From semi-synthetic to natural biomaterials: a journey to the development of an optimized in-vitro model for liver tissue engineering

Within the liver, tissue resident cells exist in a state of dynamic reciprocity with the extracellular matrix (ECM) whereby the ECM provides structural and biochemical cues to influence cellular functions and cells response by modifying the ECM. To direct cell behaviour, generation of a scaffold with similar biochemical properties to the ECM is therefore highly desirable. One of the main challenges of producing such a scaffold is to find suitable biomaterials that can replicate liver ECM microarchitecture and biochemical complexity. This study investigated the suitability of both natural and semi-synthetic biomaterials to be used as a scaffold for culturing hepatic cells for the development of a 3D in-vitro liver model to use for drug investigation. Alginate, although biocompatible, once chemically conjugated with small molecules it became unsuitable to use as a potential liver scaffold, due to the compound’s cytotoxicity. Both HepG2 and primary hepatocytes encapsulated in the chemically conjugated alginates died within 24 and 16 hours, respectively. Also, DNA content and both Protein and Albumin secretion were highly effected. Porcine livers were decellularized using an enzymatic and detergent washing process. More than 89% of cellular components were removed and sulphated GAGs were still present post decellularisation. Decellularised livers were digested and solubilised to form ECM-derived hydrogels. Both mechanical and viscoelastic proprieties from a range of liver ECM concentration were tested and compared with the derived Basement Membrane Matrix (BME2) matrix, which is the gold standard material to culture and expand progenitor liver organoids. No significant difference in terms of storage moduli and stiffness was found between 2, 3 mg/ml liver ECM gels and BME2 matrix. Therefore, these concentrations were used to culture and expand progenitor liver cells. Adult progenitor liver cells were taken from human liver biopsies and sorted by the presence of the EpCAM marker. Progenitor cells were encapsulated in a variety of liver ECM concentrations and compared to the animal-derive gold standard BME2. Progenitor live cells proliferated in lower concentration ECM hydrogels (2 and 3mg/ml ECM), but fail to proliferate in higher concentration of liver ECM hydrogels, indicating the significant role of stiffness in cell culture. Ultimately, utilising the lower ECM hydrogels concentrations and home-made differentiation media, progenitor liver cells were differentiated towards both a hepatic and cholangiocyte phenotype. This present work show that the liver ECM matrix has a major role in directing progenitor cells differentiation towards a hepatocyte’s lineage. Many hepatic genes were upregulated at the RNA level when hepatic progenitor cells were encapsulated in liver ECM hydrogels. Cells encapsulated in the BME2 matrix were found to be biased towards a cholangiocyte phenotype with cholangiocyte markers upregulated at the RNA level. This study emphasises the importance of the careful considerations of biomaterials composition that is required for differentiating liver progenitor cells. The combination of matrix composition and differentiation medium thereby influence the successful development of an in-vitro liver model