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A technique has been developed which takes advantage of the spherical symmetry of EMT6/Ro multicellular tumor spheroids to isolate subpopulations of cells originating from various locations in the spheroid structure. The method involves gently exposing spheroids to a dilute trypsin solution at 18 to 20° in specially designed dishes on a rotary shaker. Under these conditions, cells are released only from the outer spheroid surface; successive trypsin treatments dissociate cells from increasing depths in the spheroid. Measurements of the spheroid diameter and rate of cell dissociation demonstrated the reproducibility of the technique. Observations of histological sections showed that trypsin was active only over a small distance into the spheroid. Characterization of the cells isolated indicated that cell volume, membrane integrity, and clonogenic capacity all decreased for cells located in inner spheroid regions …