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

TL;DR

This paper introduces a novel in situ method for real-time 3D measurement of soluble biomarkers in organoid cultures, enabling detailed spatiotemporal analysis of biomarker dynamics within complex cell environments.

Contribution

The study presents a new bead-based immunoassay technique for continuous, in situ quantification of biomarkers in 3D cultures, allowing for dynamic and spatially resolved concentration profiling.

Findings

Measured cytokine concentration gradients in 3D cultures.

Estimated cytokine production rates per cell.

Validated assay with osteosarcoma tumoroids.

Abstract

Advanced cell culture techniques such as 3D bio-printing and hydrogel-based cell embedding techniques harbor many new and exciting opportunities to study cells in environments that closely recapitulate in-vivo conditions. Researchers often study these environments using fluorescence microscopy to visualize the protein association with objects such as cells within the 3D environment, yet quantification of concentration profiles in the microenvironment has remained elusive. Here, we present a method to continuously measure the time-dependent concentration gradient of various biomarkers within a 3D cell culture assay using bead-based immunoassays to sequester and concentrate the fluorescence intensity of these tagged proteins. This assay allows for near real-time in situ biomarker detection and enables spatiotemporal quantification of biomarker concentration. Snapshots of concentration profiles can be taken, or time series analysis can be performed enabling time-varying biomarker production estimation. Example assays utilize an osteosacroma tumoroid as a case study for a quantitative single-plexed gel encapsulated assay, and a qualitative multi-plexed 3D bioprinted assay. In both cases, a time-varying cytokine concentration gradient is measured. An estimation for the production rate of the IL-8 cytokine per second per osteosarcoma cell results from fitting an analytical function for continuous point source diffusion to the measured concentration gradient and reveals that each cell produces approximately two IL-8 cytokines per second. Proper calibration and use of this assay is exhaustively explored for the case of diffusion-limited Langmuir kinetics of a spherical adsorber.