Drug-induced mitochondrial toxicity has been studied for many decades in both academic and industrial settings. Recently, research on mitochondrial function has gained more momentum as it has become evident that mitochondria are major contributors to drug-induced organ injury. The blockbuster drug troglitazone, for example, had to be withdrawn from the market in 1997 because of severe liver toxicity that at least in part was caused by mitochondrial toxicity. This example has prompted industry-wide focus on the development of better mechanistic-based cell models for testing of mitochondrial liabilities in vitro.
The Seahorse XFe96 platform (Agilent Technologies, CA, USA) enables direct measurement of mitochondrial activity in whole cells by measuring oxygen flux (OCR) and extracellular acidification (ECAR) using a pair of fluorescent sensors. The Seahorse XF Cell Mito Stress Test (MST) Kit uses modulators of cellular respiration that specifically target components of the electron transport chain (ETC) to reveal key parameters of metabolic function in response to drugs on a wide variety of cell types.
A few years ago, we teamed up with Seahorse Bioscience to investigate the use of 3D microtissues to evaluate the impact of drugs on mitochondrial function. Our 3D InSight™ Human Liver Microtissues are known to maintain a differentiated state and stable phenotype as well as metabolic activity for up to 4 weeks in culture and therefore allowing to test not only short-term effects but also effects of mitochondrial active metabolites and long-term effects. Practically, the measurement is made possible by the important feature of the 3D microtissue format, with which treatment and measurement can be fully decoupled from each other as the non-adhering spherical microtissues can be transferred from the culturing plate to the Seahorse assay plate.
In the recently published book chapter, we present this novel 3D mitotoxicity assay for assessment of mitochondrial impairment by combining 3D microtissues with the Seahorse XFe96 analyzer. The high SRC of microtissues allows it to be used as an early marker for mitochondrial stress and has shown to be a sensitive and robust endpoint for mitochondrial impairment across three different tissue types.
InSphero offers a Mitochondrial Toxicity Testing Service that applies the same robust and highly predictive two-step assay described in this book chapter. By combining 3D InSight™ Human Liver Microtissues with Seahorse XFe96 technology, we can assess potential for mitochondrial liabilities with high sensitivity and specificity with higher SRC than possible in 2D monolayer hepatocyte cultures.