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 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 μm). Though the relatively small cell volume fractions were the most enriched (82%) in quiescent cells, such cells were also observed in significant numbers (≃20%) even in the largest cell fractions. The cell clonogenicity of the various elutriation fractions was also assessed and shown to be lowest (plating efficiency ≃20%) in the small spheroid cells but relatively constant in fractions containing intermediate and large cells (plating efficiency ≃ 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 findings indicate the utility of centrifugal elutriation for quiescent cell characterization in in vitro tumor systems.
This research was supported by NIH grants CA 11198, CAO9363, and CA 11051 and Contract DE-ACO2-76EV03490, United States Department of Energy. Part of the research was supported by the Cell Separation Facility of the University of Rochester Cancer Center. Flow cytometry was performed at the University of Rochester Cell Sorting Facility under NIH Grant GM-23088. Presented in part at the Eighth Conference on Analytical Cytology and Cytometry, Wentworth-by-the-Sea, N. H., May 1981.