Institute of Technical Chemistry, Gottfried Wilhelm Leibniz University Hannover, Hannover, Germany.
Faculty of Biomedical Engineering, Technion, Haifa, Israel.
Biotechnol Bioeng. 2020 Nov;117(11):3265-3276. doi: 10.1002/bit.27503. Epub 2020 Jul 27.
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% O ). 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.
天然氧梯度存在于生物组织中，也存在于三维（3D）体外培养环境中。氧扩散限制和嵌入细胞的代谢耗氧会导致组织/基质中出现缺氧区。然而，可靠的系统来检测 3D 细胞培养系统中的氧梯度和细胞对缺氧的反应仍然缺失。在这项研究中，我们开发了一种使用人脂肪组织来源的间充质干细胞（hAD-MSCs）的 3D 氧梯度可视化系统，这些细胞被稳定表达荧光基因工程缺氧传感器 HRE-dUnaG 修饰。修饰后的细胞在增殖和分化能力方面保留了其干细胞特性。在不同氧水平（2.5%、5%和 7.5% O ）下，通过荧光显微镜和流式细胞术对缺氧报告细胞进行了评估。我们证明了报告 hAD-MSCs 的输出对不同的氧水平敏感，并在再氧合后显示出快速的衰减动力学。此外，报告细胞被封装在具有可变细胞数量的块状水凝胶中，以研究模型 3D 细胞培养应用中的传感器响应。使用基于 MSC 的缺氧报告细胞代表了一种有价值的工具，可以接近真正的体内细胞微环境，并将有助于更好地理解 AD-MSCs 的再生潜力。
