Abstract The purpose of this study was to characterize a three-dimensional calcium alginate culture system for real-time monitoring of cancer cell metabolism and radiation response. HCT116 colorectal cancer cells were encapsulated in 2.5% calcium alginate using a custom electrostatic rig adapted from a previously published system. Bead diameter was a function of voltage and alginate concentration: 4 x 107 cells/mL encapsulated at 4 and 8 kV resulted in mean diameters of 880 and 584 µm, respectively. The distribution of bead radii for all voltages showed positive excess kurtosis (4 kV = 1.6, 8 kV = 3.0) and 72.9% of 4 kV and 77.2% of 8 kV bead diameters fell within one standard deviation of the mean. Encapsulated cell viability was assessed by staining with Trypan Blue and clonogenic survival assays (CSAs). The baseline viability of cells immediately after encapsulation was 93.2%, which decreased 2 days after a single 10 Gy dose (90.7%, p<0.05). After two weeks of continuous culturing, 84.1% of non-irradiated and 73.8% of irradiated encapsulated cells remained viable (p<0.003). Pimonidazole staining demonstrated the presence of hypoxic cores proportional to bead size. CSAs of 8 kV beads showed a dose modifying factor (DMF) of 1.06 relative to cells grown in a monolayer, while larger 4 kV beads showed a DMF of 1.58. Mean oxygen consumption rate rose with increasing number of 8 kV beads/well: 1 bead, 35.5; 2 beads, 97.4; 3 beads, 175.5; 4 beads, 209.9; 2.0 x 104 monolayer cells, 92.6 (all pmol/min, ~8.2 x103 cells/bead, p<0.005). Hyperpolarized 13C-NMR spectroscopy of 400 uL of 8 kV beads containing 1.2 x 108 cells/mL showed a detectable conversion of [1-13C]-pyruvate to [1-13C]-lactate; 1 hour after a single 10 Gy dose, the lactate:pyruvate ratio decreased by 25%. Unpaired 2-tailed Student’s t-tests were used to determine significance between and one-way ANOVA was used to determine significance among groups with α set at p≤0.05. These data demonstrate the versatility of alginate hydrogels for real-time metabolic and radiation response studies which are non-invasive, higher throughput, and lower cost compared to in vivo systems. Future directions include additional metabolic flux analysis and hyperpolarized 13C-NMR spectroscopy to further investigate the cancer cell metabolic response to drug and/or radiation therapy. Citation Format: Aparna H. Kesarwala, Jenna L. Carter, Graham H. Read, Natsuko Miura, Kazutoshi Yamamoto, James B. Mitchell, Murali C. Krishna. Alginate hydrogels for three-dimensional culture and real-time monitoring of cancer cell metabolism and radiation response [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 833. doi:10.1158/1538-7445.AM2017-833