Natural oxygen gradients occur in tissues of biological organisms and also in the context of three‐dimensional (3D) in vitro cultivation. Oxygen diffusion limitation and metabolic oxygen consumption by embedded cells produce areas of hypoxia in the tissue/matrix. However, reliable systems to detect oxygen gradients and cellular response to hypoxia in 3D cell culture systems are still missing. In this study, we developed a system for visualization of oxygen gradients in 3D using human adipose tissue–derived mesenchymal stem cells (hAD‐MSCs) modified to stably express a fluorescent genetically engineered hypoxia sensor HRE‐dUnaG. Modified cells retained their stem cell characteristics in terms of proliferation and differentiation capacity. The hypoxia‐reporter cells were evaluated by fluorescence microscopy and flow cytometry under variable oxygen levels (2.5%, 5%, and 7.5% O₂). We demonstrated that reporter hAD‐MSCs output is sensitive to different oxygen levels and displays fast decay kinetics after reoxygenation. Additionally, the reporter cells were encapsulated in bulk hydrogels with a variable cell number, to investigate the sensor response in model 3D cell culture applications. The use of hypoxia‐reporting cells based on MSCs represents a valuable tool for approaching the genuine in vivo cellular microenvironment and will allow a better understanding of the regenerative potential of AD‐MSCs.

Interphase microtubules are organized into a radial array with centrosome in the center. This organization is a subject of cellular regulation that can be driven by protein phosphorylation. Only few protein kinases that regulate microtubule array in interphase cells have been described. Ste20-like protein kinase LOSK (SLK) was identified as a microtubule and centrosome-associated protein. In this study we have shown that the inhibition of LOSK activity by dominant-negative mutant K63R-ΔT or by LOSK depletion with RNAi leads to unfocused microtubule arrangement. Microtubule disorganization is prominent in Vero, CV-1, and CHO-K1 cells but less distinct in HeLa cells. The effect is a result neither of microtubule stabilization nor of centrosome disruption. In cells with suppressed LOSK activity centrosomes are unable to anchor or to cap microtubules, though they keep nucleating microtubules. These centrosomes are depleted of dynactin. Vero cells overexpressing K63R-ΔT have normal dynactin “comets” at microtubule ends and unaltered morphology of Golgi complex but are unable to polarize it at the wound edge. We conclude that protein kinase LOSK is required for radial microtubule organization and for the proper localization of Golgi complex in various cell types.

Genetically-encoded fluorescent sensors have been actively developed over the last few decades and used in live imaging and drug screening. Real-time monitoring of drug action in a specific cellular compartment, organ, or tissue type; the ability to screen at the single-cell resolution; and the elimination of false-positive results caused by low drug bioavailability that is not detected by in vitro testing methods are a few of the obvious benefits of using genetically-encoded fluorescent sensors in drug screening. In combination with high-throughput screening (HTS), some genetically-encoded fluorescent sensors may provide high reproducibility and robustness to assays. We provide a brief overview of successful, perspective, and hopeful attempts at using genetically encoded fluorescent sensors in HTS of modulators of ion channels, Ca²⁺ homeostasis, GPCR activity, and for screening cytotoxic, anticancer, and anti-parasitic compounds. We discuss the advantages of sensors in whole organism drug screening models and the perspectives of the combination of human disease modeling by CRISPR techniques with genetically encoded fluorescent sensors for drug screening.