TY - JOUR

T1 - The Dual Luminescence Lifetime pH/Oxygen Sensor: Evaluation of Applicability for Intravital Analysis of 2D- and 3D-Cultivated Human Endometrial Mesenchymal Stromal Cells

AU - Litvinov, Ilia K.

AU - Belyaeva, Tatiana N.

AU - Salova, Anna V.

AU - Aksenov, Nikolay D.

AU - Chelushkin, Pavel S.

AU - Solomatina, Anastasia I.

AU - Tunik, Sergey P.

AU - Kornilova, Elena S.

PY - 2023/10/26

Y1 - 2023/10/26

N2 - The oxygenation of cells and tissues and acidification of the cellular endolysosomal system are among the major factors that ensure normal functioning of an organism and are violated in various pathologies. Recording of these parameters and their changes under various conditions is an important task for both basic research and clinical applications. In the present work, we utilized internalizable dual pH/O 2 lifetime sensor (Ir-HSA-FITC) based on the covalent conjugation of human serum albumin (HSA) with fluorescein isothiocyanate (FITC) as pH sensor and an orthometalated iridium complex as O 2 sensor. The probe was tested for simultaneous detection of acidification level and oxygen concentration in endolysosomes of endometrial mesenchymal stem/stromal cells (enMSCs) cultivated as 2D monolayers and 3D spheroids. Using a combined FLIM/PLIM approach, we found that due to high autofluorescence of enMSCs FITC lifetime signal in control cells was insufficient to estimate pH changes. However, using flow cytometry and confocal microscopy, we managed to detect the FITC signal response to inhibition of endolysosomal acidification by Bafilomycin A1. The iridium chromophore phosphorescence was detected reliably by all methods used. It was demonstrated that the sensor, accumulated in endolysosomes for 24 h, disappeared from proliferating 2D enMSCs by 72 h, but can still be recorded in non-proliferating spheroids. PLIM showed high sensitivity and responsiveness of iridium chromophore phosphorescence to experimental hypoxia both in 2D and 3D cultures. In spheroids, the phosphorescence signal was detected at a depth of up to 60 μm using PLIM and showed a gradient in the intracellular O 2 level towards their center.

AB - The oxygenation of cells and tissues and acidification of the cellular endolysosomal system are among the major factors that ensure normal functioning of an organism and are violated in various pathologies. Recording of these parameters and their changes under various conditions is an important task for both basic research and clinical applications. In the present work, we utilized internalizable dual pH/O 2 lifetime sensor (Ir-HSA-FITC) based on the covalent conjugation of human serum albumin (HSA) with fluorescein isothiocyanate (FITC) as pH sensor and an orthometalated iridium complex as O 2 sensor. The probe was tested for simultaneous detection of acidification level and oxygen concentration in endolysosomes of endometrial mesenchymal stem/stromal cells (enMSCs) cultivated as 2D monolayers and 3D spheroids. Using a combined FLIM/PLIM approach, we found that due to high autofluorescence of enMSCs FITC lifetime signal in control cells was insufficient to estimate pH changes. However, using flow cytometry and confocal microscopy, we managed to detect the FITC signal response to inhibition of endolysosomal acidification by Bafilomycin A1. The iridium chromophore phosphorescence was detected reliably by all methods used. It was demonstrated that the sensor, accumulated in endolysosomes for 24 h, disappeared from proliferating 2D enMSCs by 72 h, but can still be recorded in non-proliferating spheroids. PLIM showed high sensitivity and responsiveness of iridium chromophore phosphorescence to experimental hypoxia both in 2D and 3D cultures. In spheroids, the phosphorescence signal was detected at a depth of up to 60 μm using PLIM and showed a gradient in the intracellular O 2 level towards their center.

KW - Fluorescein-5-isothiocyanate

KW - Humans

KW - Hydrogen-Ion Concentration

KW - Iridium/chemistry

KW - Luminescence

KW - Mesenchymal Stem Cells

KW - Oxygen

KW - hypoxia

KW - endolysosomes

KW - dual sensor

KW - lifetime imaging

KW - mesenchymal stromal cells

KW - phosphorescence

KW - autofluorescence

KW - oxygen sensing

KW - FITC

KW - pH sensing

KW - fluorescence

KW - Ir(III) complexes

KW - spheroids

UR - https://www.mendeley.com/catalogue/74109080-1080-397a-ba16-8e72a51dc8ba/

U2 - https://doi.org/10.3390/ijms242115606

DO - https://doi.org/10.3390/ijms242115606

M3 - Article

C2 - 37958592

VL - 24

JO - International Journal of Molecular Sciences

JF - International Journal of Molecular Sciences

SN - 1422-0067

IS - 21

M1 - 15606

ER -