Apoptosis and Its Suppression in Hepatocytes Culture

Abstract

In order to achieve the goal of developing extracorporeal liver support devices, it is necessary to optimise bioprocess environment such that viability and function are maximised. Optimising culture medium composition and controlling the constitution of the cellular microenvironment within the bioreactor have for many years been considered vital to achieving these aims. Coupled to this is the need to understand apoptosis, the prime suspect in the demise of animal cultures, including those of hepatocytes. Results presented here show that absent nutrients including glucose and amino acids play a substantial part in the induction of apoptosis. The use of chemical apoptosis inhibitors was utilised to investigate key components of hepatic apoptosis where caspases, predominantly caspase 8, were implicated in staurosporine (STS)-induced HepZ apoptosis. Caspase 9 and 3 activation although recorded was of less significance. Interestingly, these results were not consistent with those of mitochondrial membrane depolarisation where inhibition of caspase activation appeared to drive depolarisation. Inhibition of mitochondrial permeability transition and use of anti-oxidants was unsuccessful in reducing apoptosis, caspase activation and mitochondrial membrane depolarisation. In further studies, the anti-apoptotic gene bcl-2 was over-expressed in HepZ, resulting in a cell line that was more robust and resistant to death induced by glucose and cystine deprivation and treatment with STS. Bcl-2 did not however show significant cytoprotectivity where apoptosis was stimulated by deprivation of glutamine and serum. Overall, results indicated that although apoptosis can be curbed by use of chemical inhibitors and genetic manipulation, their success is dependent on apoptotic stimuli.

Impact of alginate type and bead diameter on mass transfers and the metabolic activities of encapsulated C3A cells in bioartificial liver applications

“…A comparative study of the effects of medium components on hepatocyte functionality in suspension culture highlighted a decrease in ATP/ADP ratio in glucose-free medium (Elaut et al, 2005). Mukwena showed that the absence of glucose inhibited hepatocyte proliferation without any observable effect on cell viability for 96 h culture (Mukwena and Al-Rubeai, 2004). Our results confirm these findings, with culture conditions in beads preserving cell viability higher than 83% after 24 or 48 h of fluidisation.…”

Novel approach for the prediction of cell densities and viability in standardized translucent cell culture biochips with near infrared spectroscopy

“…In a second approach, we developed a calibration model for the pooled samples analyzed by flow cytometry, which were treated with staurosporine, a protein‐kinase inhibitor and known inducer of apoptosis .…”