Primary hepatocytes and their cultures in liver apoptosis research

Abstract

Apoptosis not only plays a key role in physiological demise of defunct hepatocytes, but is also associated with a plethora of acute and chronic liver diseases as well as with hepatotoxicity. The present paper focuses on the modelling of this mode of programmed cell death in primary hepatocyte cultures. Particular attention is paid to the activation of spontaneous apoptosis during the isolation of hepatocytes from the liver, its progressive manifestation upon the subsequent establishment of cell cultures and simultaneously to strategies to counteract this deleterious process. In addition, currently applied approaches to experimentally induce controlled apoptosis in this in vitro setting for mechanistic research purposes and thereby its detection using relevant biomarkers are reviewed.

Fig. 1

Mechanisms of hepatocyte apoptosis. In the intrinsic pathway, which can be activated by p53 upon DNA damage, cytochrome C is released from mitochondria. This process is mediated by B cell lymphoma 2 (Bcl-2) proteins which can act either proapoptotic (e.g. Bid and Bax) or anti-apoptotic (e.g. Bcl-xL and Bcl-2). Liberated cytochrome C forms an apoptosome with apoptotic protease activating factor 1 (APAF-1), deoxyadenosine triphosphate (dATP) and procaspase 9. The subsequent activation of caspase 9 can be counteracted by the inhibitor of apoptosis (IAP) family, which itself can be blocked by the smac protein. Caspase 9 then triggers caspase 3, which is a major executor of apoptosis by cleavage of a broad spectrum of cellular proteins. In the extrinsic pathway, specific ligands, such a Fas ligand, bind to their corresponding receptors at the cell plasma membrane surface, which induces the recruitment and cleavage of procaspase 8. In hepatocytes, this pathway can as such not lead to activation of caspase 3 and needs mitochondrial amplification, a process that relies on the actions of Bid

Fig. 2

Activation of spontaneous apoptosis in hepatocyte cultures. During 2-step collagenase perfusion isolation of hepatocytes from liver tissue, cellular contacts are abolished and ischaemia/reperfusion occurs. These deleterious events trigger an inflammatory reaction, mediated by nuclear factor-kappa beta (NF-κβ), and a proliferative response, mediated by mitogen-activated protein kinase (MAPK). In turn, this results in the progressive deterioration of the differentiated phenotype, which ultimately burgeons into the onset of spontaneous apoptosis (adapted from Paine and Andreakos 2004; Vinken et al. 2012b)