Adala, Inchirah
(2022)
Development of a coaxial electrospun scaffold for hepatic progenitor cells culture, differentiation and maturation.
PhD thesis, University of Nottingham.
Abstract
Liver disease represents one of the fastest growing disease classes in the world, with an increasing mortality of 400% since 1970 in the UK. According to the National Health Service, it is one of the top five causes of premature death in the UK, with incidence rising sharply by 20% over the last decade. The only curative therapy for end-stage liver disease is liver transplantation. One of the most promising strategies to overcome the shortage of liver transplants is the development of a bioengineered livers for transplantation. However, limited access to primary liver tissue, reduced survival time and the inability of primary hepatocytes to proliferate in culture has drawn researchers to look for an alternative cell source of mature hepatocytes. In severe liver injury, bipotential hepatocyte progenitor cells (HPCs) located next to the bile duct are able to proliferate and subsequently differentiate into either hepatocytes or cholangiocytes. The aim of this thesis was to develop a 3D coaxial electrospun scaffold in order to mimic some of the physical features of the liver ECM environment and deliver a combination of signalling molecules to aid in HPCs culture, differentiation and maturation in vitro.
The first part of this study describes how proteins were physically and chemically loaded into a single coaxial electrospun fibre scaffold to obtain bi-phasic release profiles. Cyto-compatible polymers were used to construct the scaffold, using polyethylene oxide (PEO) for the core and polycaprolactone (PCL) reacted or mixed with (bis-aminopropyl)polyether (Jeffamine ED2003; JFA) for the shell. Horseradish peroxidase (HRP), a model protein, was loaded in the core and functionalised onto the scaffold surface by coupling of protein carboxyl groups to the available polymer amine groups. Fibre morphologies were evaluated by scanning electron microscopy (SEM) and transmission electron microscopy (TEM) and functional group content was determined using X-ray photoelectron spectroscopy (XPS) and time-of-flight secondary ion mass spectrometry (TOF SIMS). Hydrophobicity profiles of the fibres before and after protein loading were evaluated by water contact angle (WCA) and the mechanical properties of the electrospun scaffolds were determined by performing tensile tests. The electrospun fibre scaffolds generated by reacting PEO/PCL with 1,6-diaminohexane and those from mixing PEO/PCL with JFA were further characterised for protein conjugation and release. Fibres prepared by the mixed PEO/PCL/JFA system were found to be the most appropriate for the simultaneous release of protein from the core and the immobilisation of another protein on the shell of the same scaffold. Moreover, JFA enhanced scaffold properties in terms of porosity and elasticity. Finally, the cytocompatibility and cell response to protein-loaded and -conjugated scaffolds were successfully demonstrated using HepG2 cells.
In the second part of this study human EPCAM positive HPCs were isolated, expanded as organoids, and their culture, differentiation and maturation was explored on a PEO/PCL/JFA scaffold. Cell viability was evaluated with Live/Dead staining, the Presto blue assay, DNA quantification, SEM imaging and Ki67 immunostaining. Cells differentiation was evaluated by measuring gene expression levels. Finally, CYP Glo assay and ELISA assay were performed to quantify respectively CYP3A4 activity and Albumin as indicators of hepatic function and cell differentiation/maturation. Scaffold cytocompatibility was successfully demonstrated with HPCs and SEM imaging showed that the cells formed multicellular adherent layers on the scaffold. Moreover, it was found that the scaffold supported HPCs proliferation reaching confluency at day 4 post-seeding and supported cells differentiation and maturation towards hepatic lineage. Finally, the combined effect of scaffold substrate and an increase in DAPT was found to increase further the level of HPCs differentiation.
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