To better understand the consequences of creatine deficiency in the brain, short-hairpin RNAs (shRNAs) were used to silence the expression of AGAT in the rat oligodendroglia-glioma hybrid cell line (ROC). Our results show an average of 88% decrease in AGAT enzyme expression in normal conditions. We show that in ROC cells, cell survival was not affected by AGAT silencing, both in normal conditions and under creatine deficiency (serum deprivation). Adeno-Associated Viruses (AAV) transducing shRNAs targeting AGAT mRNA are currently produced to test the effect of creatine deficiency in 3D organotypic primary cultures of brain cells. As creatine (Cr) plays an essential role for the maintenance of ATP levels in tissues with high demand, such as brain, creatine deficiency syndromes could result in energy depletion. This depletion appears as a plausible mechanism for causing overproduction of reactive oxygen species (ROS) that can lead to loss of the cell redox control resulting in cell aberrant proliferation even apoptosis. We selected two CRTR fibroblast cell lines (genotypes: p.F360del and p. W154X) with low intracellular Cr levels, overproduction of ROS, increased apoptosis and aberrant proliferation. The aim of this study was to explore the effect of Cr supplementation (500 μM 24 h) on intracellular Cr levels, and stress parameters. Results showed that intracellular Cr levels, measured by MS-MS, increased in both cell lines (7.9 and 4.6 fold) without reaching control levels. In addition, significant reduction of ROS levels, decrease of the apoptotic population (G0 phase) and progression of cells to S-G2/M stages were also detected by flow cytometry.