1 Introduction in vivo Padgham et al., 1993 Padgham & Paine, 1993 Padgham et al., 1993 Agius, Chowdhury, & Alberti, 1986 Enat et al., 1984 LeCluyse, Bullock, Parkinson, & Hochman, 1996 Waxman, Morrissey, Naik, & Jauregui, 1990 Bissell, Arenson, Maher, & Roll, 1987 Gomez-Lechon et al., 1998 Rogiers & Vercruysse, 1993 Vallette et al., 1998 In the present study we have introduced a new medium for the long-term culture of differentiated hepatocytes. This is keratinocyte serum-free medium (KSFM). It was tested both alone, and in combination with supplements (dexamethasone, EGF and pituitary gland extract), in comparison with the standard Williams’ medium E, and was used to maintain rat hepatocytes in culture for up to 28 days. We performed immunohistochemical, Western blotting and RT-PCR analysis of the cells under the different culture conditions. We chose hepatic markers which are known to be either rapidly switched off after hepatocyte isolation or are important for detoxification. Expression of a particular gene or protein is normally taken as an indication of intact liver function. However, it is difficult to know simply from expression of a gene or protein that the associated function remains intact. For this reason we also assayed for hepatocyte ureagenesis, glycogen synthesis and response to xenobiotics. Lastly, to gain some mechanistic insight we determined the expression of several liver-enriched transcription factors. Our results suggest that KSFM, in combination with dexamethasone, EGF and pituitary gland extract can maintain the liver phenotype for between 21 and 28 days and it is likely that the ability to do this depends on the sustained expression of liver-enriched transcription factors. 2 Materials and methods 2.1 Materials l Shen, Slack, & Tosh, 2000 Tosh, Shen, & Slack, 2002 2.2 Rat hepatocyte isolation and culture Tosh, Alberti, & Agius, 1988 d d 2 g d 2 5 l l 2.3 Immunostaining and antisera Shen et al., 2000 Table 1 2.4 Western blotting ® Table 2 Table 2 2.5 Qualitative and real-time RT-PCR Li, Horb, Tosh, & Slack, 2005 Table 3 @ 2.6 Periodic acid–Schiff's (PAS) staining PAS staining was performed to detect glycogen. The cells were seeded onto coverslips and cultured for up to 4 weeks in KDS medium. The cells were then incubated with KDS plus 25 mM glucose for 24 h, fixed with 4% PFA and then permeabilised with 1% Triton X-100 at room temperature for 20–25 min. The cells were washed with tap water for 1–2 min and transferred to 1% periodic acid solution for 30 min. Next, the cells were washed with running tap water for 3 min and then incubated in the Schiff's reagent at room temperature for 30 min to develop. After washing in running tap water for 10 min, the slides were mounted in Gel/Mount mounting medium. 2.7 Urea cycle assays Meng, Zhang, & Wu, 2004 Corraliza, Campo, Soler, and Modolell (1994) 2 l 2 4 3 4 2 iso 2.8 Image collection Fluorescent images were collected using a Zeiss LSM 510 confocal microscope and figures compiled using Adobe Photoshop 7.0. For cell counting and PAS staining experiments, numbers of random fields were selected using the 40× objective lens of a Leica DMRB microscope. The cell numbers were visualised by 4,6-diamidino-2-phenylindole (DAPI) staining. Coverslips were incubated with DAPI (500 ng/ml) in PBS for 30 min at room temperature before being mounted on to slides in Gel/Mount mounting medium. 2.9 Statistical analysis t 3 Results 3.1 Expression of hepatic differentiation markers in rat hepatocytes cultured in Williams’ medium E and KSFM in vitro Andl et al., 2003 Chen, Chang, Lee, Javier, & Azar, 2002 Vallette et al., 1998 Katsura et al. (2002) 2 Fig. 1 Fig. 1 Fig. 1 Fig. 1 Blaheta et al., 1998 Fig. 1 Kumar & Gilula, 1986 Nicholson et al., 1987 Krysko, Leybaert, Vandenabeele, & D’Herde, 2005 Cheng et al., 2004 Luebeck, Buchmann, Schneider, Moolgavkar, & Schwarz, 2005 Fig. 2 Fig. 2 3.2 Rat hepatocytes cultured in KDS medium retain their differentiated properties for up to 28 days Fig. 3 Fig. 3 Fig. 3 Fig. 3 Fig. 3 Fig. 3 Fig. 4 Fig. 4 Fig. 4 Fig. 4 Fig. 4 Fig. 4 3.3 Glycogen storage and urea cycle activity is preserved in KSFM-cultured rat hepatocytes Fig. 5 Fig. 6 Fig. 6 Kang, Berthiaume, Nath, & Yarmush, 2004 3.4 Hepatic metabolising enzymes are induced by phenobarbital treatment in KDS-cultured hepatocytes cis cis Cyp2b10 Kakizaki et al., 2003 Fig. 7 UGT Cyp2B12 Cyp3A1 Cyp7A1 Fig. 7 4 Discussion in vitro Michalopoulos, Bowen, Mule, & Luo, 2003 Miyazaki et al., 1998 Lazaro et al., 2003; Runge et al., 2000 Michalopoulos et al., 2003; Miyazaki et al., 1998 Mitaka, 1998 Kojima et al., 1996 Shelly, Tynan, Schmid, Schutz, & Yeoh, 1989 Rana, Mischoulon, Xie, Bucher, & Farmer, 1994 Costa, Kalinichenko, Holterman, & Wang, 2003 Naiki, Nagaki, Asano, Kimata, & Moriwaki, 2005 Kurash, Shen, & Tosh, 2004 Shen et al., 2000; Tosh et al., 2002 Sheen et al., 2004 Stoehr & Isom, 2003 Fig. 4 Kano-Sueoka et al., 1979; Platica et al., 1992 in vitro