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

• Applicant: Privatdozentin Dr. Antonina Lavrentieva
• Subject Area: Technical Chemistry
• Term: from 2018 to 2024
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 398007461

The proposed project builds upon work undertaken in the ongoing grant on 3D dual gradient systems for functional cell screening. The aim of the follow-up project is to create a biomolecular sensor platform for elucidating hypoxic signatures in 2D and 3D in vitro culture systems. In the ongoing project, we successfully modified human mesenchymal stem cells (MSCs) with genetically encoded hypoxia sensors for gradient hydrogels characterization. We anticipated that the modified cells would provide qualitative (yes/no) information about the onset of hypoxia. We made the surprising observation that hypoxia-reporter MSCs were also sensitive to different oxygen levels: fluorescence intensity was in strict correlation with available oxygen concentrations. Thus, reporter cells (RC) can serve as a reliable and valuable tool for further studies in various 3D constructs.The first phase of the follow-up project will focus on the modification of additional cell types (chondrocytes, endothelial and neural progenitor cells) with hypoxia sensors and investigation of their “hypoxic signatures” in various 3D cultivation systems (cellular aggregates, hydrogels and scaffolds). For the first time, critical size and cell densities for 3D constructs will be detected for different cell types. Preliminary experiments with modified chondrocytes demonstrated an onset of hypoxia at much lower oxygen concentrations than for MSCs. This shows that different cell types not only have different oxygen consumption rates, but have different requirements on oxygen availability. Since chondrocytes and endothelial cells and MSCs are the most widely used cell types in bioregenerative medicine, new insights will contribute to better understand and improve cell survival and functionality in 3D constructs. Neural progenitor cells are used for cellular therapies in central nervous system damages and in 3D in vitro models for degenerative diseases. Since neurons consume high amounts of oxygen, exact knowledge of in vitro oxygen limitations will facilitate the development of reproducible 3D models. RC fluorescence and in situ oxygen concentrations will be monitored. Fluorescence imaging analysis will be developed to allow the prediction of in situ oxygen concentrations based directly on cell fluorescence. Hypoxia triggers switch to glycolysis and critical drop in pH. Cells also increase production of reactive oxygen species (ROS) in hypoxia, causing oxidative stress and DNA damage. Therefore, cells will be modified with novel pH and ROS biosensors and in situ pH shift and ROS production will be monitored for all 3D systems where hypoxia occurs. In sum, the proposed extension will deliver valuable information on the onset of hypoxia in 3D in vitro systems and the response of different types of human cells. The various RC created in this project will also present a versatile, transferable toolkit for further studies in e.g. bioprinting, novel biomaterials or disease modeling.

• DFG Programme: Research Grants
• International Connection: Russia, United Kingdom
• Cooperation Partners: Professor Vsevolod Belousov, Ph.D., until 3/2022; Professor Nicholas Forsyth, Ph.D.