Mitochondrial Function Assay

Mitochondrial Function Assay2022-11-29T10:53:36-05:00

Background

  • Drug induced liver injury (DILI) represents the most probable cause for toxicity-related withdraw of a drug from the market, with cholestatic mechanisms of DILI representing a particularly severe and prevalent subset of cases, accounting for approximately half of all hepatic drug toxicity.
  • Cholestatic drugs can inhibit bile secretion and bile acid transport by inhibiting or affecting the function or expression of transporters like BSEP, MDR1, and MRP2, thus affecting canalicular efflux of bile acid. The subsequent accumulation of bile acids is thought to induce oxidative stress by damaging hepatocellular mitochondria in a manner which leads to apoptosis.
  • 3D cell culture models are increasingly being used to study this process, since the transporter proteins and enzymes that facilitate drug metabolism and bile acid secretion in in vivo animal models can exhibit crucial functional differences from their human analogues and since 2D in vitro models from human cells similarly can lack expression of these transporters and enzymes.
  • Since cell death in cholestatic mechanisms of DILI is likely predicated on mitochondrial dysfunction, an indicator of mitochondrial membrane potential can be used in the assessment of DILI mechanism.

Protocol

InstrumentMolecular Devices ImageExpress Micro Confocal
Analysis MethodHigh content screening
MarkersThermo Fisher MitoHealth stain (mitochondrial membrane potential)
Thermo Fisher Image-iT DEAD stain (cell viability)
DAPI (total cell count)
Cell Types AvailableHepaRG, HepG2, primary human hepatocytes, with or without NPCs
Other cells upon request
Test Article Concentration8 point assay (i.e. 0.05, 0.1, 0.5, 1, 5, 10, 50, 100 µM)
(custom concentrations available)
Number of Replicates3 replicates per concentration
Quality Controls0.5% DMSO (vehicle control)
Acetaminophen (positive control)
Test Article Requirements50 uL of 20 mM solution or equivalent amount of solid
Data DeliveryDose response curves, EC50 values, total cell counts, viable and non-viable cell counts

General Procedure

  1. Liver spheroids grown by seeding 1000 cells/well into ULA U-bottom plates
  2. Liver spheroids grown to approximately 200 μm in diameter
  3. Treatment with test compounds
  4. After 48 hours (7 and 14 day timepoints also available upon request), liver spheroids are labeled with MitoHealth and Image-iT DEAD stains, fixed and labeled with DAPI.
  5. Tissue clearing is applied to render liver spheroids transparent
  6. High content imaging is conducted on well plates
  7. Images are analyzed to quantify fluorescence intensity of the MitoHealth stain as a measure of mitochondrial potential and to determine the frequencies of viable (Image-iT DEAD negative) versus non-viable (Image-iT DEAD positive) cells.

Data

Figure 1. Visikol Open Liver HepaRG/NP 3D treated with acetaminophen (triangle indicates dose) and labeled with MitoHealth stain (red; accumulates in active mitochondria) and Image-iT Dead (green; accumulates in non-viable cells).

Figure 2. Dose-response curves indicating mitochondrial health in Visikol OpenLiver HepaRG/NP 3D models treated with acetaminophen (Acet.) with or without bile acid media (+/-BA) for 48 h.

References:

  1. Rabinowich, R., & Shibolet, O. (2015). Drug Induced Steatohepatitis: An Uncommon Culprit of a Common Disease. BioMed Research International.
  2. Perez, M., et al. “Bile-acid-induced cell injury and protection.” World J Gastroenterol. 14 (2009): 1677-1689.
  3. Li, Z., et al. “Application of periodic acid-Schiff fluorescence emission for immunohistochemistry of living mouse renal glomeruli by an in vivo cryotechnique.” Arch Histol Cytol. 69.3 (2006): 147-161.
  4. Gaskell, H., et al. “Characterization of a functional C3A liver spheroid model.” Toxicology research4 (2016): 1053-1065.
  5. Anderson, N., et al. “Molecular mechanisms and therapeutic targets for steatosis and steatohepatitis.” Pharmacological Reviews 60 (2008): 311-357.