Fibrosis Assay

Fibrosis Assay2019-02-12T16:09:24-05:00

Background

  • Non-alcoholic fatty liver disease (NAFLD), which originates from disordered lipid metabolism attributed to the activation of de novo lipogenesis from high sugar diets, affects 20-50% of the population (1). The consequential hepatocellular accumulation of lipid droplets (steatosis) can lead to enlargement of the liver which, in non-alcoholic steatohepatitis (NASH), is accompanied by inflammatory events that form the basis for fibrosis and liver damage (1,2).
  • In later phases of NASH, steatosis induces inflammatory events, which in turn lead to the activation of Kupffer cells and hepatic stellate cells (HSCs) (3). More specifically, the transition of HSCs from a quiescent to proliferating, fibrogenic, myofibroblast-like phenotype is facilitated by cytokines such as PDGF, TGFβ, and IL-1 that are released by already damaged HSCs or by infiltrating immune cells. Left unchecked, the extracellular matrix remodeling events facilitated by HSCs in fibrotic stages of the disease can impair normal liver functionality and lead to cirrhosis (4,5).
  • Given the differences in how humans versus animal models manifest liver disease, in vitro models are increasingly being used in the development of therapeutics to prevent or treat fibrosis.
  • Given the necessary interplay of multiple cell subtypes in fibrogenesis, 3D cell cultures of mixed hepatocytes or hepatocyte-like cells with nonparenchymal cells (NPCs) provide the greatest utility in this assay.
  • High content, high throughput imaging of collagen deposition as well as stellate cell activation enables for a more complete understanding of the pathogenesis of fibrosis while still facilitating the screen of several compounds in a single assay plate.

Protocol

InstrumentThermoFisher CX7 LZR
Analysis MethodHigh content screening
MarkersPan collagen (collagen I, III, IV, or others available as an alternative or addition)
Alpha-SMA (stellate cell activation marker)
Live/Dead stain (viability indicator)
DAPI (total cell count)
Cell Types AvailableHepaRG or primary human hepatocytes with NPCs.
Other/custom liver models available upon request.
Test Article Concentration8 point assay (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)
ALK-5 inhibitor (positive control)
Test Article Requirements50 uL of 20 mM solution or equivalent amount of solid
Data DeliveryDose response curves and EC50 values for cell viability, collagen deposition, and stellate cell activation in response to anti-fibrotic agents.
Alternative EndpointsELISA (collagens, inflammatory cytokines)

General Procedure

  1. For generation of liver spheroids, 2000 cells/well (60% hepatocytes, 40% NPCs) are seeded into ULA U-bottom plates and maintained under standard culture conditions for 5-6 days to enable spheroid aggregation to a size of approximately 200 μm in diameter.
  2. Spheroids are treated with test compounds. If test compounds are intended to ameliorate rather than prevent fibrosis, test compounds will be added after induction of fibrosis.
  3. Following 1 h of pre-treatment (other timepoints available upon request), fibrosis is induced via addition of 100 nM TGF-β for an additional 48 h. Test article concentration is maintained through induction of fibrosis.
  4. Following treatment, liver spheroids are labeled with a viability indicator, fixed, and labeled with antibodies against α-SMA and pan collagen.
  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 α-SMA, fluorescence intensity and thresholded area of collagen staining, and viability.

Data

Figure 1. Visikol OpenLiver HepaRG/NP 3D spheroids were either pretreated with vehicle or an ALK-5 inhibitor for 1 h prior to inducing fibrosis with 100 nM TGF-β for 48 h. DAPI in blue, viability indicator in green, pan collagen in red.

Figure 2. Visikol OpenLiver HepaRG/NP 3D spheroids were either pretreated with vehicle or an ALK-5 inhibitor for 1 h prior to inducing fibrosis with 100 nM TGF-β for 48 h. Collagen fluorescence intensity (A) and collagen volume fraction (B) was quantified for each condition.


References:

  1. Kozyra, M., et al. “Human hepatic 3D spheroids as a model for steatosis and insulin resistance.” Scientific Reports 8 (2018): 14297.
  2. Lauschke, V., et al. “Novel 3D culture systems for studies of human liver function and assessments of the hepatotoxicity of drugs and drug candidates.” Chemical Research in Toxicology. 12 (2016): 1936-1955.
  3. vanGrunsven, L. “3D in vitro models of liver fibrosis.” Advanced Drug Delivery Reviews. 121.1 (2017): 133-146.
  4. Basaranoglu, M., et al. “From fatty liver to fibrosis: A tale of ‘second hit.’” World J Gastroenterol. 8 (2013): 1158-1165.
  5. Hinz, B. “The extracellular matrix and transforming growth factor-β1: Tale of a strained relationship.” Matrix Biology. 47 (2015): 54-65.
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