Custom mechanistic and investigative toxicology services

are available on request to systematically evaluate mechanisms of toxicity using 3D InSight™ Human Liver Microtissues. Work with our liver services team to design a custom study plan based on your desired dosin scheme (short-term: 3-7 days; long-term: 14-28 days), and desired endpoints:

• Hepatotoxicity: ATP content, α-GST (released/intracellular), Albumin secretion, LDH release
• Kupffer cell function/toxicity: IL-6 production, TNF-α
• Reactive metabolites: GSH content
• Metabolic competence: Cytochrome activation/induction
• Apoptosis: Caspace 3/7 activation
• Steatosis, phospholipidosis: Lipid accumulation
• Cholestasis: Efflux transporter inhibition
• Tumorigenicity: DNA synthesis
• Unbiased discovery of mechanism of action (MOA): Transcriptomics, proteomics

ATP – This cell viability assay is a bioluminescent ATP detection assay with an optimized protocol and improved formulation that has been expressly designed to measure the viability/metabolic activity of 3D microtissues. This single-component liquid reagent has significant lytic capacity and exhibits high ATP recovery.

LDH – This cytotoxicity assay is bioluminescent plate-based, for quantifying lactate dehydrogenase (LDH) release into the culture medium upon plasma membrane damage. The bioluminescent detection is more sensitive than colorimetric or fluorescent methods, allowing accurate detection of LDH from a small number of cells, including primary cells and 3D cell cultures. LDH quantification has been demonstrated to be equivalent to the clinical parameter AST for the assessment of liver injury in vitro. This LDH assay involves removing only a small amount of cell media (2–5µl) from each treated well, allowing you to get more data by sampling the same well over time, and by using the remaining media and cells for other cell-based assays (such as ATP at the end of a 7 or 14-day experiment).

Causality Assays

There are numerous causality assays that can help characterize compound-specific mechanisms of toxicity.

Efflux transporter/cholestasis: Lowering of bile acid secretion into the supernatant (measured by mass spectrometry), can be indicative of blockage of hepatocyte efflux transporters and cholestasis. 

Metabolism-related toxicity: Concurrent exposure of microtissues to Aminobenzotriazole (ABT) will irreversibly inhibit CYP enzymes, and therefore serve as a tool to determine whether a metabolite or the parent compound is the toxic moiety. Likewise, concurrent exposure of microtissues to buthionine sulfoximine (BSO) will sequester protective GSH, to see if depleting this protective pathway increases toxicity. 

Inflammation-related toxicity: Concurrent incubation with LPS (a membrane component of gram-negative bacteria) can imply whether an idiosyncratic immune reaction is a factor of the toxicity.

Vendor: InSphero AG