In situ 3D spatiotemporal measurement of soluble biomarkers in spheroid culture

Advanced cell culture techniques such as 3D bioprinting and hydrogel-based embedding harbor many new exciting opportunities to study cells in environments that closely recapitulate vivo conditions. Researchers often these using fluorescence microscopy visualize the protein association with objects within environment, yet quantification of concentration profiles microenvironment has remained elusive. Demonstrate an assay enables near real-time situ biomarker detection spatiotemporal culture. A distributed bead-based immuno-assay was used continuously measure time-dependent gradient various biomarkers by sequestering soluble target molecules concentrating intensity tagged proteins. Timelapse confocal profile a calibration curve separately generated. Application transfer function data is quantify concentration. Example assays utilize osteosarcoma spheroid case for quantitative single-plexed gel encapsulated assay, qualitative multi-plexed 3D-bioprinted assay. In both cases, time-varying cytokine measured. An estimation production rate IL-8 per second results from fitting analytical continuous point source diffusion measured reveals approaches nearly 0.18 fg/s after 18 h Theoretical experimental demonstration immunoassays diffusion-limited shown, includes example measurements cytokines produced spheroid. Proper use this exhaustively explored Langmuir kinetics spherical adsorber.