Identification of lymphoid cells in cultured of murine leukocytes and thymus.

Several culture conditions and media were studied in an effort to establish long-term cultures of murine lymphoid cells from blood and thymus. Cultures vessels included small glass bottles and rubber-stoppered tubes. Media such as Roswell Park Memorial Institute 1640, 1700, 1701, 1715, GEM 1717, NCTC, fetal calf and horse serum supplements, and conditioned medium were tried. Lymphoid cells in mouse leukocyte cultures survived as long as eight months before dying out. However, lymphoid cells in thymus cell cultures, strarted and maintained with GEM 1717 medium with 20% fetal calf serum supplementation, gave rise to cell lines that continued to yield subcultures for more than 2 years. Mcroscopic examination of thymus cell subcultures revealed lymphoid and thymic epighelioid cells. Tumorigenicity studies of one cell line were negative. Chromosomal preparations of this cell line often contained near-normal karyotypes but were complicated by the presence of binucleated cells. Live cell fluorescent antibody assays for surface theta-antigen and immunoglobulin revealed immunoglobulin-negative cells possessing barely detectable theta determinants. Functional assays for thymus-derived lymphoid cell activity suggested that these cells were mitogen responsive and weakly reactive in one-way mixed lymphocyte culture. On the basis of this evidence it was sugguested that these cells represent a class of T-cells (thymus-derived lymphocytes) that have all but lost theta antigen, possibly due to prolonged culture.