture systems should help to delineate potential sites and mechanisms of developmental toxicity.