Propagation and functional characterization of serum-free cultured porcine hepatocytes for downstream applications

Abstract

Hepatocyte transplantation is considered an alternative to whole organ transplantation. However, the availability of human cadaveric livers for the isolation of transplantation-quality hepatocytes is increasingly restricted. Xenogeneic porcine hepatocytes may therefore serve as an alternate cell ressource. The propagation of hepatocytes is often necessary to yield a sufficient cell number for downstream applications in xenotransplantation and in, for example, bioartificial liver support or pharmacological and toxicological studies. Our goal has been to propagate primary porcine hepatocytes in vitro and to determine the functional maintenance of the propagated cells. Porcine hepatocytes were cultured under serum-free conditions in the presence of hepatocyte growth factor and epidermal growth factor and passaged several times. The viability, proliferation and maintenance of liver-specific functions were determined as culture proceeded. Total cell number increased by 12-fold during four sequential passages, although the proliferative capacity was higher in primary cells and early passages as compared with late passages. Xenobiotics metabolism and urea synthesis gradually decreased with ongoing culture but could be restored by treatment with appropriate stimuli such, as beta-naphthoflavone and cAMP. The expression of hepatocyte-specific genes was generally lower at the beginning than at later time-points of culture of individual passages. Porcine hepatocytes can thus be propagated in vitro. The partial loss of hepatocyte function may be restored in vitro by appropriate stimuli. This may also be achieved in a recipient liver after hepatocyte transplantation provided that the proper physiological environment for the maintenance of the differentiated hepatocyte phenotype is present.

Profiling the Impact of Medium Formulation on Morphology and Functionality of Primary Hepatocytes in vitro

“…Both M199 and M1640 show differential effects on urea synthesis capacity, the latter significantly greater than all media tested (Fig. 1), and substantially higher than previously published reports631. Total CYP450 content remained stable in 2-day-old cultures, despite a 66% decrease compared with fresh cells although ML-15 maintained day 2 tCYP450 levels up to culture day 4.…”

“…Previous studies have shown that the type of medium formulation modulates morphology and function of rat and sheep hepatocyte cultures2930, as well as porcine hepatocytes on biomatrix-coated culture dishes with or without serum131.…”

Three-dimensional perfusion cultures of mouse and pig fetal liver cells in a packed-bed reactor: Effect of medium flow rate on cell numbers and hepatic functions

“…4). In this regard, it is known that pig mature hepatocytes proliferate to some extent even in vitro, whereas mouse and rat mature hepatocytes scarcely proliferate (Cheng et al, 2005;Schneider et al, 2006). From these facts, the different performances of pig and mouse FLCs might be due mainly to differences in the intrinsic properties of these cells, in addition to differences in the developmental process of these species.…”

Xenotransplantation Literature Update November–December 2005

“…Schneider et al [17] studied the isolation, culture and expansion of primary porcine hepatocytes. Porcine hepatocytes were cultured under serum‐free conditions in the presence of hepatocyte growth factor (HGF) and epidermal growth factor (EGF), and could be passaged several times.…”