Development of a serum-free medium for lymphocyte cultures.

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The regulatory mechanisms of immune responses have been investigated by using in vitro culture systems of murine lymphocytes. Fetal calf serum(FCS)-containing culture medium has been usually used in these cultures. But, it has often been pointed out that the magnitude and the properties of immune responses varied markedly depending on the lot of FCS used. In order to overcome these problems, we attempted to develop a serum-free medium that is able to induce a primary antibody responses as effectively as the serum-containing medium.1. Based on the discovery that beta-cyclodextrin is effective as a serum-substitute, we first devised a basal serum-free RPMI-1640 medium that was supplemented with beta-cyclodextrin, albumin, transferrin and insulin. The addition of ehter-soluble fraction of FCS resulted in a marked enhancement of the immune response. The active substance was purified and identified as cholesterol. In addition, low density lipoprotein (LDL) was found to be more effective than cholesterol itself.2. The screening was carried out to find substances that ard useful to further improve the efficiency of the above medium. We found that the simultaneous addition of L-alanine and putrescine was the most effective. By determining the optimal concentrations of these additives, we finally succeeded in establishing a serum-free medium that can induce a primary antibody response as effectively as FCS-containing culture medium.3. This serum-free medium was also applicable to investigate the proliferation and the differntiation of T cells, suggesting a wide applicability of this medium in various lymphocyte culture systems.