Isolation of quiescent cells from multicellular tumor spheroids using centrifugal elutriation

Abstract

A quiescent (nonproliferating) subpopulation was identified by flow cytometric analysis using two-step acridine orange staining in the EMT6/Rochester, N. Y. subline multicellular tumor spheroid, an in vitro culture system which provides a cellular microenvironment which mimics that of many of in vivo tumors. To isolate a viable quiescent cell subpopulation, centrifugal elutriation which allows for cell separation mainly on the basis of size was used. This technique provided single cells of relatively homogeneous cell volume which varied over a wide range (approximately 100 to 5000 cu microgram). Though the relatively small cell volume fractions were the most enriched (82%) in quiescent cells, such cells were also observed in significant numbers (congruent to 20%) even in the largest cell fractions. The cell clonogenicity of the various elutriation constant in fractions was also assessed and shown to be lowest (plating efficiency congruent to 20%) in the small spheroid cells but relatively constant in fractions containing intermediate and large cells (plating efficiency congruent to 50%). Continuous [3H]thymidine labeling indicated a slower rate of accumulation of labeled cells in the small spheroid cells, which may result from the transition of proliferating spheroid cells to the quiescent compartment during the course of labeling. These finding indicate the utility of centrifugal elutriation for quiescent cell characterization in in vitro tumor systems.