Influence of growth phase, nutrition and hypoxia on heterogeneity of cellular buoyant densities in in vitro tumor model systems.

EMT6/Ro multicellular spheroids were sequentially dissociated into outer, middle and inner layers, and the buoyant densities of these subpopulations were compared to aerobic or exponential or plateau-phase monolayers by isopycnic density centrifugation using Percoll as the gradient medium. Spheroids were more heterogeneous in buoyant density distributions than monolayer cultures. The outer-layer cells of spheroids and fed plateau monolayers peaked at a similar density; the majority of middle-layer cells banded close to unfed plateau and exponential hypoxic monolayers, while most inner-layer cells banded where fed or unfed plateau hypoxic monolayer peaked in density. Our data suggest that metabolic heterogeneity due to growth phase, together with altered cellular microenvironment brought about by differences in oxygenation and external nutrient supplies between spheroids and monolayers, can be manifested in one biophysical parameter such as buoyant density.