Feeder layer and nutritional requirements for the establishment and cloning of human malignant lymphoma cell lines

Cell lines were successfully established in continuous suspension culture from 10 patients with a histopathological diagnosis of diffuse histiocytic lymphoma (SU-DHL-1 to SU-DHL-10), two with North American Burkitt's lymphoma (SU-AmB-1 and SU-AmB-2), and one with acute lymphoblastic leukemia (SU-ALL-1). By screening a variety of parameters, including media, sera, effusion fluids, feeder layers, and chemical supplements, the nutritive growth requirements of lymphoma cells obtained from malignant effusions and lymph node biopsies were determined for each tumor. Most of these cell lines initially required human skin fibroblast or epithelial cell feeder layers from which they could be weaned after one to six weeks in culture and maintained in Roswell Park Memorial Institute Tissue Culture Medium 1640 containing 20% fetal calf serum and 10% pooled human serum. Several of these cell lines were successfully cloned on 0.5% Noble agar substrates. In the presence of human serum and selected feeder monolayers, cloning efficiencies increased significantly from less than 1% to 15 to 25%. In addition, the cloning efficiencies of certain cell lines showed a concentration-dependent increase with specific chemical supplements including L-cysteine and dithiothreitol. Placental colony-stimulating factor, nerve growth factor, epithelial growth factor, and fibroblastic growth factor were ineffective in augmenting the cloning efficiencies of the human lymphoma cell lines. After a single passage on agar, cells subpassaged from visible colonies showed markedly increased cloning efficiencies to levels as high as 50%. Such cloning efficiencies, coupled with the use of replica plating, make this technique applicable to genetic and quantitative radiobiological, immunological, and chemotherapeutic studies. Although these methods have thus far been used only with lymphoreticular tumors, they may also be applicable to the cell culture of other human neoplasms and normal tissues.