Analysis of Hypoxia in U87 Glioblastoma Suspension Cultures

Expanding cancer stem cell (CSC) populations while maintaining their vital stem cell character represents a significant challenge in modern biotechnology. CSCs are radio- and chemo-resistant, and further study requires a reproducible method for propagating them in vitro. Suspension culture holds promise as a paradigm for the expansion of cancer stem cells, since cells grown in non-adherent culture tend to maintain stemness more efficiently than those grown in adherent media. However, as cell spheres grow, their size can lead to hypoxic microenvironments in larger sphere aggregates, which can potentially preserve stem cell character, but can also be deleterious if the low oxygen levels lead to necrosis. Because of this, it is important for researchers to know the degree to which cells are exposed to hypoxia in culture. This work presents the implementation of novel methods for the characterization of hypoxia within multicellular tumor spheroids. A quantitative strategy was devised for determining the degree to which cells had been exposed to hypoxic conditions based on sphere volumes, as calculated by pre-processing images in ImageJ, then analyzing them using ReViSP, an open-source volume-estimation application for 2D images. Five samples of U87 glioblastoma cells were cultivated in suspension culture and incubated in ROS-ID� Hypoxia Probe Reagent. The cells were then imaged and assayed for the presence of hypoxia using fluorescence microscopy, microplate spectrophotometry, and flow cytometry. A MATLAB-based script file was written and implemented to predict the degree to which cells had been exposed to hypoxia, based on the images collected. Sphere size distributions were built from the volume projections and used to predict hypoxia exposure for the cell populations, which was then compared to the experimental data. A correlation was found between the hypoxia detected by the fluorescence assays and the computational predictions. In the future, this strategy could be implemented in order to evaluate overall suspension culture hypoxia without performing labor-intensive assays.