Intracellular label-free detection of mesenchymal stem cell metabolism within a perivascular niche-on-a-chip

Abstract

The stem cell niche at the perivascular space in human tissue plays a pivotal role in dictating the overall fate of stem cells within it. Mesenchymal stem cells (MSCs) in...

Microfluidic organ-on-chip system for multi-analyte monitoring of metabolites in 3D cell cultures

“…2b. Previous studies demonstrated for adherent cell monolayers 53 and matrix-embedded cancer cells 54 that such gradients lead to nutrient-dependent metabolic profiles and cell viability. Consequently, spheroids developing closer to the supply were exposed to more favourable growth conditions than cells growing more distant, as reflected in their structure.…”

Non-Invasive classification of macrophage polarisation by 2P-FLIM and machine learning

“…Recapitulating the sequence of metabolic enzyme inhibition which formed a basis of the extracellular flux analysis, sequential 2P-FLIM micrographs of IFNγ- or IL-4-polarised macrophages subjected to the same regimes were acquired (Figure 3A). A reduced τ avg , observed with IFNγ-M1 macrophages reflects a higher relative amount of free NAD(P)H (which has characteristic short fluorescence lifetimes), indicative with glycolysis (Perottoni et al 2021, Walsh et al 2013). In contrast IL-4-M2 macrophages had higher τ avg - reflective of OxPhos, due to increased proportion of longer lifetime, protein-bound NAD(P)H (Okkelman et al 2019, Walsh et al 2020) (Figure 3B).…”

“…NAD(P)H is noted as having a long fluorescent lifetime when enzyme-bound and a short fluorescence lifetime when free in the cytoplasm with 2P-FLIM facilitating quantification of these fluorescence lifetimes and their respective proportions. This information has been harnessed to spatially assess the metabolism of murine tumour tissues, murine-derived organoid cultures, human T-cells, human B-cells, murine macrophages and cultures subjected to bioreactor engineered nutrient gradients in which fluorescence intensity and lifetime of NAD(P)H and FAD + were influenced by microenvironmental metabolites (Alfonso-Garcia et al 2016, Floudas et al 2020, Okkelman et al 2019, Perottoni et al 2021, Skala et al 2007, Szulczewski et al 2016, Walsh et al 2020). NADH and NADPH fluorescence properties are identical, hence NAD(P)H refers to these intracellular pools combined (Huang et al 2002).…”

Luminescence lifetime imaging of three-dimensional biological objects

“…FLI and PLIM in tissue engineering frequently deal with 'top-tobottom' engineering approaches, such as organizing fluidic flow (tissues and organ-on-a-chip) (Perottoni et al, 2021;Zirath et al, 2018;Ahmed et al, 2019), scaffold materials (Appel et al, 2013;Sud et al, 2006;Neto et al, 2020) and, more recently, direct bioprinting of biologics (Ozturk et al, 2016(Ozturk et al, , 2020Kingsley et al, 2019). Autofluorescence FLIM has been used as a readout parameter to monitor the successful biofabrication of dermal matrix engineered scaffolds (Formigli et al, 2012) and scaffold composition during bioengineered cartilage growth (Fite et al, 2011).…”