Generation of Hepatic Organoids from Primary Cells of the Human Liver

Abstract

The ability to isolate cells from the human liver and culture them in the laboratory has enabled numerous practical applications in the biotechnology and medical sectors. These range from the generation of in vitro models of various liver pathologies, to the preclinical screening of novel therapeutics for efficacy and safety before clinical trials. In addition to their use ex vivo, isolated cells have also been used to produce advanced therapy medicinal products for regenerative medicine applications. However, despite the countless use cases of cell culture models and cell-based therapeutics, these systems and modalities are still in their adolescence, often lacking the complexity required to accurately model a specific tissue, or the efficacy and safety required for routine clinical use. The present thesis builds on the large body of work relating to hepatic cell culture and the generation of organoids from primary cells of the human liver. This dissertation focuses on (1) the expansion of primary hepatic epithelial cells as organoids, and (2) the establishment of advanced models of the human liver for in vitro modeling and regenerative medicine applications. Chapter 1 begins with an introduction to hepatic cell culture, highlighting its potential to revolutionize the biotechnology and medical sectors, as well as the limitations of current organoid-based cell culture models of the liver. Chapter 2 provides an overview of early liver development and regeneration of the adult liver, processes which share many of the same signalling pathways and molecular players that researchers take advantage of to generate and expand organoids. Chapter 3 introduces the various types of liver organoids that can be generated using current techniques and provides a detailed overview of the state of the liver organoid field (as of 2021). Chapter 4 describes the development of a novel miniaturized bioreactor system, the RPMotion, for scaling up organoid cultures in a time, labour and cost-effective manner. Chapter 5 describes development of a novel biofunctionalized polyethylene glycol-based bioink optimized for 3D bioprinting and tailored to promote the growth and migration of cells, facilitating tissue formation. In chapter 6 human ICOs are co-cultured with stromal stem cells following their mass expansion to generate hepatic multicellular spheroids, which are then used as the hepatic building blocks in 3D bioprinted constructs generated employing the bioink developed in chapter 5. In chapter 7 there is a departure from ICO-based cell culture systems. Here, the mass generation, expansion and maturation of a novel human hepatocyte organoid system is described. The aim of this chapter was to develop a scalable platform for the efficient mass generation of human hepatocyte organoids from adult primary human hepatocytes for in vitro modelling and regenerative medicine applications. The dissertation ends with a cross-study transcriptomic comparison of the various liver organoid models established from primary cells of the human liver and a perspective future outlook for the field of hepatic organoid biology.