An investigation of mechanisms responsible for modulated biosynthetic function in 3-dimensional cultures of a human hepatocyte cell line, for potential use in a bioartificial liver support system
The thesis describes the performance of the HepG2 cell line, a proliferating liver cell line, potentially to be used as the cellular component of a bioartificial liver (BAL). HepG2 cells in 3-Dimensional (3-D) spheroidal culture demonstrate dramatically improved function, compared to monolayer culture. The main aims of the thesis were to investigate the phenomenon in this culture system, whereby function in 3-D culture is optimal between Days 8-10, and thereafter there is a decline in function in spite of continued proliferation and viability. For optimal use in a BAL, it is necessary to increase function per cell either at later time points of 3-D HepG2 culture when cell numbers are greater, and/or increase cell number at times of peak function i.e. Days 8-10 of 3-D culture. The initial hypothesis was that the downregulation of activity observed from Day 11 occurred at the transcriptional level. Approaches to determine expression of liver-enriched transcription factors at times of peak and diminished function in 3-D culture and in monolayer culture were not conclusive. To understand the mechanisms underlying the temporal change in function in 3-D culture, the hypothesis that hypoxia and/or other forms of stress were responsible for the drop in function observed was explored. Microarray data and Western blot analysis highlighted the expression of genes and proteins known to be upregulated by hypoxia at Day 15. Ambient oxygen concentration was increased to attempt to increase cell performance but was ineffective. Genes and proteins implicated in oxidative stress were expressed. Results from assays to measure oxidative stress in HepG2 culture demonstrated an increase at Day 15 compared with Day 8 spheroidal cultures. Attempts to alleviate this stress by supplementing the culture medium with additional anti-oxidants at later times of 3-D culture did not enhance cell performance. Some stress-related proteins and genes were more strongly expressed in Day 8 cultures, as an adaptive response to increased metabolic activity during peak function, while others in Day 15 were turned off. These genes were investigated further at a functional level and results reflected the pattern observed. 3-D culture was manipulated with the addition of extracellular matrix (ECM) in order to enhance cell performance. Cell proliferation was measured by total nuclei quantification, incorporation of BrdU as a measure of DNA synthesis and Ki-67 labelling, as a measure of the total growth fraction. Positively labelled cells were seen throughout the spheroid indicating that even at the centre of the spheroids; there was maintenance not only of viability, but also the capacity to proliferate, indicating other microenvironmental changes may be responsible for the diminished function observed at Day 15. The thesis has highlighted the possible causes for the downregulation of function observed and modulation of the 3-D environment to overcome this, and emphasised the complex relationship between cell performance and the stress response, in order to improve a system which could provide the basis for the biological component of a BAL.