InSphero and Agilent discuss the need for metabolically competent models with stable metabolic activity for mitochondrial toxicity testing in a new textbook chapter.

Mitochondrial Dysfunction Caused by Drugs and Environmental Toxicants, a new textbook co-edited by Yvonne Will of Pfizer and James Dykens of Eyecyte Therapeutics, is an exhaustive two-volume reference that belongs on the bookshelf of any researcher and student interested in mitochondrial pharmacology and toxicology. The book covers topics ranging from basic concepts of mitochondrial toxicity, dysfunction, and etiology to the latest methods and models for detecting mitochondrial toxicity. The editors asked InSphero and Agilent to write Chapter 18, “Using 3D Microtissues for Identifying Mitochondrial Liabilities,” in which we review the current tools and technology available for detecting mitochondrial liabilities and describe our recommended protocol for spare-respiratory capacity (SRC)-based detection of mitochondrial liabilities.

The Role of Mitochondria in Drug-Induced Organ Injury

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 The blockbuster drug troglitazone, for example, had to be withdrawn from the market in 1997 because of severe liver toxicity that at least in part was caused by mitochondrial toxicity.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 an industry-wide focus on the development of better mechanistic-based cell models for testing mitochondrial liabilities in vitro.

The Seahorse XF^e96 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 Perfect Partnership of 3D Cell Culture Technology

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 active mitochondrial metabolites and long-term effects. Practically, the measurement is made possible by the important feature of the 3D spheroid 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 the assessment of mitochondrial impairment by combining 3D spheroids with the Seahorse XF^e96 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.

Assess Mitochondrial Liabilities with 3D InSight™ Toxicology Services

InSphero offers a Mitochondrial Toxicity Testing Service that applies the same robust and highly predictive assay described in this book chapter. By combining 3D InSight™ Human Liver Microtissues with Seahorse XF^e96 technology, we can assess the potential for mitochondrial liabilities with high sensitivity and specificity with higher SRC than possible in 2D monolayer hepatocyte cultures.