Introduction: Liver fibrosis is caused by progressive accumulation of extracellular matrix (ECM) coupled with chronic inflammation. Advanced liver fibrosis results in increased risk of liver cancer, cirrhosis, portal hypertension and liver failure, resulting in the need for liver transplantation. Studies of the mechanisms that promote fibrosis are necessary to understand this multi-faceted disease and for the development of novel therapeutic targets. Traditional cell culture models often lack the immune cell compartment and the ECM, failing to recapitulate the complex fibrotic microenvironment, which is highly immune-mediated. Bioengineering allows for the development of disease models to study complex diseases, such as liver fibrosis, where both cellular and extracellular microenvironment components contribute to the pathology. Here, we describe two novel bioengineered models which incorporate the dynamic co-culture of circulating immune cells in decellularised liver ECM-scaffolds, supported by two custom-made perfusion bioreactors, and we demonstrate how these models allow to explore immune responses to fibrotic liver ECMs.
Methodology: We developed two custom-made bioreactors for whole rat livers or human tissue segment culture (WL and HuTS bioreactor respectively). Decellularised normal and fibrotic rat and human liver tissues were obtained following established detergent and enzymatic treatment protocols. Human peripheral blood mononuclear cells (PBMCs) from healthy donors, were perfused in WL or HuTS bioreactors in absence (baseline characterization) or presence of decellularised normal or fibrotic liver ECM-scaffolds. Circulating cell viability, phenotype, and cytokine production were assessed (via FACS and Luminex) in comparison to static culture conditions. The gene expression and phenotype of PBMCs present inside the scaffolds were examined via qPCR and immunofluorescence.
Results: The custom-made bioreactors supported perfusion of PBMCs for up to 7-days without altering cell viability and phenotype in comparison to conventional static culture conditions. Bioreactor culture also improved cell distribution inside the scaffolds compared to static cultures, suggesting that perfusion culture better promotes cell-ECM interactions.
FACS analysis of circulating PBMCs showed that co-culture with both healthy or fibrotic liver matrix-induced an increase in the relative proportion of NKT cells and B cells and that this increase was greater when cultured with fibrotic livers. When cultured with fibrotic scaffolds, the number of circulating T cells decreased and interestingly, monocytes sub-populations changed in response to healthy or fibrotic liver matrices.
Immunofluorescent staining on sections of perfused ECM-matrices revelated that immune cells infiltrated the ECM-scaffolds. These were mostly composed of monocytes and macrophages, with higher relative proportion in fibrotic livers, indicating fibrotic ECM-induced homing of monocyte-derived macrophages, an event that recapitulates the in vivo fibrotic microenvironment.
Cytokine analysis revealed that the ECM triggers the release of innate and adaptive immune system cytokines and those related to pro-regenerative immune responses.
Conclusion: Here we show the validation of two innovative bioreactor-based systems which allow for the perfusion of immune cells through decellularised liver matrices. This perfusion system proved to be suitable for the study of immune cell interaction with normal or fibrotic ECM, and more in general, with bioengineered multi-cellular liver constructs, and showed that mechanisms of chronic inflammation observed in fibrosis can be replicated in vitro.