A study into the relationship between physical environment and the functionality, structure and viability of hepatocytes in-vitro

Liver failure is a life threatening condition. A large problem in the manufacturing of synthetic liver tissue is maintaining the viability and differentiation state of the hepatocytes in-vitro. Cells are profoundly influenced by the stiffness of their ECM and deviations in in-vitro models from the in-situ environment can impact on their functionality. The aim of this project was to investigate the cellular structure and shape, the actin cytoskeleton, the metabolic activity and viability of cells cultured in a collagen sandwich bioreactor and observe the differences compared with monolayer cultures. HepG2 cells were chosen to model hepatocytes in comparisons between collagen sandwich bioreactors and monolayer conformations. This was done by sandwiching HepG2 cells between layers of collagen gels and visualising microscopically 24 hours after seeding them in monolayer. To visualise distribution and aggregation of cells they were treated with MTT, colouring viable cells blue. To determine overall cell structure the cell cultures were stained with Phalloidin-FITC (binds to actin), Calcein-AM and CFDA (a live dead stain) and fluorescent microscopy was performed. The results presented show that human HepG2 cell morphology and intercellular communication is strongly influenced by both the environment and cellular densities of the culture. The findings in this research suggest that lower concentrations (0.5, 1 and 5 x10⁴ cells/cm²) of HepG2 cells seeded in collagen sandwiches are able to maintain a rounded in-situ morphology while culturing cells at a high density 10 x10⁴ cells/cm² in a 3D scaffold allows for increased intercellular interactions and aggregation. This in turn may improve retention of in-vivo-like functionality and viability when culturing in-vitro. The increased cell to cell interaction and maintenance of in-vivo morphology could possibly help retain liver function in-vitro. This could be beneficial for future research aimed at improving the impact and efficiency of studies modelling detoxification of candidate pharmaceuticals and development of synthetic livers tissues.Liver failure is a life threatening condition. A large problem in the manufacturing of synthetic liver tissue is maintaining the viability and differentiation state of the hepatocytes in-vitro. Cells are profoundly influenced by the stiffness of their ECM and deviations in in-vitro models from the in-situ environment can impact on their functionality. The aim of this project was to investigate the cellular structure and shape, the actin cytoskeleton, the metabolic activity and viability of cells cultured in a collagen sandwich bioreactor and observe the differences compared with monolayer cultures. HepG2 cells were chosen to model hepatocytes in comparisons between collagen sandwich bioreactors and monolayer conformations. This was done by sandwiching HepG2 cells between layers of collagen gels and visualising microscopically 24 hours after seeding them in monolayer. To visualise distribution and aggregation of cells they were treated with MTT, colouring viable cells blue. To determine overall cell structure the cell cultures were stained with Phalloidin-FITC (binds to actin), Calcein-AM and CFDA (a live dead stain) and fluorescent microscopy was performed. The results presented show that human HepG2 cell morphology and intercellular communication is strongly influenced by both the environment and cellular densities of the culture. The findings in this research suggest that lower concentrations (0.5, 1 and 5 x10⁴ cells/cm²) of HepG2 cells seeded in collagen sandwiches are able to maintain a rounded in-situ morphology while culturing cells at a high density 10 x10⁴ cells/cm² in a 3D scaffold allows for increased intercellular interactions and aggregation. This in turn may improve retention of in-vivo-like functionality and viability when culturing in-vitro. The increased cell to cell interaction and maintenance of in-vivo morphology could possibly help retain liver function in-vitro. This could be beneficial for future research aimed at improving the impact and efficiency of studies modelling detoxification of candidate pharmaceuticals and development of synthetic livers tissues