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Senescence of hepatocytes isolated from rainbow trout (Oncorhynchus mykiss) in primary culture

An ultrastructural study

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Summary

In order to evaluate the suitability of isolated fish hepatocytes for toxicological studies, hepatocytes were isolated from rainbow trout (Oncorhynchus mykiss) by liver collagenase perfusion. Isolated hepatocytes were investigated for seven days in primary culture on uncoated petri dishes using light and electron microscopy. Viability of isolated hepatocytes, as estimated from trypan blue exclusion, declined from >90% at the beginning of the incubation to ⩽80% after eight days of primary culture. Survival of hepatocytes was best at an incubation temperature of 14°C, and addition of fetal calf serum failed to improve cell performance. Freshly isolated hepatocytes appeared as solitary spherical cells with numerous microvilli at the outer surface. Except for a 30% reduction in cell size due to stress-induced glycogen reduction, the ultrastructure of freshly isolated hepatocytes closely resembled that of rainbow trout hepatocytes in vivo, which is characterized by distinct cytoplasmic segregation into a perinuclear portion containing rough endoplasmic reticulum (RER) arranged in extensive stacks, mitochondria, peroxisomes and the peribiliary complex (dictyosomes, lysosomes), and large peripheral glycogen fields occasionally interspersed with small lipid inclusions. Within one day of culture, about 60–80% of the isolated hepatocytes sedimented to form a monolayer attached to the culture dishes, whereas up to 20% remained in suspension forming hepatocyte aggregates. Cell adhesion was weak, and during prolonged culture increasing amounts of cell detached, whereas the floating cell accumulations grew to aggregates of more than 100 cells. Cell viability and ultrastructure was similar in monolayers and spheroids, and only from the fifth day in culture, did hepatocytes in the centre of floating aggregates become necrotic. From day 1 to day 5, hepatocytes in primary culture displayed only minor cytological alterations. Excellent cytoplasmic compartmentation, restoration of hepatocytic glycogen stores, high secretion rates of very low density lipoproteins by dictyosomes, establishment of cell-to-cell contacts, restitution of cellular polarity and the epithelial character of the cells, as well as formation of bile canaliculi documented recovery of the hepatocytes in primary culture. From day 5 in culture, an increasing number of cells detached from the substratum, and cell senescence was indicated by a marked increase in ultrastructural heterogeneity, with progressive vesiculation and fractionation of the RER, transformation of RER stacks into huge membrane whorls, aggregation and proliferation of peroxisomes and SER, lack of dictyosomal VLDL production, drastic accumulation of lysosomes, myelinated bodies and autophagic vacuoles, as well as successive exhaustion of cellular glycogen deposits. Whereas with conventional methods for assessing cell viability, most hepatocytes appeared intact for up to ten days in culture, cytological investigations revealed severe deterioration of cellular integrity from day 7. However, for incubation periods of up to five days, isolated rainbow trout hepatocytes can be recommended as an excellent model for physiological and toxicological studies.

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Abbreviations

BME:

basal medium Eagle

DAB 3:

3′-diaminobenzidine

EDTA:

ethylendiamine tetraacetic acid

MEM:

minimum essential medium

HEPES:

N-(2-hydroxyethyl)piperazine-N′-2-ethanesulfonic acid

PVP:

polyvinylpyrrolidone

RER:

rough endoplasmic reticulum

SER:

smooth endoplasmic reticulum

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  1. Zoologisches Institut I, Universität Heidelberg, Im Neuenheimer Feld 230, D-W-6900, Heidelberg, Federal Republic of Germany

    T. Braunbeck & V. Storch

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Braunbeck, T., Storch, V. Senescence of hepatocytes isolated from rainbow trout (Oncorhynchus mykiss) in primary culture. Protoplasma 170, 138–159 (1992). https://doi.org/10.1007/BF01378789

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