Development of a serum-free supplement for primary neuron culture reveals the interplay of selenium and vitamin E in neuronal survival

Abstract

Serum-free media require a number of supplements in order to support long-term neuronal survival. Commercially available B27, in combination with Neurobasal medium, supports neuronal survival and suppresses glial proliferation. However, B27 contains many biological antioxidants as well as catalase and superoxide dismutase, eventually demanding the application of unphysiologically high peroxide concentrations in survival assays. Moreover, optimal amounts of selenium (Se) are included in "B27 supplement minus antioxidants", a commercially available supplement used for the study of the role of antioxidants. Hence, Se-dependent enzymes like glutathione peroxidase are maximally expressed when this supplement is used and Se-depletion studies are not possible without changing the medium composition. We have therefore developed a modified serum-free media supplement which allows for free variation of all constituents. Our supplement was comparable to B27 with regard to cell survival and expression of neurochemical markers. Reduction of Se content in the supplement reduced selenoprotein expression and made cortical neurons more sensitive towards challenges with peroxides. Withdrawal from the medium supplement of vitamin E alone did not alter the survival of neurons in response to peroxides, while simultaneous reduction of Se and vitamin E rendered neurons hypersensitive towards peroxide challenge. This finding implied that adequate Se supply of neurons is required to minimize lipid peroxidation. Our medium supplement is easily prepared, inexpensive, and should be applicable to the analysis of survival mechanisms beyond peroxide challenge.