Surgery (St Vincent's) - Theses
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ItemDeveloping liver specific vasculature to support the growth of liver progenitor cells for liver tissue engineering( 2015)Tissue engineering is the combination of organ/tissue specific cells in matrices or scaffolds to grow new tissues. Tissue engineering and the related area of cell therapy hold much promise for the repair, replacement and regeneration of organs and tissues damaged by disease and trauma. To date, liver tissue replacement research has focused on cell therapies – particularly hepatocyte transplantation into the diseased liver which specifically aims to reduce the need for liver transplantation, and aims to treat the myriad of end stage liver diseases and metabolic disorders. Despite the technological advances, a major limitation to the clinical success of hepatocyte cell therapy and other liver tissue engineering strategies is the inability to generate a vascular supply capable of supporting the engineering of large, three-dimensional (3D) tissues and organs. The liver sinusoidal endothelial cell (LSEC) makes up the liver specific microvascular network (sinusoids) and plays an integral role in liver development, liver homeostasis and liver regeneration. Vascularisation itself is rarely addressed in liver tissue engineering; let alone the incorporation of LSECs in liver constructs. In this study murine LSECs were used to construct liver specific blood vessels and were identified by their surface markers (LYVE1+/ CD31-), as opposed to capillary endothelial cells (LYVE1-/ CD31+), and lymphatics (LYVE1+/ CD31+). LSECs were tested in vitro and in vivo with and without the addition of murine liver progenitor cells (LPCs). Liver progenitor cells are a native liver progenitor cell, capable of differentiating into both hepatocytes and cholangiocytes. Whilst hepatocytes are the gold standard for liver tissue engineering, LPCs offer an alternative, rarely investigated source of hepatocytes for tissue engineering. LPCs are responsible for liver regeneration during end stage liver disease, when hepatocyte proliferation has been impaired. As LSECs and LPCs play important roles in liver regeneration, both cell types were investigated for their possible applications in liver tissue engineering. LSECs and LPCs were cultured as 3-D multicellular spheroids of one cell type-termed homospheres, or co-cultured together as heterospheres in vitro, for subsequent in vivo implantation in a vascularized tissue engineering chamber. This thesis demonstrates that LSECs and LPCs are capable of forming homogeneous homospheres, and heterogeneous spheroids of co-cultured heterospheres. Furthermore, LSECs form vascular structures in vitro when cultured as homospheres heterospheres. The ability to generate vascular structures through co-culturing with endothelial cells in vitro is termed pre-vascularization, and is currently at the forefront of vascular tissue engineering. The thesis also demonstrates that LSECs were capable of integrating into native vasculature when implanted in vivo to form a liver specific vasculature at an ectopic site in healthy SCID mice. The liver specific vasculature was identifiable as LYVE1+/ CD31- and through the detection of DiI labeled LSECs. Implantation of LSECs also increased the generation of native neo-vasculature (LYVE1-/CD31+)- this was significant when the matrix Matrigel was used in the tissue engineering chamber. However, the generation of liver specific vasculature and increased native vasculature did not support significant survival of LPCs. Whilst the results indicate that heterospheres of LSECs and LPCs improve the survival and spontaneous hepatocyte differentiation of LPCs, the overall survival was inconsistent throughout the in vivo experimental groups in SCID mice. Finally, LPC spheroids were implanted in a mouse model of the metabolic disorder methylmalonic aciduria (MMA). LSECs were not used in the final study; as LPC survival was inconsistent and low regardless of the presence/absence of LSECs. Instead, the total number of LPCs implanted was increased 4 fold. Again, survival of LPCs was low and appeared to elicit an immune reaction in MMA mice. Ultimately, the final study did not demonstrate any functional disease reversal, due to poor LPC survival. This is the first liver tissue engineering study that has used LSECs with LPCs. Isolated LSECs demonstrated significant promise in their ability to generate a liver-specific vasculature in vivo for liver tissue engineering, however the use of LPCs presented a number of issues, which require further investigation – the most significant of which was their inconsistent and generally poor survival in vivo.