MONITORING OF LIVE SPHEROID OXYGENATION USING FLUORESCENCE MICROSCOPY AND NANOSENSORS

Jun 30, 2022, 11:40 AM
20m
Room: S4 C

Room: S4 C

Speaker

Okkelman, Irina (Tissue Engineering and Biomaterials Group, Department of Human Structure and Repair, Faculty of Medical and Health Sciences, Ghent University)

Description

"Introduction
Multicellular spheroids can be a powerful model mimicking the physiological environment of the tissue in a microscale format. They are often seen as ‘microscale-bioreactors’ providing an appropriate environment for cell differentiation and stem and cancer cell niche, which makes them important for physiological studies and biofabrication. However, this advantage becomes a problem when it comes to the question of characterization / standardization of spheroids produced in different labs, by different methods or even cell passage number1. The gradients of cell proliferation, oxygenation, cell death and other biomarkers are thus largely unexplored and ignored.
Methodology
Here we describe a nanosensor-based analysis of live spheroid oxygenation helping to routinely estimate O2 gradients on a conventional fluorescence microscope. Described method helps spheroid phenotype characterization (size, relative hypoxia), informing on their metabolism and viability. We optimized generation of spheroids loaded with ratiometric red (650 nm reference) / near infrared- (760 nm O2-sensing channel) emitting O2-sensing biocompatible nanoparticles. Presented visualization of oxygenation can be combined with multiparametric analysis in available ‘blue’ and ‘green’ channels such as cell death staining or advanced imaging modalities such as FLIM and PLIM2.
Results
Presented approach was tested in different experiments with homo- and heterocellular (human dental pulp stem hDPSC with endothelial HUVEC cells) spheroids produced from stem and cancer cells: (1) we performed long-term monitoring of individual spheroids oxygenation for more than 14 days; (2) we detected changes in oxygenation upon adding mitochondrial uncouplers / inhibitors; (3) we performed ‘endpoint’ multiparameter analysis of oxygenation coupled with labeling cell death by SYTOX Green. To standardize the analysis of oxygenation in spheroids we looked at oxygenation at spheroid core and periphery, value of O2 gradient and their ‘steepness’.
We found that in contrast to hDPSC spheroids, ‘addition’ of HUVEC cells to spheroids provided higher oxygenation and significantly steeper gradient. Heterocellular spheroids were also statistically larger, suggesting that their oxygenation was caused by cell composition-related differences in bioenergetics agreeing with the known data on HUVEC and hDPSC metabolism.
To illustrate the applicability of the approach for biofabrication we compared O2 gradients in hDPSC spheroids before and on a day 1 after bioprinting in GelMA. Bioprinted hDPSC spheroids had significant changes in periphery which affected the range and steepness of their periphery-to-core O2 gradients. The dead cell staining was more profound in bioprints.
Conclusions
We demonstrated that spheroid oxygenation reflects the bioenergetic state and viability of cells in 3D, allowing application of ratiometric oxygenation analysis for standardization of spheroid phenotype. Usage of ratiometic analysis versus phosphorescence lifetime calculation enables for more ‘cost-efficient’ O2 gradients studies with almost all types of conventional fluorescence microscopes. The method is compatible with multi-parameter physiological measurements (e.g., cell death, proliferation, and cell composition) and downstream assays (immunofluorescence, FACS etc.), and long-term monitoring, essential for bioprinted constructs containing spheroids.
References
1 Peirsman, A. et al., Nature methods. 18, 1294-1303 (2021).
2 Dmitriev, R. I. et al., Biomaterials. 34, 9307-9317 (2013)."
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