April 4, 2016 — Mitochondrial dysfunction plays a critical role in the development of chemical and pharmaceutical toxicity. However, current methods to evaluate mitochondrial activity still rely on traditional tests called end-point assays, which provide limited prognostic information.
Microfluidic organ-on-a-chip technology, which uses microchip-manufacturing methods to arrange living cell cultures to simulate the physiology of tissues and organs, is poised to replace drug toxicity testing in animals, but thus far has demonstrated few advantages over traditional methods.
Now, researchers from The Hebrew University of Jerusalem describe a new generation of Liver on Chip devices, in which the researchers add glucose and lactate micro-sensors, allowing them to measure minuscule changes in central carbon metabolism in real time (seconds to minutes).
The robust microfluidic platform is composed of sub-millimeter human tissues, characterized as “bionic” since they contain optoelectronic sensors for oxygen and are maintained under conditions simulating the human physiological environment. The platform includes a computer-controlled switchboard and permits the automated measurement of glucose and lactate using clinical-grade micro-sensors. The sensor-integrated platform permits real-time tracking of the dynamics of metabolic adaptation to any type of mitochondrial damage for over a month in culture.
The study, entitled “Real-time Monitoring of Metabolic Function in Liver-on-Chip Microdevices Tracks the Dynamics of Mitochondrial Dysfunction,” appears in the Proceedings of the National Academy of Sciences.
“Central carbon, or glucose and amino acid, metabolism is by far the most important source of energy and materials for our cells. It is the backbone of cellular function,” said the study’s lead author Professor Yaakov Nahmias, Director of the Alexander Grass Center for Bioengineering at The Hebrew University of Jerusalem. Otto Warburg recognized the importance of changes in central carbon metabolism to cancer development back in 1924, and more recent studies tied alterations in this pathway to the emergence of stem cells. “This metabolic pathway is very sensitive, and any toxic damage will either directly or ultimately lead to changes in glucose metabolism,” said Nahmias.
This approach already enabled Nahmias and his team to identify a new mode of acetaminophen (Tylenol®) toxicity back in June, suggesting the drug could directly block respiration in the kidneys and skin.
In the current research, the team used an array of micro-sensors to measure small changes in metabolic fluxes. In other words, the sensor-integrated “bionic” micro-livers actively told researchers how they change their metabolic pathways (what they “eat,” “digest” and “spew out”) when exposed to new drugs.
This allows researchers to identify new causes for idiosyncratic toxicity, one of the biggest problems in drug discovery and the main cause for post-market drug withdrawal.
Idiosyncratic toxicity occurs without obvious reason to about 1:100,000 in the population, forcing new warning labels or complete withdraw of the drug (costing billions).
The researchers tested the new technology on troglitazone (Rezulin®), an anti-diabetic and anti-inflammatory drug which was removed from the U.S. market in 2000 due to severe drug-induced liver injury, costing Pfizer Inc. over $750 million in lawsuits.
The results showed that even at low troglitazone concentrations previously regarded as safe, in which traditional tests don’t reveal any damage to the cells, the new liver-on-chip technology was able to detect mitochondrial stress that forces the liver to increase its reliance on glucose metabolism.
By revealing the dynamics of cellular adaptation to mitochondrial damage, this novel organ-on-chip technology permits detection of chemical toxicity before any effects on cell or tissue viability can be observed.
Professor Nahmias said: “The ability to measure metabolic fluxes using small numbers of cells under physiological conditions can redefine the study of neurodegenerative disease, stem cells and cancer, in addition to drug discovery. In contrast to other instruments, such as the SeaHorse Flux Analyzer, our sensors do not require calibration and our cells do not undergo hypoxia or become exposed to decaying chemical signals due to non-specific absorption. Our microfluidic system could be of significant interest to biological and clinical laboratories, whose work can range from the study of mitochondria and oxidative stress in neurological disorders (Alzheimer’s, Parkinson’s), to metabolic disease (diabetes, obesity), stem cell biology (pluripotency), virology and cancer.”
Dr. Daniel Duche, former head of the Early Safety Laboratory, unit of the Predictive Models and Methods Development Department of L’Oréal Research & Innovation, said: “The work performed by Y. Nahmias’s team demonstrates that it becomes possible to monitor dynamically in real time metabolic functions of cells exposed to different drug concentrations over a long period using organ-on-chip microdevices. That helps to define Adverse Outcome Pathways and brings a real breakthrough in the in vitro alternative methods to animal experimentation for evaluating toxicity of chemicals.”
Yissum, the technology transfer company of the Hebrew University, recently filed two provisional patent applications covering the promising Liver-on-Chip technology, that will be the basis of a new company to be established together with Professor Yaakov Nahmias. This new company will provide diagnostic and prognostic analysis for pharmaceutical and cosmetics companies to help define drug safety and screen out idiosyncratic drug toxicity.
Participating institutions in this research include: The Alexander Grass Center for Bioengineering at The Hebrew University of Jerusalem; Department of Cell and Developmental Biology, Silberman Institute of Life Sciences, The Hebrew University of Jerusalem; Fraunhofer Institute for Cell Therapy and Immunology, Branch Bioanalytics and Bioprocesses (Fraunhofer IZI-BB), Potsdam, Germany; Department of In Vitro Toxicology and Dermato-Cosmetology, Vrije Universiteit Brussel, Brussels, Belgium; Centre for Microsystems Technology, IMEC and Ghent University, Ghent, Belgium; and Federal Institute for Risk Assessment (BfR), Berlin, Germany.
Funding: The research was funded by ERC Starting Grant TMIHCV (N° 242699), the British Council BIRAX Regenerative Medicine award (N° 33BX12HGYN) and the HeMibio consortium funded by the European Commission and Cosmetics Europe as part of the SEURAT-1 cluster (N° HEALTH-F5-2010-266777). Additional funding was provided by the generous gift of Sam and Rina Frankel.