The culture of mouse thymic reticulum cells: The effect of homologous serum on the establishment of primary cultures

Monolayer cultures of reticular cells from mouse thymus are grown from dispersed cell suspensions with no enzymatic treatment. Cell suspensions were cultured in medium containing mouse serum for 4 days (the establishment phase). The resulting nonlymphoid adherent cells were then cultivated in the medium containing fetal calf serum (the propagation phase). Mouse serum in the establishment phase is essential. Neither fetal calf serum nor horse serum resulted in adherent cells capable of proliferation. Serum from 2- to 6-week-old mice was more effective than serum from older mice, whereas serum from AKR mice of preleukemic and leukemic age did not effectively promote adherence and growth of thymus reticulum cells into monolayers. Allogeneic serum was not markedly different from syngeneic serum in its ability to establish adherent reticular cells. Heat treatment of serum at 56°C had no effect, but the use of dialyzed serum in the establishement phase prevented growth in the propagation phase. Adherent cell monolayers consisted of large squamous cells with ruffled surface characteristics and smaller dendritic cells. Distinct multilayered morphologic structures were also observed in most cultures and were apparently mesenchymal in nature. Fibroblast outgrowths and differentiating fat cells were sometimes seen associated with these structures. Ultrastructurally, two cell types were observed, the first being secretory, and the second being characterized by extensive vacuolation. Large squamous cells from monolayer areas were characterized by numerous filamentous cytoplasmic projections, an undulating or ruffled surface, and numerous vesicles and inclusion bodies. Thymic reticulum cells can be easily established so that the role of these cells in thymocyte differentiation and leukemogenesis can be readily studied.