Evaluation of cell subpopulations isolated from human tumor xenografts by centrifugal elutriation.

Human epidermoid tumor cells (Coll2, ME180, A431, HEp3) grown as xenografts in nude mice, were dissociated into single cell suspensions using an enzyme cocktail containing 0.025% collagenase, 0.05% pronase, and 0.04% DNase. The dissociated cell suspensions were separated by centrifugal elutriation into fractions containing homogeneous cell subpopulations primarily based on the differences in the rates of sedimentation. The quality of separation was evaluated by several techniques including flow cytometry, cell volume distributions, in vitro colony forming assay and morphological examination of Wright-Giemsa stained cells. In each separated fractions, the host to neoplastic cell ratio, the DNA ploidy, the plating efficiency and the cell cycle distribution were determined. After an initial separation of non-neoplastic host cells from malignant cells, a purity of greater than 95% host cells was obtained from the four xenografts studied. DNA analysis of tumor suspensions showed that neoplastic cells of different xenografts contained aneuploid cells with a DNA index of 1.51 to 1.95. The neoplastic cells were further separated into fractions according to their positions in the cell cycle. Fractions containing greater than 95% G1, 65% S, and 72% G2M cells were obtained from HEp3 xenografts. Less efficient separation with respect to cell cycle was attained with cells derived from Coll2, ME180, and A431 xenografts. Colony forming abilities of the neoplastic cells were determined at different phases of the cell cycle and found to be similar to those of the unseparated cell suspensions after corrections for non-neoplastic host cells were made. These investigations indicate that centrifugal elutriation is an effective technique of obtaining homogeneous subpopulations of cells from human tumor xenografts for various tumor biology and cell kinetics studies.