Balance between the cell viability and death in 3D

Debruyne, Angela C.; Okkelman, Irina A.; Dmitriev, Ruslan I.

doi:10.1016/j.semcdb.2022.09.005

Cited by 6 publications

(6 citation statements)

References 230 publications

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“…107,108 On the other hand, spheroids used as tissue building blocks in 3D printing, bioassembly, and biofabrication also require monitoring of their viability, cell death, and oxygenation. 109,110 Our "discovery" of the inverted oxygenation gradients with the presented approach and its correlation with autofluorescence label-free two-photon FLIM highlights the utility of the method, as more affordable, direct, and highly compatible with other multi-parameter micro-and mesoscopy modalities of 3D tissue models.…”

Section: Discussionmentioning

confidence: 83%

“…The oxygenation gradients depend upon multiple factors, including spheroid size, extracellular viscosity, and spheroid formation and handling methods. This emphasizes the importance of oxygenation monitoring in studies involving the development and validation of targeted drug-/radiotherapies with cancer cells, tumor organoids, and “tumor avatars”. , On the other hand, spheroids used as tissue building blocks in 3D printing, bioassembly, and biofabrication also require monitoring of their viability, cell death, and oxygenation. , Our “discovery” of the inverted oxygenation gradients with the presented approach and its correlation with autofluorescence label-free two-photon FLIM highlights the utility of the method, as more affordable, direct, and highly compatible with other multi-parameter micro- and mesoscopy modalities of 3D tissue models.…”

Section: Discussionmentioning

confidence: 86%

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Live Microscopy of Multicellular Spheroids with the Multimodal Near-Infrared Nanoparticles Reveals Differences in Oxygenation Gradients

Debruyne,

Okkelman,

Heymans

et al. 2024

ACS Nano

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Assessment of hypoxia, nutrients, metabolite gradients, and other hallmarks of the tumor microenvironment within 3D multicellular spheroid and organoid models represents a challenging analytical task. Here, we report red/near-infrared (NIR) emitting cell staining with O 2 -sensitive nanoparticles, which enable measurements of spheroid oxygenation on a conventional fluorescence microscope. Nanosensor probes, termed "MMIR" (multimodal infrared), incorporate an NIR O 2 -sensitive metalloporphyrin (PtTPTBPF) and deep red aza-BODIPY reference dyes within a biocompatible polymer shell, allowing for oxygen gradient quantification via fluorescence ratio and phosphorescence lifetime readouts. We optimized staining techniques and evaluated the nanosensor probe characteristics and cytotoxicity. Subsequently, we applied nanosensors to the live spheroid models based on HCT116, DPSCs, and SKOV3 cells, at rest, and treated with drugs affecting cell respiration. We found that the growth medium viscosity, spheroid size, and formation method influenced spheroid oxygenation. Some spheroids produced from HCT116 and dental pulp stem cells exhibited "inverted" oxygenation gradients, with higher core oxygen levels than the periphery. This contrasted with the frequently encountered "normal" gradient of hypoxia toward the core caused by diffusion. Further microscopy analysis of spheroids with an "inverted" gradient demonstrated metabolic stratification of cells within spheroids: thus, autofluorescence FLIM of NAD(P)H indicated the formation of a glycolytic core and localization of OxPhos-active cells at the periphery. Collectively, we demonstrate a strong potential of NIR-emitting ratiometric nanosensors for advanced microscopy studies targeting live and quantitative real-time monitoring of cell metabolism and hypoxia in complex 3D tissue models.

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Section: Discussionmentioning

confidence: 83%

Section: Discussionmentioning

confidence: 86%

Live Microscopy of Multicellular Spheroids with the Multimodal Near-Infrared Nanoparticles Reveals Differences in Oxygenation Gradients

Debruyne,

Okkelman,

Heymans

et al. 2024

ACS Nano

Self Cite

View full text Add to dashboard Cite

show abstract

“…This emphasizes the importance of oxygenation monitoring in studies involving the development and validation of targeted drug/ radio-therapies with cancer cells, tumour organoids, and ‘tumour avatars’. 107,108 On the other hand, spheroids used as tissue building blocks in 3D printing, bioassembly, and biofabrication, also require monitoring of their viability, cell death, and oxygenation 109,110 . Our ‘discovery’ of the inverted oxygenation gradients with the presented approach and its correlation with autofluorescence label-free two-photon FLIM, highlights the utility of the method, as more affordable, direct, and highly compatible with other multi-parameter micro- and mesoscopy modalities of 3D tissue models.…”

Section: Discussionmentioning

confidence: 99%

Live microscopy of multicellular spheroids with the multi-modal near-infrared nanoparticles reveals differences in oxygenation gradients

Debruyne,

Okkelman,

Heymans

et al. 2023

Preprint

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Assessment of hypoxia, nutrients, metabolite gradients, and other hallmarks of the tumor microenvironment within 3D multicellular spheroid and organoid models represents a challenging analytical task. Here, we report red/near-infrared emitting cell staining O2-sensitive nanoparticles, which enable measurements of spheroid oxygenation on a conventional fluorescence microscope. Nanosensor probes, termed ‘MMIR’ (multi-modal infrared), incorporate a near-infrared O2-sensitive metalloporphyrin (PtTPTBPF) and a deep red aza-BODIPY reference dyes within a biocompatible polymer shell, allowing oxygen gradients quantificationviafluorescence ratio emission and phosphorescence lifetime readouts. We optimized staining techniques and evaluated nanosensor probe characteristics and cytotoxicity. Subsequently, we applied nanosensors to the live spheroid models based on HCT116, DPSCs, and SKOV3 cells, at rest and treated with drugs affecting cell respiration. We found that the growth medium viscosity, spheroids size, and formation method influenced spheroid oxygenation.Unexpectedly, some spheroids (produced from HCT116 and dental pulp stem cells) exhibited ‘inverted’ oxygenation gradients, with higher core oxygen levels than the periphery. This contrasted with the frequently encountered ‘direct’ gradient of hypoxia towards the core caused by diffusion. Further microscopy analysis of spheroids with an “inverted” gradient demonstrated metabolic stratification of cells within spheroids: thus, autofluorescence FLIM of NAD(P)H revealed the formation of glycolytic core, and localization of OxPhos-active cells at the periphery. Collectively, the presented methodology demonstrates strong potential for advanced microscopy studies targeting live and quantitative real-time monitoring of cell metabolism and hypoxia in complex 3D tissue models.Graphical abstract

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“…Studying metabolism alone has its benefits but often adding other biosensors and sensing moieties helps integrating the biochemical processes and linking e. RNA dynamics and others [181,182]. Many additional imaging probes and modalities can be integrated into multi-parameter FLIM of cell metabolism, such as probes informing on cell mechanical properties, e.g.…”

Section: Other Biomarkers and Physical Parametersmentioning

confidence: 99%

Probing organoid metabolism using fluorescence lifetime imaging microscopy (FLIM): The next frontier of drug discovery and disease understanding

Barroso,

Monaghan,

Niesner

et al. 2023

Advanced Drug Delivery Reviews

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Balance between the cell viability and death in 3D

Cited by 6 publications

References 230 publications

Live Microscopy of Multicellular Spheroids with the Multimodal Near-Infrared Nanoparticles Reveals Differences in Oxygenation Gradients

Live Microscopy of Multicellular Spheroids with the Multimodal Near-Infrared Nanoparticles Reveals Differences in Oxygenation Gradients

Live microscopy of multicellular spheroids with the multi-modal near-infrared nanoparticles reveals differences in oxygenation gradients

Probing organoid metabolism using fluorescence lifetime imaging microscopy (FLIM): The next frontier of drug discovery and disease understanding

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