Abstract. In this article a technique is described for the cryopreservation of human lymphocytes employing a programmed cooling device. A preset temperature curve was used which compensated for the heat exchange during crystallization. The influence of this method of lymphocyte preservation on a variety of the in vitro functions of these cells was evaluated by means such as the lymphocyte transformation test, CML and investigations for lymphocyte membrane markers. All the lymphocyte functions that were tested were found to be retained in full.
Experiments were performed to establish optimal conditions for specific and non‐specific lymphocyte transformation in vitro, in cultures containing 1–3 × 105 human lymphocytes. The stimulation as measured by 14C‐thymidine incorporation was evaluated when using various methods of lymphocyte purification and different numbers of contaminating erythrocytes, different kinds of tubes, serum from different sources, and varying PHA concentrations.
Furthermore, the dependence of the 14C‐thymidine incorporation on the thymidine concentration in the medium and on the total number of lymphocytes present per culture was determined.
Two methods of harvesting the cells to measure the 14C‐thymidine incorporation were compared.
On the basis of this analysis, conditions for maximal sensitivity and reproducibility could be defined in measuring lymphocyte reactivity to PHA, allogeneic lymphocytes and antigens in a miniaturized culture system.
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