Animal nutrition and breeding conditions modify the physiology of isolated primary cells.

Animal primary cell cultures are widely used in biomedical research to investigate cell metabolism, diseases and to devise novel treatments. Modern animal breeding techniques are developed to unify, control and reduce the amount of microorganisms that the animals are being exposed to. Furthermore, health monitoring and strict caging and handling protocols allow animals to be exposed only to a selected spectrum of microbes. We are starting to appreciate that nutrition can influence composition of gut microbiota that can impact hosting organism's physiology and can even result in development of pathological changes. Evidence is also emerging that acute as well as chronic stresses can profoundly influence the physiology of certain organs, especially heart and liver. Our preliminary data imply that changes in animal nutrition and stress levels initiated up to minutes before the cell isolation could alter the cell stress response of cultured primary hepatocytes after isolation, leading to differences in sensitivity of apoptosis triggering. Therefore, we propose the hypothesis that conditions of animal breeding, especially diet and stress levels, are reflected in the physiology of the isolated primary cells. Variations in animal breeding conditions may influence experimental results on isolated cells and their applicability for studying human disorders.

DMSO modulates the pathway of apoptosis triggering.

We demonstrate here that distribution of caspase-9 influences the pathway of apoptosis triggering, since caspase-9 is activated efficiently only when it is distributed solely in the cytosol. Caspase-9 moves to the nuclei in a response to cell stress during isolation of primary hepatocytes; this is called preapoptotic cell stress response. The dimethyl sulfoxide (DMSO) treatment cannot prevent the migration of caspase-9 into the nuclei when it is added to primary hepatocytes immediately after isolation; however, it can trigger redistribution of caspase-9 from the nuclei into the cytosol when added 1 day post-isolation. This redistribution is temporary, since caspase-9 returns to the nuclei within 48 hours of DMSO treatment. Thereafter, some caspase-9 is retained in the nuclei of DMSO-treated hepatocytes for longer than in the nuclei of untreated hepatocytes. By measuring caspase activities, we demonstrate that the addition of DMSO to cell culture medium can temporarily normalize the susceptibility of hepatocytes for apoptosis triggering through the intrinsic pathway. DMSO contributes also to the prolonged pathway inactivation, i.e., by extending preapoptotic cell stress response. We propose that DMSO extends the survival of primary hepatocytes by modulating preapoptotic cell stress response, which could be exploited for extending the lifespan of other primary cell cultures.

Preapoptotic cell stress response of primary hepatocytes.

UNLABELLED: Primary hepatocytes are an important in vitro model for studying metabolism in man. Caspase-9 and Bcl-2-associated X protein (Bax) are regulators of the apoptotic pathway. Here we report on the translocation of procaspase-9 and Bax from cytoplasm to nuclei as well as on dispersion of mitochondria; these processes occur after isolation of primary hepatocytes. The observed changes appear similar to those at the beginning of apoptosis; however, the isolated hepatocytes are not apoptotic for the following reasons: (1) cells have a normal morphology and function; (2) the mitochondria are energized; (3) there is no apoptosis unless it is induced by, e.g., staurosporine or nodularin. Staurosporine does not trigger apoptosis through activation of caspase-9, as its activity is detected later than that of caspase-3. We propose that the translocation of procaspase-9 and Bax into the nuclei reduces the ability to trigger apoptosis through the intrinsic apoptotic pathway. The shifts of procaspase-9 and Bax are reversible in the absence of the apoptotic trigger; the spontaneous reversion was confirmed experimentally for procaspase-9, whereas Bax shifted from the nuclei to the cytosol and mitochondria after the initiation of apoptosis. To distinguish this process from apoptosis, we call it preapoptotic cell stress response. It shares some features with apoptosis; however, it is reversible and apoptosis has to be induced in addition to this process. CONCLUSION: Knowledge on preapoptotic cell stress response is important for assessing the quality of the cells used in cell therapies, in regenerative medicine, and of those used for modeling metabolic processes.