Development of a serum-free medium for lymphocyte cultures.

利用小鼠淋巴细胞体外培养系统研究了免疫应答的调节机制。含胎牛血清（FCS）的培养基通常用于这些培养物。但是，人们经常指出，免疫反应的大小和性质根据所用FCS的数量而明显不同。为了克服这些问题，我们试图开发一种无血清培养基，其能够与含血清培养基一样有效地诱导一抗应答。基于β-环糊精作为血清替代物是有效的这一发现，我们首先设计了补充有β-环糊精、白蛋白、转铁蛋白和胰岛素的基础无血清RPMI-1640培养基。添加FCS的水溶性部分导致免疫应答的显著增强。对活性物质进行纯化并鉴定为胆固醇。此外，低密度脂蛋白（LDL）被发现比胆固醇本身更有效。进行筛选以发现对进一步提高上述培养基的效率有用的物质。我们发现同时添加L-丙氨酸和腐胺是最有效的。通过确定这些添加剂的最佳浓度，我们最终成功地建立了一种无血清培养基，它可以像含FCS的培养基一样有效地诱导一次抗体反应.该无血清培养液也适用于研究T细胞的增殖和分化，表明该培养液在各种淋巴细胞培养系统中具有广泛的适用性。

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.