Real-Time Biomonitoring of Cytotoxicity in 3D Autobioluminescent Human Tissue Culture Models

Current optimal imaging technologies (e.g., fluorescence and firefly luciferase-based bioluminescence) require an external stimulation be applied prior to signal generation. To overcome this limitation, a ‘humanized’ bacterial luciferase reporter operon (lux) has been developed for expression in eukaryotic organisms, resulting in human cell lines capable of self-producing bioluminescent output without external stimulation. These cells can be programmed to continuously produce an autobioluminescent signal with declining intensity correlating with exposure to toxicants detrimental to cellular health or to auto-initiate bioluminescent production only in response to the detection of specific target agents. To evaluate the bioluminescent response to toxic compounds, constitutive autobioluminescent human embryonic kidney (HEK293) cells were either encapsulated in collagen hydrogel or seeded as a monolayer in 96-well plates, and exposed to the antibiotic Zeocin. Significant decreases in bioluminescence were observed after 48 hours of treatment with 1000 µg/ml and 400 µg/ml Zeocin concentrations in 3D and 2D conditions, respectively. HEK293 cells expressing the lux system under the regulation of the Tet-On inducible promoter responded to doxycycline stimulation similarly under both 3D and 2D growth conditions, with cells grown under 2D conditions producing a higher fold increase in bioluminescence induction compared to those grown in 3D conditions, suggesting that 3D-grown cells are more resistant to toxicants than cells grown in 2D. The results demonstrated the utility of a reagent-free autobioluminescent cellular system for continuous, real-time toxicity monitoring in 3D tissue culture models.