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Identification of Lymphoid Cells in Cultures of Murine Leukocytes and Thymus¹

Roswell Park Memorial Institute, Springville Laboratories, Springville, New York 14141 [T. R. B.]; Department of Surgical Research, Denver General Hospital, Denver, Colorado 80204 [G. E. M.]; and Department of Immunology, Mayo Clinic, Rochester, Minnesota 55901 [J. D. S.]

Several culture conditions and media were studied in an effort to establish long-term cultures of murine lymphoid cells from blood and thymus. Culture 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, started 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.

Microscopic examination of thymus cell subcultures revealed lymphoid and thymic epithelioid 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 -antigen and immunoglobulin revealed immunoglobulin-negative cells possessing barely detectable 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 suggested that these cells represent a class of T-cells (thymus-derived lymphocytes) that have all but lost -antigen, possibly due to prolonged culture.

¹ This work was supported in part by Award DT-27 from the American Cancer Society.

² To whom requests for reprints should be addressed.

Received 1/29/74. Accepted 11/20/74.