Acute liver failure (ALF) and acute chronic liver failure (ACLF) are life-threatening conditions with mortality rates of up to 85%. The only reliable treatment is orthotopic liver transplantation but there is a shortage of organ donors. Bioartificial livers (BALS) have been developed to overcome the limitations associated with non-biological devices and to replace the metabolic activities of the liver as a bridge to transplant or organ recovery after liver decompensation. These comprise hepatocytes within a biocompatible matrix. However, there is no evidence for enhanced patient survival, for unknown reasons, contact with the plasma of individuals with liver failure severely reduced hepatocytes key metabolic functions. Although overall cell viability was unaffected this loss of function completely compromised device function and represents an important barrier to the development of working BAL systems. Since senescence contributes to altered hepatocyte function in cirrhosis and the combination of an altered phenotype and unaffected viability in response to physiological stressors seen in BAL hepatocyte populations is highly reminiscent of cellular senescence. The aim of this study was therefore to investigate the role and significance of senescence on bio-artificial liver performance using an alginate modified p (HEMA)-MBA cryogel prototype seeded with hepatocytes and consider mechanisms by which to mitigate any effects on bio-artificial liver function using protective resveralogues. In this study, HEMA-MBA cryogels modified with alginate were used as a hepatocyte extracellular matrix to provide a 3-D environment for human hepatocyte studies. A model of hepatocyte senescence induction was developed using etoposide-treated HepG2 cells. To understand the role of senescence on bioartificial liver function, HepG2 cells were exposed to a non-cytotoxic concentration of senescence-inducing drug such as etoposide and analysed for potential markers of cellular senescence. The impact of hepatocyte senescence on key metabolic functions was analysed by measuring albumin and urea production. The data showed that etoposide treatment induced a senescent phenotype in HepG2 cells that combines growth arrest and loss of key functional phenotypes. The percentage of cell proliferation markers including 5-ethynyl-2'-deoxyuridine (EdU) (p< 0.001) and Ki67 (p< 0.0001) labelling decreased significantly after 48h treatment with 10µM etoposide. In addition, HepG2 senescence caused a significant reduction in albumin (p˂0.05) and urea production (p˂0.001) at the 3-D surface. HepG2 cells were treated with a liver toxins cocktail at physiological doses and over clinically relevant periods (6h) and the effect of liver toxins on HepG2 cells proliferstion was measured. Also, the effect of senescence inhibitor compounds (resveratrol and resveralogues) on rescuing HepG2 cells from liver toxins effects and reversion of senescence effects were studied. The results of this study showed that HepG2 cells enter a senescent state in response to the liver toxin and inflammatory cytokine exposure which was confirmed by a significant reduction in the percentage of EdU and Ki67 positive cells (p< 0.001), and a considerable increase in cytokines production including IL-6 (p< 0.01) and IL-8(p < 0.001). However, resveratrol at 5 µM concentration either before or during exposure to liver toxins cocktail preserves their capacity to produce both urea and albumin and suppresses cytokine production. In conclusion, this study showed that senescence plays a significant role in lost metabolising hepatocyte cell fraction over time and in the reduction of BAL synthetic and detoxification functions. However, this can be prevented by the use of polyphenolic compounds to protect the cells in the device from senescence-associated phenotypic changes, via both SIRT1-dependent and independent pathways.
The impact of senescence on bioartificial organ design and performance
Heidari, N. (Author). Aug 2022
Student thesis: Doctoral Thesis