Kawai T, Yasuchika K, Ishii T, Katayama H, Yoshitoshi EY, Ogiso S, Kita S, Yasuda K, Fukumitsu K, Mizumoto M, Hatano E, Uemoto S. grow in monolayer culture for months and were not tumorigenic culture, including continuous medium and oxygen supply, and metabolite removal [4, 5]. In addition, various hollow fiber bioreactor systems were developed using hepatocytes of several species . In these systems, cells attach to the surface of fibers or membranes and reorganize themselves into three-dimensional structures that may result in a hepatocyte microenvironment closely resembling the physiological one. Unfortunately, the described cell culture systems are not yet standardized and cannot be easily transferred to other laboratories. To overcome limitations that negatively regulate human hepatocyte viability and functionality, isolated rodent hepatocytes have been increasingly used as a tool to identify pharmacological and toxicological responses to drugs. Primary rat hepatocytes represent a useful experimental model as their isolation is a relatively easy procedure, guaranteeing a good success rate and an adequate degree of reproducibility. In addition, this Mouse monoclonal to CD45RO.TB100 reacts with the 220 kDa isoform A of CD45. This is clustered as CD45RA, and is expressed on naive/resting T cells and on medullart thymocytes. In comparison, CD45RO is expressed on memory/activated T cells and cortical thymocytes. CD45RA and CD45RO are useful for discriminating between naive and memory T cells in the study of the immune system procedure provides a large number of cells from a single rat liver. Nevertheless, their use cannot be exploited for studying the biochemical/molecular events leading to cell transformation, as primary hepatocytes survive in culture no longer than 1 week. Even though long-lasting HCC cell lines are useful for drug screening and/or molecular manipulation of gene expression, a major limit in their use is the lack of a normal counterpart for reference. In the present study, taking advantage of the Resistant Hepatocyte model of rat hepatocarcinogenesis (R-H) , we generated and characterized a long-term, non-tumorigenic hepatocyte cell line (RNT), and the corresponding fully transformed cell line (RH). These matched cell lines represent a valuable model to study hepatocarcinogenesis, through genetic engineering aimed at reproducing the multistep process of liver cancer development. RESULTS Isolation and characterization of RNT and RH cell lines The R-H model consists of a single injection of DENA followed by a brief exposure to a promoting environment (2-AAF + PH). HCCs arise 10-14 months after DENA treatment (the protocol SR-3029 scheme is shown in Supplementary Figure 1). Control rats exposed to 2-AAF + PH in the absence of DENA, do SR-3029 not develop tumors. RH and RNT cells were obtained from a rat exposed to the full R-H protocol and from a rat not exposed to DENA, respectively. Briefly, cells were isolated SR-3029 from liver rats through collagenase perfusion by portal vein and maintained SR-3029 in culture. Both cell lines were vital after more than 50 passages in conventional 2D culture dishes, and did not change their morphology and behavior. Therefore, they can be defined as spontaneously immortalized cells. RNT cells exhibit a clear hepatocyte morphology, as they show a typical polygonal architecture and big rounded nuclei; these cells are serum-dependent and show contact inhibition when growing in monolayer (Figure 1A, 1C, 1E). On the opposite, a more elongated morphology (fibroblast-like) characterizes RH cells (Figure 1B, 1D), that are able to proliferate under suboptimal culture conditions (low serum, Figure ?Figure1E),1E), losing cell-cell contact inhibition and continuing to divide and forming multilayered foci. Open in a separate window Figure 1 Morphological characterization and growth rate of RNT and RH cellsPhase-contrast microscopy and H&E staining of cultured RNT A., C. and RH B., D. cells. Magnification 20x. For the experimental procedure followed to obtain the cell lines, see Materials and Methods. E. The growth rate of the two cell lines in adherent conditions, in optimal (10% serum) and suboptimal (2% serum) growing conditions, was measured at the indicated times. Cells were fixed and stained with crystal violet; the dye retained by the cells was solubilized in 10% acetic acid and the Optical Density (570nm) was measured. On the X axis is shown the fold change increase of cell number, compared to time zero. ** P 0.01; ****P 0.0001. Next, we further characterized RNT and RH cells for the expression of hepatocyte and non-hepatocyte markers. Both cell lines were positive for glycogen (as shown by PAS staining), a classical marker of hepatocyte function (Figure ?(Figure2A).2A). Immunofluorescence and flow cytometry analysis showed SR-3029 that.