Abstract
Brain diseases including Down syndrome (DS/TS21) are known to be characterized by changes in cellular metabolism. To adequately assess such metabolic changes during pathological processes and to test drugs, methods are needed that allow monitoring of these changes in real time with minimally invasive effects. Thus, the aim of our work was to study the metabolic status and intracellular pH of spheroids carrying DS using fluorescence microscopy and FLIM. For metabolic analysis we measured the fluorescence intensities, fluorescence lifetimes and the contributions of the free and bound forms of NAD(P)H. For intracellular pH assay we measured the fluorescence intensities of SypHer-2 and BCECF. Data were processed with SPCImage and Fiji-ImageJ. We demonstrated the predominance of glycolysis in TS21 spheroids compared with normal karyotype (NK) spheroids. Assessment of the intracellular pH indicated a more alkaline intracellular pH in the TS21 spheroids compared to NK spheroids. Using fluorescence imaging, we performed a comprehensive comparative analysis of the metabolism and intracellular pH of TS21 spheroids and showed that fluorescence microscopy and FLIM make it possible to study living cells in 3D models in real time with minimally invasive effects.
Highlights
Induced pluripotent stem cells are attractive objects for scientific research in various fields of cell biology, one of which is the study of human hereditary diseases
We demonstrated for the first time the differences in cellular metabolism and spheroids
We demonstrated for the first time the differences in cellular metabolism and intracellular pH between normal karyotype (NK) neural spheroids and trisomy of chromosome 21 (TS21) spheroids, using both fluoresintracellular pH between NK neural spheroids and TS21 spheroids, using both fluorescent and FLIM methods
Summary
Induced pluripotent stem cells (iPSCs) are attractive objects for scientific research in various fields of cell biology, one of which is the study of human hereditary diseases. It has been shown that iPSCs from individuals with hereditary diseases can develop into the disease-associated cell types and that the derived cells reproduce the metabolic hallmarks of the respective diseases, for example, Down syndrome, Alzheimer’s disease, Parkinson’s disease many other disorders [1,2,3,4,5]. Traditional two-dimensional cell cultures do not adequately reflect the microenvironment of such cells in vivo. To address this and better mimic the complex environment of human tissues, great numbers of protocols have been developed for the generation of 3D cell models from iPSCs. 3D models can help in the study of the tissues of patients with hereditary diseases Several studies using cells differentiated from TS21 human iPSCs have shown that these objects reflect the molecular and cellular phenotypes of primary cells obtained from patients with
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