Separation of Cells From a Murine Fibrosarcoma on the Basis of Size. I. Relationship Between Cell Size and Age as Modified by Growth In Vivo or In Vitro23

Abstract

Cells from a murine fibrosarcoma (FSa) that had been Induced by 3-methylcholanthrene were separated and synchronized on the basis of cell size by centrifugal elutriation. More than 10⁸ tumor cells were elutriated either immediately following disaggregation of a solid tumor or after 48 hours of incubation in vitro. Among the tumor cells elutriated immediately after disaggregation, the tumor suspension contained up to 32% normal diploid cells. Normal cells were not detected, however, in the suspension obtained after in vitro incubation. Recovery of cells after elutriation was 85%, and cell viability was greater than 95%. Selected fractions of cells were pooled, and average cell volumes were determined. The (modal) size of FSa cells after incubation in vitro for 48 hours increased from 980 to 1,620 μ³. These size differences were also reflected in the size of the cells comprising the pooled elutriated subpopulations. Modal volumes ranged from 740 to 1,300 μ³ for FSa cells separated without prior in vitro incubation as compared with 880-2,150 μ³ for those cells separated with prior in vitro incubation. With the use of the method of flow microfluorometry, the cell cycle parameters and the relative synchrony of each of the separated populations were determined. Cells incubated in vitro for 2 days were more efficiently synchronized than were cells separated immediately following excision of the tumor. The latter situation reflects the heterogeneity of size of cells in the same phase of the division cycle present in the tumor growing in vivo. The increased efficiency of elutriation in separating synchronized cohorts from cell populations incubated in vitro was probably due to the induction of a more homogeneous class of cells as a result of exposure to a uniform environment.