Context of Use or Disease: Endothelial-to-mesenchymal transition (EndMT) in liver fibrosis 

DOI: In vitro models 2022, bioRxiv 2026

Platform: Emulate - Liver-on-a-Chip (LOAC) co-culture kit

Description: Using the commercially available liver-chip co-culture chip from Emulate following their standard protocol, we developed live cell fluorescent reporters to assess the behaviour of EndMT during liver fibrosis. Human LSEC were transduced using a lentiviral packaged CNN1-eGFP construct as an inducible EndMT-Rep (CNN1-Rep). We demonstrate high-resolution single-cell EndMT tracking by live cell time-lapse microscopy for up to 24 hours to perform a comparative study of EndMT and healthy liver sinusoidal endothelial cells (LSECs) within a Metabolic Dysfunction-Associated Steatohepatitis (MASH)-like LOAC microenvironment.

Fig. 1. A. Schematic representation of time-lapse live cell imaging workflow in LOAC using the MuviCyte system. B. Example of programmable single-field and stitched whole-LOAC imaging with CNN1-Rep reporter showing EndMT induction following TGFβ2 and TNFα treatment. C. Static quantitation of CNN1-Rep⁺ EndMT induction between treatments with representative 24-h time-lapse imaging stills showing individual cell tracking after 48-h FAA and TGFβ2 + TNFα treatment under MASH-like conditions. D. Quantitative comparison of migratory behaviour between RFP⁺ LSECs and eGFP CNN1-Rep⁺ EndMT LSECs under the same environmental conditions per field. **P < 0.01 or ***P < 0.001 by unpaired t-test.

Characterisation & Validation: We validated the induction of our CNN1-Rep showing that the GFP signal co-localised with known EndMT markers such as SMA following TGFb2+TNFα treatment. These EndMT cells were shown to be further increased in the presence of free fatty acids and TGFb2+TNFα treatment in the Emulate LOAC but also have an enhanced migratory phenotype compared to healthy LSECs. These findings are in line with the idea that these cells destabilise the microvascular network in the liver during tissue fibrosis.  

Ongoing Research: Molecular and functional benchmarking of on-chip EndMT-reporter to in vivo mice equivalents

Research Team: Neil Dufton, Christina Gkantsinikoudi, Joshua Dignam, Meenakshi Rana

Lead Contact: Neil Dufton

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