Density distributions of human squamous carcinoma cells: Influence of growth conditions, proliferative status and DNA content

Human squamous carcinoma cells (A43I, CaSki) were grown in vitro as multicellular spheroids or as exponential and plateau‐phase monolayer cultures. Single‐cell suspensions were obtained by disaggregating the spheroids and monolayer cultures with trypsin‐EDTA. Their buoyant density distributions were then compared by centrifuging the suspensions for 30 min at about 800 g ave and 4°C in linear, continuous Percoll gradients. We found differences in buoyant density related to the growth condition ( i.e. cells grown as spheroids or plateau‐phase cultures were more dense than those from exponential cultures) and to the cell‐line ( i.e. CaSki cells were more dense than the A43I cells). Cells isolated from the inner layers of the spheroids appeared to be less dense than those from the outer layers. In addition, quiescent cells separated from the A431 spheroids by centrifugal elutriation, a technique based on cell size, were more dispersed in buoyant density than the corresponding proliferating cells recovered from the same spheroids. Flow cytometric analysis with mithramycin was performed to measure the DNA content of the cells. We found that the CaSki cells contained relatively more DNA than the A431 cells whereas, for the same cell line, growth as monolayer cultures or as multicellular spheroids did not influence their relative DNA content. We conclude that the growth conditions, the proliferative status of the cells and possibly their relative DNA content may influence the density distribution of these cells.