Design and Validation of Bioluminescent Assays for 3D Cell Culture Models Poster
Poster Oct 09, 2015
Terry L. Riss, Michael P. Valley, Chad A. Zimprich, Andrew L. Niles, Kevin R. Kupcho and Dan F. Lazar
Cells cultured in 3D model systems often acquire relatively large in vivo-like structures compared to the thickness of a 2D monolayer of cells grown on standard plastic plates. Multicellular 3D culture systems containing more than one cell type and exhibiting formation of a complex extracellular matrix represent a more physiologically relevant environment, yet provide a challenge for assay chemistries originally designed for measuring events from monolayers of cells. There is an unmet need for guidelines for design and verification of convenient and effective assays useful for larger 3D microtissues. Critical factors to consider for each model system include effective penetration of detection reagents and/or complete lysis of microtissue structures using combinations of detergent and physical disruption. We have developed an improved reagent formulation for bioluminescent detection of ATP for measuring cell viability. The improved formulation demonstrates more effective lysis of large microtissues. Results from modifying assay procedures to include more rigorous physical disruption of microtissues will be presented for measuring caspase activity for detecting apoptosis and cell stress assays to detect mechanisms leading to cytotoxicity.
Multiplexing cell-based assays is possible using 3D culture models that are larger and more complex than monolayers
Real-time detection methods to measure live or dead cells provide much flexibility for multiplexing
All multiplexed assay combinations should be verified using appropriate controls for each 3D cell culture model.
Basic fibroblast growth factor (bFGF) is widely used in vitro for the maintenance and stimulation of a variety of cells. However, use of native bFGF in cell biology is limited by the fact that bFGF rapidly degrades at physiological temperatures. We have addressed this problem with an engineered form of bFGF, named Heat Stable bFGF (HS bFGF), which is stable at 37 degrees Celsius.READ MORE