A versatile multiplexed assay to quantify intracellular ROS and cell viability in 3D on-a-chip models

Redox Biology, vol. 57, pp. 102488

Abstract

Reactive oxygen species (ROS) have different properties and biological functions. They contribute to cell signaling and, in excessive amounts, to oxidative stress (OS). Although ROS is pivotal in a wide number of physiological systems and pathophysiological processes, direct quantification in vivo is quite challenging and mainly limited to in vitro studies. Even though advanced in vitro cell culture techniques, like on-a-chip culture, have overcome the lack of crucial in vivo-like physiological aspects in 2D culture, the majority of in vitro ROS quantification studies are generally performed in 2D. Here we report the development, application, and validation of a multiplexed assay to quantify ROS and cell viability in organ-on-a-chip models. The assay utilizes three dyes to stain live cells for ROS, dead cells, and DNA. Confocal images were analyzed to quantify ROS probes and determine the number of nuclei and dead cells. We found that, in contrast to what has been reported with 2D cell culture, on-a-chip models are more prone to scavenge ROS rather than accumulate them. The assay is sensitive enough to distinguish between different phenotypes of endothelial cells (ECs) based on the level of OS to detect higher level in tumor than normal cells. Our results indicate that the use of physiologically relevant models and this assay could help unravelling the mechanisms behind OS and ROS accumulation. A further step could be taken in data analysis by implementing AI in the pipeline to also analyze images for morphological changes to have an even broader view of OS mechanism.

Authors 8

1. Camilla Soragni corresponding Aachen Mimetas BV

   Maastricht University · Mimetas (Netherlands)

   Affiliation as printed

   MIMETAS BV, Leiden, the Netherlands; Department of Cardiology, Maastricht University, Maastricht, the Netherlands. Electronic address: c.soragni@maastrichtuniversity.nl

   Department of Cardiology, Maastricht University, Maastricht, the Netherlands

   MIMETAS BV, Leiden, the Netherlands

2. Gwenaëlle Rabussier Aachen Mimetas BV

   Maastricht University · Mimetas (Netherlands)

   Affiliation as printed

   MIMETAS BV, Leiden, the Netherlands; Department of Cardiology, Maastricht University, Maastricht, the Netherlands

   Department of Cardiology, Maastricht University, Maastricht, the Netherlands

   MIMETAS BV, Leiden, the Netherlands

3. Henriëtte L. Lanz Aachen Mimetas BV

   Mimetas (Netherlands)

   Affiliation as printed

   MIMETAS BV, Leiden, the Netherlands

4. Kristin M. Bircsak Aachen Mimetas BV

   Mimetas (Netherlands)

   Affiliation as printed

   MIMETAS BV, Leiden, the Netherlands

5. León J. De Windt

   Maastricht University

   Affiliation as printed

   Department of Cardiology, Maastricht University, Maastricht, the Netherlands

6. Sebastiaan J. Trietsch Aachen Mimetas BV

   Mimetas (Netherlands)

   Affiliation as printed

   MIMETAS BV, Leiden, the Netherlands

7. Colin E. Murdoch

   University of Dundee

   Affiliation as printed

   Systems Medicine, School of Medicine, University of Dundee, Dundee, Scotland, UK

8. Chee Ping Ng Aachen Mimetas BV

   Mimetas (Netherlands)

   Affiliation as printed

   MIMETAS BV, Leiden, the Netherlands