Human Liver Tissue Engineering - From Organoids to Tissues

Abstract

A healthy liver has an extraordinary ability to regenerate upon acute damage, however, once this regenerative capacity gets exhausted due to chronic injuries, end-stage liver diseases occur. Currently, the only effective treatment for end-stage liver diseases is liver transplantation. While less than 10% of patients obtain a suitable donor liver for transplantation, many patients are dying on the waiting list. Therefore, there is an urgent need to find alternatives for donor livers for transplantation. This thesis focuses on the development of methods to engineer human liver tissue for transplantation purposes. For the engineering of human liver tissue, it is important to develop suitable biomaterials to mimic the extracellular matrix (ECM) for tissue formation, which is the focus of Part I. The ECM forms the three-dimensional (3D) microenvironment of cells in vivo and provides the support and biological signals for cells to form tissues. Currently, one of the most promising biomaterials to mimic the ECM for human liver tissue engineering are hydrogels. In Chapter 2, the most commonly used hydrogels for liver tissue engineering were reviewed. Following this review, we developed a chemically defined hydrogel, PIC-Laminin, for human liver organoid culture in Chapter 3. PIC-Laminin is an animal-free alternative to the commonly used animal-derived hydrogel Matrigel (or BME) for organoid culture, and promising for clinical applications. Gelatin-based hybrid hydrogels were developed to induce further hepatic maturation in Chapter 4. Organoids cultured in hybrid hydrogels showed an overall trend of improved differentiation, which indicates the potential of this hydrogel for engineering functional liver tissues. Part II focuses on the liver cell production for liver tissue engineering. As the liver is the largest internal organ of the human body, the number of cells (mostly hepatocytes) needed is extremely high, amounting to approximately 200 billion cells. Therefore, in Chapter 5, we established a protocol for large-scale production of liver organoids in suspension using commercial spinner flasks. The cell mass produced in spinner flasks in a short timeframe makes it possible to provide enough cells for whole liver tissue engineering. To exploit the advantages of suspension cultures for research and development, where fewer cells are needed, we developed a miniature spinning bioreactor, RPMotion, in Chapter 6. The RPMotion is an easy-to-use and cost-effective technology to rapidly produce organoids in standard 50mL vessels. In Part III, we focused on the development of a strategy to incorporate major aspects of liver tissue engineering, such as cellular complexity, mimicry of the ECM, fluidic stimuli, and vascularization. In Chapter 7, we engineered human liver tissues with epithelial cells (liver organoids) and different mesenchymal cells (HSCs and MSCs) embedded in optimized PIC hydrogels. The results showed that different types of liver cells can interact with each other and spontaneously form tissues in vitro and the tissues can be vascularized on chicken chorioallantoic membranes. To summarize, all results together establish a roadmap to engineer liver tissues by combining several aspects of tissue engineering, including but not limited to multicellular organization, hydrogels as ECM mimicry, dynamic fluidic stimuli, and potential vascularization.