Morphology and lysosomal enzyme activity of primary cultures of rat liver sinusoidal cells

Adult rat liver sinusoidal cells were isolated by enzymatic perfusion and incubation of the liver, and cultured in Dulbecco's medium supplemented with 17% fetal calf serum. The structure of the cultured cells was studied after various incubation periods by means of phase contrast microscopy and transmission electron microscopy. Cultured sinusoid cells were incubated with latex particles (0.8 μm) to distinguish between the phagocytic Kupffer cells and other cells. Within 24 hours the Kupffer cells in the sinusoid cell suspension adhere to the plastic surface of the culture dish. Endothelial cells, pit cells and fat storing cells rarely become attached to the bottom of the dish. The number of endothelial cells decreased rapidly during prolonged culture, because of cell death and phagocytosis by Kupffer cells and their removal with the renewal of the medium. Phase contrast microscopy of the cultured Kupffer cells shows that they resemble other macrophages after adherence to plastic, although Kupffer cells assume more irregular shapes. With regard to the nucleus, mitochondria, rough endoplasmic reticulum and the Golgi apparatus, the ultrastructure of Kupffer cells in these cultures is similar to Kupffer cells in situ. The erratic shape of the membrane of the cultured Kupffer cells, with numerous microvillous, cytoplasmic extensions, sometimes resembling pseudopods, also shows similarities with Kupffer cells in situ. The cell coat, visualized with ruthenium red, appears less thick, while worm-like structures present in situ are absent. Specialized changes in the membranes can occur at sites of contact between Kupffer cells. The most striking difference from Kupffer cells in situ can be found in the vacuolar and lysosomal systems. Enormous digestive vacuoles, the result of ingestion of cellular material, are present in every Kupffer cell after the first day of culture. During prolonged culture, the digestive vacuoles bacome translucent. Determination of lysosomal enzyme activities after different periods of culture reveals a strong increase in the activities per mg cellular protein of cathepsin D and N-acetyl-β-glucosaminidase, while that of β-glucuronidase remains constant during the first 4 days of culture. The activity per mg cellular protein of acid phophatase at day 4 is decreased to 60% of the values of day 1. The amount of cellular protein in the cultures decreases steadily during the first 4 days of culture. © Copyright 2017 Elsevier B.V., All rights reserved.