Study of morphological and compositional influence on bioartificial hepatic microenvironments within electrospun polycaprolactone scaffolds

Abstract

Liver disease is a leading cause of death throughout the world and has seen rising mortality rates since the 1970s in contrast to other leading causes of death such as cardiovascular disease and cancer. A quarter of the global population is predicted to have non-alcoholic fatty liver disease (NAFLD) and are at risk of developing chronic conditions such as fibrosis and hepatocellular carcinoma (HCC). There currently exist no approved pharmaceutical therapeutics for the treatment of liver disease and disease management is particularly complex to negotiate. End-stage liver disease presents a significant threat to life and the only cure is to replace the chronically damaged liver with a transplant. To ease the burden on liver disease patients and health workers, it is necessary to find pharmaceutical and regenerative therapies that can effectively hinder disease progression. Reaching such treatments implies the use of in-vitro methods to produce liver models for testing target and drug molecules, for the expansion of cells with regenerative capacity and for bioartificial liver devices. In-vitro hepatocyte culture methods have progressed in recent decades from standard 2D tissue culture to 3D organoids and scaffolded cultures which provide a biomimetic environment and elicit phenotypic responses from hepatocytes in-vitro. This thesis has sought to optimise electrospun polycaprolactone (PCL) scaffolds for the culture of hepatocytes, with a focus on morphology and composition, through three methods: 1) assessment of the influence of electrospun PCL fibre diameter and morphology on hepatocyte culture 2) the inclusion of rat liver extracellular matrix (rLECM) into hybrid PCL:rLECM scaffolds 3) the inclusion of human liver ECM (hLECM) into hybrid PCL:hLECM scaffolds with a comparison of donor-to-donor variance. All fabricated scaffolds were subject to physical and chemical analyses and cultured with an immortalised hepatic cell line (HepG2) or Primary Mouse Hepatocytes (PMHs). The biological influence of the scaffolds on cell cultures was assessed through proliferation analysis, immunohistochemistry and RT-qPCR gene expression analysis of cultured cells. Our results show that fibre morphology affects the attachment, morphology and proliferation of hepatocytes. The architecture of the cells upon the scaffolds also shows to affect cell function through significant upregulation of key hepatic phenotypic markers on scaffolds with higher porosity. Incorporation of both rLECM and hLECM into electrospun scaffolds with morphological consistency has been demonstrated. rLECM at concentrations of 5 w/w% and 10 w/w% shows to significantly increase the proliferative activity of HepG2 cultures. The incorporation of hLECM at a 1 w/w% concentration demonstrated variable proliferative activity of HepG2 between tissue donors whilst functional gene expression is maintained across donors. Biological differences between the immortalised cell line (HepG2) and Primary Mouse Hepatocytes are reflected in functional response differences observed upon different scaffold microenvironments. These studies show that the morphology and composition of electrospun polycaprolactone scaffolds can have a measurable impact on hepatocyte cultures and our methods hold potential for the development of utile in-vitro hepatic microenvironments.