Decellularized liver extracellular matrix as a 3D scaffold for bioengineering applications

Abstract: The increasing global burden of end-stage liver disease has increased the need for liver transplantation, the definitive cure. However, there is a huge discrepancy between the number of available organ donors and the number of patients waiting for transplantation, resulting in the deaths of a significant number of patients on the waiting list as only 10% of the global need for transplantation is met. Liver tissue engineering is a promising alternative solution to this problem, which utilizes bioengineering techniques to create an ex vivo microenvironment niche for liver cells embedded in a liver-specific extracellular matrix (ECM) for cell growth and function. Despite many advances in this field, the scarcity of appropriate ECM-mimicking biomaterials with good mechanical properties for biofabrication technique remains limited. To address this, different biofabrication techniques, such as bioprinting and biomaterial scaffolds, are studied to simulate liver microarchitecture for different applications. This thesis presents the development and application of a decellularized liver extracellular matrix hydrogel combined with the liver cell line HepG2 (papers 1-3). It also focuses on the decellularized whole liver scaffold to differentiate amniotic epithelial cells (paper 4). The decellularized liver extracellular matrix (dLM) is a cell-free scaffold that retains liver-specific components to direct cell growth and functions. The dLM can be digested to form hydrogel for 3D bioprinting applications, or it can be used as a biomaterial scaffold to seed the cells directly. In paper I, porcine dLM hydrogel was modified with gelatin and a PEG-based crosslinker to induce a cytocompatible gelation mechanism to generate a robust bioink with a 16-fold increment in viscosity and a 32-fold increment in storage modulus as compared to unmodified dLM hydrogel. This work established the application of dLM with other biofabrication methods, such as Indirect bioprinting, where a sacrificial biopolymer is 3D printed, and the scaffold material is subsequently added. In paper II, a 3D-printed polyvinyl alcohol framework resembling the liver lobules was used as a sacrificial scaffold to impart its structure to the dLM hydrogel modified with PEG-based crosslinker and mushroom tyrosinase. The crosslinked dLM hydrogel with co-culture of HepG2 and NIH 3T3 fibroblasts cell line retained the structure of PVA to create a scaled-up liver-like microarchitecture with lobules. The PVA dissolved with cell culture media leaving behind a robust 3D construct of dLM hydrogel. In paper III, cellulose nanofibril-coated HepG2 spheroids incorporating dLM hydrogel were studied for tumor modeling. The dLM incorporation affected the spheroid formation and growth depending on the time of addition. In paper IV, the functional differentiation of amniotic epithelial cells into hepatocyte-like cells was performed in a decellularized rat liver scaffold in a perfusion bioreactor with dynamic oxygenation and media exchange. This dLM perfusion technology supported the maturation and proliferation of amniotic epithelial cells into hepatocyte-like cells. This is a preliminary step into developing a liver-like organ model in a laboratory setting. To conclude, this thesis presents different bioengineering approaches, such as 3D bioprinting and perfusion decellularization, to study the 3D dLM scaffolds for HepG2 and amniotic epithelial cell culture. 3D bioprinting technique utilized a robust dLM hydrogel to create a scaled-up microarchitecture, whereas perfusion decellularization retained the natural 3D architecture of the whole liver ECM and the native vascular system for recellularizing the scaffold with stem cells. We successfully modified and characterized the dLM hydrogel to enhance its printability to develop complex structures such as liver lobules and microchannels. We utilized different cell systems, including monoculture, co-culture, and spheroids, to analyze the biocompatibility, cell proliferation, and liver-specific functions of the dLM scaffold. Ultimately, the advancement of dLM as a biomaterial presented in this thesis could improve the application and modification of various decellularized tissues to generate larger-scale models for in vitro testing and organ transplantation.