Isolation of koala lymphoid cells and their in vitro responses to mitogens

Baseline parameters have been established for the successful in vitro culture of mononuclear cells from the peripheral blood (PMC) of koalas. To minimise stress-related influences and allow repeated testing of cells from the same animals, most studies were performed using blood samples from captive koalas which had become accustomed to regular handling. Ficoll-Paque density gradient fractionation of whole blood was required to prepare cell suspensions which responded well to the T-lymphocyte mitogens phytohaemagglutinin, concanavalin A and pokeweed mitogen. In contrast, very low or negligible proliferative responses were induced by the B-lymphocyte mitogens lipopolysaccharide, jacalin and protein A, even when purified PMC were cultured with a wide range of concentrations of these molecules. Using the standard approaches established with T-lymphocytes of eutherian animals, it was shown that concanavalin A-stimulated PMC produced an interleukin 2-like growth factor. The significance of these findings is discussed in the context of current knowledge and understanding of similar studies carried out using the lymphoid cells of eutherian and other metatherian animals.