Comparison of five cell culture media.

Tested on Tu44 (thoracic PPGL, SDHD p.(D92Y)) and Tu46 (carotid body PGL, SDHD p.(L95P)), stained with 3 marker antibodies and scored double blinded in duplo. Percentages of positive cells, with standard error of the mean (SEM), are presented in 10log scale. P values calculated with one-way ANOVA (p S1 Table for details) when sufficient tissue was available. We assessed cultures for the presence of synaptophysin as a marker for chromaffin cells and GFAP as a marker for sustentacular cells. In addition, we estimated the proportion of proliferating cells based on Ki-67 expression. In general, serum-free medium supported the highest proportion of synaptophysin-positive cells, but no long-term serum-free cultures including these cells were noted, probably because serum-free culture medium leads to the slow depletion of cells. Chromaffin cells visibly fail to prosper in this medium, often typified by poor filopodia development and poor adhesion. Media including some bovine serum, at either 1% or 5%, seemed to achieve a better balance between proliferation of fibroblasts, which may have a supportive function in these cultures, and survival of chromaffin tumour cells. Richer media such as Izal and PC12 led to a predominance of fibroblasts that appeared detrimental to the survival of chromaffin cells, possibly due to the rapid consumption of available metabolites in the media.