Published online Feb 27, 2021. doi: 10.4254/wjh.v13.i2.187
Peer-review started: August 26, 2020
First decision: October 21, 2020
Revised: November 4, 2020
Accepted: December 30, 2020
Article in press: December 30, 2020
Published online: February 27, 2021
Liver fibrosis is frequently associated with viral infection [Hepatitis C virus (HCV) and Hepatitis B virus] infection, chronic inflammation, and excessive alcohol consumption. Despite effective antiviral treatment, morbidity and hepatitis-related mortalities are still increasing. Moreover, the number of non-viral liver diseases such as nonalcoholic steatohepatitis and alcoholic liver disease is steadily growing.
In previous studies, we developed a three dimensional (3D) ex vivo model of HCV replication using human liver slice cultures that were followed for 10 days to evaluate a new antiviral drug.
We aimed to establish a 3D ex vivo liver slice model viable in vitro for 21 days allowing us to examine human liver fibrogenesis by fibrosis inducers and anti-fibrotic therapies.
The adult human liver tissue samples from twenty patients were collected after liver resection, and divided into three groups according to their METAVIR score (F): Non-fibrotic F0-F1, obtained during surgery for colorectal cancer liver metastases or fibrotic ranging from F2 to F4. HCV infection, alcohol (ethanol stimulation), and steatosis (palmitate stimulation) were examined in non-fibrotic F0-F1 human liver slices (HLS) compared to fibrotic (F2 to F4) liver slices (FLS) infected (or not) with HCV [Con1/C3 (genotype1b)] (INF). HLS of 350 µm (2.7 × 106 cells per slice) were cultivated for up to 21 days. At day 0, either ursodeoxycholic acid (only choleretic and hepatoprotective properties) and/or α-tocopherol (Toco, anti-oxidant properties which could reduce fibrosis progression) were added to standard of care concentrations on HLS and FLS. The following fibrosis markers expression were assayed in HLS, in FLS and in INF FLS, [tumor growth factor-beta (TGF-β1), Hsp47, Alpha smooth muscle actin, Procol1A1, Matrix metalloproteinases 2, 9 (MMP-2, 9), Vascular endothelial growth factor] and checked by real-time reverse transcription-quantitative polymerase chain reaction and the triglyceride production by enzyme-linked immunosorbent assay assays.
Here, for the first time, human LS cultures (stages F0-F4) were successfully maintained and evaluated for 21 days allowing to explore molecular fibrogenesis in more detail including the role of important factors such as HCV infection, ethanol (EtOH), or steatosis, three of the main causes of liver injury in clinical practice. In addition, it was demonstrated that LS cultures are efficient instruments to study anti-fibrotic drugs and their combination. We obtained real-time reverse transcription-quantitative polymerase chain reaction analyses of the biomarkers (TGF-β1, procol1A1, MMP-2, MMP-9, Alpha smooth muscle actin, HSP47, and Vascular Endothelial Growth Factor) involved in molecular fibrogenesis, and estimation of anti-fibrotic drugs potency, in both non-fibrotic (F0-F1) and fibrotic livers samples (F2-F3, F4). Expression of the fibrosis biomarkers and the progression to steatosis (estimated by triglyceride production) increased with the addition of HCV and /or EtOH or palmitate. We observed a significant decrease in both of the expression of TGF-β1, and procollagen1A1 as well as in the production of triglycerides observed in a combined anti-fibrotic treatment applied to the F2-F4 LS cultures infected with HCV.
The 3D ex vivo LS model provides hepatocyte-specific gene expression for 21 days, and effectively reproduces liver fibrogenesis related to HCV infection, EtOH, or lipids exposure, thus, mimicking human viral, alcoholic, and nonalcoholic steatohepatitis liver diseases. Our study is the proof of concept that this relatively easy model can be used to study human liver fibrogenesis of different origins and evaluate the potency of new anti-fibrotic therapies that are currently under development. In particular, this system might estimate unpredictable side effects when testing certain drug combinations.
Using the ex vivo model of human liver slice culture, the perspectives would be to evaluate the potency of new anti-fibrotic therapies alone or in combination and to study the immune components of liver disease.