The cells involved in the immune response of fish. III. Culture requirements of PHA-stimulation carp (Cyprinus carpio) lymphocytes

The optimalization of the culture conditions for carp peripheral blood lymphocytes was studied using the thymidine uptake technique. The quantitative and/or qualitative requirements of this culture were determined with respect to the source of serum, the concentrations of 2-ME, glutamine and PHA, and with respect to the initial cell density. High individual variations, shown along the experiments, pointed out the existence of low and high responders to the mitogenic stimulus. These individual variations were considerably reduced and the mitogenic response extremely enhanced by the addition to the culture medium of optimal concentrations of charcoal adsorbed pooled homologous serum (4%), 2-ME (125-250 μM), glutamine (8 mM), PHA (10-40 μl per ml culture, for the batch used) and by seeding the cells at the optimal density of 2 x 10⁵ per microplate well. Finally, the kinetics of cell growth were studied at optimal concentrations of all these factors. The peak response was obtained between days 6 and 7 at 28°C, and the mean doubling time at the exponential phase of the culture, at this temperature, was about 14 hours.