Quantitative analysis of U251MG human glioma cells invasion in organotypic brain slice co-cultures.

OBJECTIVE To develop an in vitro model under conditions that highly resemble the in vivo situation for searching new therapeutics targeting invasive glioma cells. MATERIALS AND METHODS We generated organotypic brain slice "co-cultures" (OBSC) from mice and cultured the models on Millicell-CM membrane inserts. U251MG glioma cells expressing enhanced green fluorescent protein (EGFP) were established. After cultured the glioma cells to form spheroids, we implanted the spheroids onto brain slice surface. Then we evaluated the invasion area and cell density after U251MG cells were treated with the Na+-K+-2Cl- cotransporter 1 (NKCC1) inhibitor bumetanide by confocal laser microscopy. RESULTS In the models, the organotypic morphology and neuronal viability were well preserved. The confocal results showed that the cell spheroid area and density of U251MG cells in bumetanide group were decreased compared to the control group in brain slices. Meanwhile, the phospho-NKCC1(p-NKCC1) protein level of U251MG cells in bumetanide-treated group was also lower than the control group. CONCLUSIONS The OBSC model is a reliable and easy-to-perform in vitro method to quantify the glioma invasion ability.