ANALYSIS OF HYPOXIA IN U87 GLIOBLASTOMA SUSPENSION CULTURE

Michael R. Doane, Seungjo (Joe) Park, Yonghyun (John) Kim

Department of Chemical Engineering, University of Massachusetts – Lowell, 220 Pawtucket St., Lowell, MA 01854, USA

Abstract

Expanding cancer stem cell (CSC) populations while maintaining their vital stem cell character represents a significant challenge in modern biotechnology. Suspension culture holds promise as a platform for the expansion of cancer stem cells, as cells are able to more efficiently maintain their ‘stemness.’ However, this can generate hypoxic microenvironments in larger sphere aggregates, leading to necrosis. This work presents the implementation of novel methods to characterize the degree of hypoxia within multicellular tumor spheroids. A quantitative strategy was devised for determining the extent of hypoxia in suspension cultures, by pre-processing images in ImageJ, then analyzing them using ReViSP, an open-source volume-estimation application for 2D images. 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 implemented to predict the degree to which cells had been exposed to hypoxia, based on the images collected. A correlation was found between the hypoxia detected by the fluorescence assays and the computational predictions. This strategy can now be implemented to evaluate overall suspension culture hypoxia without performing labor-intensive assays.

Keywords: Suspension culture, hypoxia, tumorspheres, ReViSP.