A micro-culture system for cloning human T lymphocytes in agar.

A simple and reproducible single-layer micro-agar culture system for cloning of human T lymphocytes has been described. The system consists of an agar layer, in which mononuclear cells from peripheral blood were suspended, and a liquid overlayer containing the mitogenic substance. The advantages of the described method are a low incubation volume (0.5 ml) and the liquid overlayer. The addition of different test substances to the liquid overlayer is simple and easily controllable. Depending on the agar concentration a different number of formed colonies can be found floating in the liquid phase. The morphological, cytochemical and immunological characteristics of the cells from those aggregates could be easily studied. The T-cell characteristics of formed clusters and colonies was confirmed by immunofluorescence and E rosette formation. The effects of agar, serum and cell concentrations, as well as the mitogenic activation caused by three lectins on the development and number of colonies were studied on day 7, 10 and 14 of incubation.