title: Imaging of neurosphere oxygenation with phosphorescent probes.
abstract: Multicellular spheroids are useful models of mammalian tissue for studies of cell proliferation, differentiation, replacement therapies and drug action. Having a size of 100-500 μm they mimic in vivo micro-environment and characteristic gradients of O2, pH and nutrients. We describe the use of cell-penetrating O2 probes based on phosphorescent Pt-porphyrins to perform high-resolution 2D and 3D mapping of O2 in spheroid structures by live cell fluorescence imaging technique. Optimised procedures for preparation of neurospheres from cortical neural cells isolated from embryonic rat brain, their staining with the phosphorescent O2 probes NanO2 and MM2 and subsequent analysis of oxygenation on different live cell imaging platforms, including widefield and confocal phosphorescence lifetime imaging microscopy (PLIM), conventional confocal and two-photon ratiometric intensity based O2 detection are presented. This is followed by a series of physiological experiments in which oxygenation patterns of the neurospheres are correlated with culturing conditions (atmospheric hypoxia and hyperoxia, size, growth factors), distribution of stem cells, mature neurons and astrocytes, HIF-2α stabilisation and responses to metabolic stimulation. The O2 imaging method allows multiplexing with many conventional fluorescent probes to perform multi-parametric imaging analysis of cells in 3D microenvironment. It can be applied to other types of spheroids and 3D tissue models.
authors.affiliation: Biochemistry Department, University College Cork, Cork, Ireland. Electronic address: r.dmitriev@ucc.ie.
authors.forename: Ruslan I
authors.initials: RI
authors.lastname: Dmitriev
authors.forename: Alexander V
authors.initials: AV
authors.lastname: Zhdanov
authors.forename: Yvonne M
authors.initials: YM
authors.lastname: Nolan
authors.forename: Dmitri B
authors.initials: DB
authors.lastname: Papkovsky
mesh_terms: D000818:Animals; D002470:Cell Survival; D002478:Cells, Cultured; D005456:Fluorescent Dyes / Q000737:chemistry* / Q000378:metabolism; D018613:Microscopy, Confocal / Q000379:methods; D009474:Neurons / Q000166:cytology* / Q000378:metabolism; D061848:Optical Imaging / Q000379:methods*; D009944:Organoplatinum Compounds / Q000737:chemistry* / Q000378:metabolism; D010100:Oxygen / Q000032:analysis* / Q000378:metabolism; D011166:Porphyrins / Q000737:chemistry / Q000378:metabolism; D051381:Rats; D018874:Spheroids, Cellular / Q000166:cytology* / Q000378:metabolism
chemical_list: D005456:Fluorescent Dyes; D009944:Organoplatinum Compounds; D011166:Porphyrins; D010100:Oxygen
keywords: 3D; 3D tissue model; CTX; DIV; FLIM; HBSS; HIF; HXT; Hank's balanced salt solution; Hoechst 33342; Multi-cellular spheroids; Oxygen imaging; PDL; PLIM; PQM; Phosphorescent O(2) sensitive probe; ROI; ROS; RT; Stem cell; TBST; TCSPC; TMRM; cholera toxin; days in vitro; fluorescence lifetime imaging microscopy; hypoxia inducible factor; phosphorescence lifetime imaging microscopy; phosphorescence quenching microscopy; poly-d-lysine; reactive oxygen species; region of interest; room temperature; tetramethyl rhodamine methyl ester; three-dimensional; time-correlated single photon counting; tris-buffered saline, Tween 20