Purification of Malignant Cells and Lymphocytes From a Rat Transplantable Mucinous Adenocarcinoma of the Colon by Isokinetic Sedimentation in Gradients of Ficoll 2

Abstract

The Ward transplantable rat colon carcinoma was disaggregated with 0.25% trypsin in tissue culture medium, and the resulting suspension of cells was separated by sedimentation in linear gradients of Ficoll in tissue culture medium. Prior to separation, the suspension of tumor cells contained four types of morphologically different malignant cells (32.5%, type I; 8.2%, type II; 17.9%, type III; and 14.0%, type IV) in addition to 14.2% lymphocytes, 3.4% macrophages, 0.6% granulocytes, 1.9% unidentified mononucleated cells, and 7.5% red blood cells. After sedimentation for 14 minutes at 4° C in a previously described isokinetic gradient at a centrifugal force of 97xg, malignant cells comprised 97.0% of the nucleated cells in the purest gradient fractions; after isopyknic sedimentation for 30 minutes at a centrifugal force of 2,200xg, malignant cells comprised 98.7% of the nucleated cells in the purest gradient fractions. Lymphocytes constituted 90.4% of the nucleated cells in the purest gradient fraction after isokinetic sedimentation, but were not purified by isopyknic sedimentation. Fractions 11–23 of the isokinetic gradient contained 85–95% morphologically malignant cells. When these cells were injected into rats, tumors were evident within 10 weeks. Fractions 7–9 of the gradient contained mostly red blood cells and lymphocytes and less than 5% morphologically malignant cells. Cells from these fractions failed to produce tumors even 4 months after rats were inoculated with tenfold more cells than were needed to produce tumors from fractions 11–23.