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Fetal proteins appear in the adult in beth malignant and nonmalignant conditions. Their presence has been thought to indicate a state of altered regulation or differentiation, possibly related to the "phasing" (1) of specific gene products. Where normal constraints upon growth are altered, the presence of embryonic or fetal components may be anticipated. Thus, fetal antigens have been found in the bone marrow cells of patients with erythroid hyperplasia, and pernicious anemia (2), as well as explants of human skin (3) maintained in culture for 3 wk or more.A better defined and more readily manipulated model in which to observe possible adaptive changes to altered growth is a cell culture system. Lymphoblastoid lines established from normal adults have a unique capacity to grow in suspension, to maintain some differentiated characteristics, and to remain diploid. This study reports the presence of fetal components on the surface of lymphocytes established in long-term culture from healthy adults. Materials and MethodsRabbits were immunized with perchloric acid extracts of human fetuses that had had viscera, brain, and spinal cord removed (4). The antisera were absorbed sequentially with acetone-dried powders of normal adult liver, spleen, muscle, brain, and pooled sera. Further absorption with fetal calf serum did not change the immunofluorescence results. The absorbed antisera were evaluated by indirect immunofluorescence with frozen sections or cell suspensions of normal adult tissues and with frozen sections of fetal tissues. Significant reactivity was not detected with adult tissues but widespread reactivity remained against fetal tissues. Thus "fetal antigens" are operationally defined by reactivity with these absorbed antisera. Similarly prepared antisera have been extensively evaluated in earlier reports (2-4). All cell lines were grown in suspension culture in RPMI-1640 medium supplemented with 10% heat-inactivated fetal calf serum. With the exception of MOLT (5) and Daudi (6) all lines were established from healthy adult humans. Cultured cells and fresh peripheral blood lymphocytes (PBL) prepared by the Ficoll-Hypaque method (7) were examined by indirect membrane imrnunofiuorescence with commercial goat antirabbit immunoglobulin sera (Cappel Laboratories, Inc., Downingtown, Pa.), using the microscopic system of Lewis and co-workers (2-4). In some experiments, adherent cells were removed by the method of Greaves and Brown (8). For electron microscopy, IgG fractions from the absorbed antisera were prepared by precipitation with 50% saturated ammonium sulfate followed by DEAEcellulose chromatography (9) and then conjugated with peroxidase (10). Washed cells were incubated with the antisera unfixed or after a 15 min fixation (1.25% buffered paraformaldehyde)

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Attempts were made to adapt human long-term B lymphoblastoid cell lines to prolonged growth in serum-free, chemically defined media. A newly described medium, which is an enriched modification of Dulbecco's modified Eagle's medium containing additional amino acids and vitamins, was used. The serum is totally replaced by albumin, transferrin, and soybean lipid. The cell lines were all adaptable from RPMI 1640 over a period of time during which the 10% fetal bovine serum (FBS) concentration was reduced and then eliminated in successive steps. After 3 to 6 wk minor alterations in cell shape and adhesion were noted without significant histological changes. Growth characteristics were comparable in the new medium provided a double initial inoculum was used. A panel of cell surface markers, including surface immunoglobulins, Ia antigens, Fc and complement receptors, and T and B erythrocyte rosettes, all showed no altered expression. Molecular genotyping of Ia antigens was carried out by 3-D gel electrophoresis. The antigens showed their full polymorphism without change and were shed into the new culture medium without alteration. Chromosome analysis was performed on Q-banded karyotypes from one of the lines and showed no alteration resulting from the change to serum-free conditions. Thus long-term B lymphoblastoid cell lines can be adapted to prolonged growth in serum-free medium. This will facilitate the assay and isolation of cell products regulating lymphocyte function and the identification and characterization of cell surface molecules free of interference from undefined serum components.