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Advanced cell culture techniques such as 3D bio-printing and hydrogel-basedcell embedding techniques harbor many new and exciting opportunities to studycells in environments that closely recapitulate in-vivo conditions. Researchersoften study these environments using fluorescence microscopy to visualize theprotein association with objects such as cells within the 3D environment, yetquantification of concentration profiles in the microenvironment has remainedelusive. Here, we present a method to continuously measure the time-dependentconcentration gradient of various biomarkers within a 3D cell culture assayusing bead-based immunoassays to sequester and concentrate the fluorescenceintensity of these tagged proteins. This assay allows for near real-time insitu biomarker detection and enables spatiotemporal quantification of biomarkerconcentration. Snapshots of concentration profiles can be taken, or time seriesanalysis can be performed enabling time-varying biomarker productionestimation. Example assays utilize an osteosacroma tumoroid as a case study fora quantitative single-plexed gel encapsulated assay, and a qualitativemulti-plexed 3D bioprinted assay. In both cases, a time-varying cytokineconcentration gradient is measured. An estimation for the production rate ofthe IL-8 cytokine per second per osteosarcoma cell results from fitting ananalytical function for continuous point source diffusion to the measuredconcentration gradient and reveals that each cell produces approximately twoIL-8 cytokines per second. Proper calibration and use of this assay isexhaustively explored for the case of diffusion-limited Langmuir kinetics of aspherical adsorber.

In Situ 3D Spatiotemporal Measurement of Soluble Biomarkers in Organoid Culture