Isolated trout liver cells: Establishing short-term primary cultures exhibiting cell-to-cell interactions

Abstract

Composition and interactions of cell types in rainbow trout (Oncorhynchus mykiss) liver digested with collagenase and cultured in serum-free media were investigated. Suspensions obtained after digesting trout liver with collagenase contained all the cell types present in the liver, including liver parenchymal cells (hepatocytes), biliary epithelial cells, sinusoidal endothelium, fat-storing cells of Ito, and macrophages. A major cell pellet, mainly hepatocytes but containing significant numbers of biliary epithelial cells, was obtained by centrifuging the cell suspension at 120 X g for 1 min. Cells present in this pellet quantitatively attached to culture plates coated with a trout skin extract and remain attached for 4 to 6 d with good retention of intracellular enzymes and DNA. When in culture, significant changes in and among the cells were observed. Initial preparations were rounded, single cells. Within several hours, however, cellular interactions leading to aggregation became evident and aggregates increased in size for 2 to 3 d. Scanning electron microscopy (EM) showed frequent shaftlike projections from margins of the aggregates. Transmission EM indicated that these projections represent biliary ductules forming in vitro. Adjacent hepatocytes also showed plasma membrane specializations forming junctional complexes and canaliculi characteristics of normal trout liver. After 5 to 6 d in culture, significant numbers of the cell aggregates dislodged from the plate. Analysis showed the dislodged cells were viable but vacuolated. The reestablishment in vitro of morphologic relationships resembling in situ tissue components suggest these culture preparations may have significant utility in cooperative metabolic studies of cell interactions in trout liver.

Vitellogenin synthesis in cultured hepatocytes; an in vitro test for the estrogenic potency of chemicals

“…Various techniques have been used to culture fish hepatocytes [35,37]. We chose to use the system first developed by Flouriot and Valotaire [38] and modified by Foucher et al [39], in which gentle shaking is used to encourage the hepatocyte to form small aggregates, as a first step towards tissue organization [40]. In our system, the cells are kept for 10 to 12 days (see Fig.…”

Optimization of the isolation and cultivation of Cyprinus carpio primary hepatocytes

“…The classical method of primary culture of fish hepatocytes is whole-liver perfusion. As far as we are aware, Birnbaum et al (1976) were the first to adapt the perfusion technique for preparing fresh suspension of fish liver cells and methods for primary culture of fish hepatocytes became available during the 1980s (Blair et al 1990;Klaunig et al 1985). This tool has been increasingly applied to physiological and toxicological studies, and has become the classical method (Braunbeck and Segner 2000;Mommsen et al 1994;Monod et al 1998;Pesonen and Andersson 1991;Segner 1998).…”

New Development in Aflatoxin Research: From Aquafeed to Marine Cells

“…The liver perfusion was the first important technique implemented for the preparation of primary hepatocytes (Guguen-Guillouzo, 1992). Firstly, the method was developed in rodents, (Berry & Friend, 1969;Seglen, 1976) and then improved to obtain hepatocytes from other animal sources like pig (Chen et al, 2002;Koebe & Schildberg, 1996), sheep (Clark & Vincent, 2000) and finally fish (Birnbaum et al, 1976;Blair et al 1990;Braunbeck & Segner, 2000;Mommsen et al, 1994;Segner, 1998). Since the early 70s, this was the main method largely used for aquatic vertebrates, event though the main disadvantage of this procedure is the large size of animal (approximately 100 g/body weight) required to permit the in vivo abdomen insertion of perfusion devices connected to a peristaltic pump.…”