Abstract
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.
Highlights
We developed a system for visualization of oxygen gradients in 3D using human adipose tissue–derived mesenchymal stem cells modified to stably express a fluorescent genetically engineered hypoxia sensor hypoxia‐responsive elements (HREs)‐dUnaG
We demonstrated that reporter human adipose tissue (hAD)‐Mesenchymal stem cells (MSCs) output is sensitive to different oxygen levels and displays fast decay kinetics after reoxygenation
We report for the first time the creation of a lentivirally transduced hypoxia‐reporter MSCs derived from human adipose tissue, based on this novel hypoxia‐reporter system
Summary
Medicine is their application as cell suspensions for stromal/paracrine effects (trophic rescue function and immunomodulation) as Mesenchymal stem cells (MSCs) are widely used in medicine and well as employment of MSCs in tissue engineering and related clinical research due to their promising bioregenerative potential. applications (Han et al, 2019). The applications of hypoxia‐reporter cells include the creation of hypoxic conditions in 3D cell cultures without oxygen displacement in incubators, but by a direct tuning of cell‐seeding number and hydrogel composition Using such constructs with in situ hypoxia allows a much better control of the cellular microenvironment and will be helpful in improving the reproducibility of tissue engineering protocols, as well as in vitro cell models in, for example, drug screening, tumor models, or cell behavior studies. The use of hypoxia‐reporting cells based on MSCs will allow researchers to come one step closer to the cultivation of MSCs in conditions resembling the genuine in vivo microenvironment
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