In situ 3D spatiotemporal measurement of soluble biomarkers in spheroid culture
Tóm tắt
Advanced cell culture techniques such as 3D bioprinting 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. Demonstrate an assay that enables near real-time in situ biomarker detection and spatiotemporal quantification of biomarker concentration in 3D cell culture. A distributed bead-based immuno-assay was used in 3D cell culture to continuously measure the time-dependent concentration gradient of various biomarkers by sequestering soluble target molecules and concentrating the fluorescence intensity of these tagged proteins. Timelapse confocal microscopy was used to measure the in situ fluorescence intensity profile and a calibration curve was separately generated. Application of a calibration transfer function to in situ data is used to quantify spatiotemporal concentration. Example assays utilize an osteosarcoma spheroid 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 spheroid production approaches nearly 0.18 fg/s of IL-8 after 18 h in culture. Theoretical and experimental demonstration of bead-based immunoassays in diffusion-limited environments such as 3D cell culture is shown, and includes example measurements of various cytokines produced by an osteosarcoma spheroid. Proper calibration and use of this assay is exhaustively explored for the case of diffusion-limited Langmuir kinetics of a spherical adsorber.
Tài liệu tham khảo
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