Human lymphocyte culture in semi-solid medium

We pointed out some sources of variations in the results obtained by the method of human peripheral lymphocyte culture in semi-solid medium. The influence of changes in five important parameters of the two-layer method was studied: agar concentration in the upper layer, cell concentration, mitogen dilution, type of culture medium and serum. We found that the optimal conditions for lymphocyte culture and for the evaluation of the results were 0.3% agar concentration in the upper layer, the concentration of 5 x 10(5) cells/ml, and a double dilution of phytohaemagglutinin. Of the culture media tested, MEM with 0.2% Bactotryptose supplemented with 20% inactivated mixed human serum proved most appropriate. Cells in lymphocyte colonies were characterized morphologically, cytochemically and by means of basic membrane features. The possibilities of using the method in clinical work are shown and the necessity of standardizing the culture technique is emphasized.