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Biomolecular Sensor Platform for Elucidating Hypoxic Signatures in 2D and 3D in vitro culture Systems

Subject Area Technical Chemistry
Term from 2018 to 2024
Project identifier Deutsche Forschungsgemeinschaft (DFG) - Project number 398007461
 
Final Report Year 2024

Final Report Abstract

In vitro cultivation conditions are critical to cellular physiology and response to external stimuli. Two key microenvironmental factors in these systems are three-dimensional (3D) cultivation and in situ oxygen concentrations. While 3D cultivation systems and protocols have become widely recognized in the scientific community, challenges with reproducibility and data comparison across different research groups remain common. A possible reason for this is the considerable variation in the size of 3D in vitro models and cell densities, as cell behavior is highly influenced by local oxygen concentrations in different systems. Most cells in the human body grow and function at oxygen levels lower than the ambient 21%, with each tissue having its own specific physiological oxygen range. Accurate knowledge of the oxygen concentration ranges that trigger hypoxic responses in different cell types is essential for obtaining reliable experimental data. This understanding is also crucial for producing validated therapeutic cells, developing 3D in vitro models for drug screening and disease modeling, and creating tissue-engineered constructs. The key regulator of the cellular hypoxic response is hypoxia inducible factor 1α (HIF-1α), which was shown to have 300 target genes where some of them are transcription factors themselves with hundreds of target genes. Monitoring HIF-1a stabilization is an extremely difficult task, especially in 3D cell culture systems, because this protein has a very short half-life. In this project we aimed to modify different human cell types (MSCs, two types of endothelial cells, neuronal progenitor cells and chondrocytes) with genetically encoded hypoxia sensors and evaluate hypoxic signatures of these cells in 2D and 3D cell culture systems. Furthermore, by modifying the cells with reactive oxygen species (ROS) sensors, ROS dynamics were studied in these models. We could demonstrate difference in hypoxic response between all tested cell types. For the first time we directly demonstrated that endothelial cells derived from different vessels have unequal reaction on lower oxygen levels. Surprisingly, hypoxic response of huVECs in 3D spheroids was stronger than the one for the huMEC spheroids, while in 2D cell cultures hu-VECs reacted at the same low oxygen concentrations with weaker response. We also demonstrated that different spheroid fabrication platforms lead to different hypoxic response in 3D aggregates. Moreover, with the help of hypoxia sensor cells we were able to detect the presence of oxygen gradients in microstructured plates that hat previously gone unnoticed. We demonstrated that human condrocytes have much lower hypoxic threshold than MSCs: while MSCs reported hypoxia already started with 7% O2, chondrocytes did not stabilize HIF-1α until oxygen level was reduced to 1%. This indicated that in vitro cells retain a memory of their specific physiological oxygen concentration range of their tissue of origin, highlighting the need to carefully consider the term “hypoxia” for each specific cell type. The primary objectives of the project were both, (1) to assess the hypoxic response across various cell types and (2) to develop a panel of human sensor cells for use in research by other scientific groups. Both goals were successfully achieved: the project generated important insights into cellular responses in 2D and 3D environments, and the sensor cells created were shared with other researchers and the first manuscripts utilizing these cells have already been published.

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