NOVEL ISOLATION AND PURIFICATION METHOD PERMITTING FUNCTIONAL CYTOTOXICITY STUDIES OF MACROPHAGES FROM MILKY SPOTS IN THE GREATER OMENTUM

Milky spots in the greater omentum are well organized perivascular inf iltrates of leukocytes which are probably involved in the clearance of tumor cells from the peritoneal cavity. In milky spots, macrophages a re the predominant cell type forming a distinct population of cells. T o investigate whether these macrophages have a function in the control of metastatic spread in the peritoneal cavity, a novel isolation and purification method was developed in order to study the functional cyt otoxicity of macrophages from milky spots in the greater omentum again st tumor cells in vitro. In order to obtain a cell suspension, greater omenta of unstimulated healthy male WAG/RIJ rats were incubated in co llagenase/DNase suspension and filtered. Subsequently, macrophages wer e isolated and purified using flow cytometry by sorting unstained cell s on the basis of size and internal complexity. Macrophages and other cells were identified by routine May-Grunwald-Giemsa staining and by i mmunophenotyping with the specific macrophage monoclonal antibody ED 1 . Furthermore, macrophage subtypes were characterized by ultrastructur al analysis. Functional cytotoxicity of the isolated macrophages was a ssayed against the syngeneic CC 531 tumor cell line in a colorimetric MTT assay. From three greater omenta of healthy rats 1.16 +/- 0.16 X 1 0(6) macrophages were isolated with a purity of 83 +/- 2% and a viabil ity of greater than or equal to 96%. The macrophages were of the exuda te (monocytic), exudate-resident and resident cell type and were in eq ual proportions. The contaminating cells were mainly mesothelial. A ma ximum cytotoxicity of approximately 30% was reached with the macrophag e fraction at an effector-to-target ratio of 10. Furthermore, it was e stablished that the mesothelial cells did not exhibit cytotoxicity.