Advancing glioblastoma research by establishing a whole brain slice model

Organotypic brain slice cultures are well-established and valuable three-dimensional (3D) models in neuroscience, particularly for cancer research. However, many existing 3D models rely on adult animals or are restricted to specific brain regions, such as the hippocampus. To address these limitations, we established a whole, large-format brain slice model that preserves the complex cellular architecture of the brain and its diverse cell populations. In addition, the use of 6-9-day-old postnatal mice allows a reduction in animal numbers by sectioning each brain into multiple contiguous slices of 300 µm thickness. The slices were maintained in vitro for 7-14 days under stable conditions and served as carrier tissue for brain tumor cell spheroids, thereby creating an ex vivo glioblastoma (GBM) model. To simulate tumor growth and invasion, spheroids of rat 9L/lacZ GBM cells were seeded onto the murine slices. Two different fluorescence-based methods were used to visualize and quantify the viability of the model. The AlamarBlue assay was used to identify the optimal culture medium and to longitudinally monitor whole-slice viability over a 10-day period. In parallel, the structural integrity of the brain slices and tumor invasion patterns were visualized using live/dead staining for up to 14 days post-slicing. Both methods demonstrated that the slices remained viable and stable for one week before a gradual decrease in viability. When co-cultured with GBM spheroids, the slices' viability slightly decreased, likely due to the tumor's invasive nature, which compromised the complex brain structures and cell networks. In summary, our ex vivo whole-brain slice GBM model preserves the complex organotypic brain and tumor structures essential for a robust in vitro GBM 3D model. The model stability for a minimum of 7 days, with the potential for extended culture duration, makes it a valuable tool in brain cancer research.