Characterization of uptake and efflux transporters in fresh isolated and cryopreserved primary hepatocytes of different species by using radiolabeled substrates

Abstract

Primary mammalian hepatocytes are used for several in vitro applications like testing of drug metabolism, toxicity and transporter assays, but little is known about the effect of cryopreservation on transport kinetics. Therefore, we characterized the uptake and efflux transport of different substrates in hepatocytes of different species. Human, dog and monkey hepatocytes were incubated in serum free media. After cell isolation or after thawing a concentration dependent uptake of [3H]-estrone-3-sulfate (E1S) was measured in presence or absence of bromosulfophthaleine (BSP). To measure theeffluxactivityof P-glycoprotein (P-gp) andmultidrug resistance protein (MRP) 2 cells were incubated shortly with [3H]-talinolol (Tal) or [3H]-estradiol-17 -glucoronide (E17 -Gln), respectively. Intracellular concentration of Tal and E17 -Gln were measured in presence or absence of P-gp inhibitor PSC833 or MRP2 inhibitor MK571. E1S revealed affinity to human, monkey and dog hepatocytes with Km-values of 11.6-20.8 mol/l and Vmax-values of 415–926pmol/mg×min. E1S uptake was inhibited in the presence of BSP. Cryopreserved cells showed significant differences in Vmax-values of E1Suptake inmonkeyhepatocytes. Inhumanhepatocytes, Tal efflux was decelerated in cells incubated with PSC833. While monkey hepatocytes showed a decelerated efflux of Tal and E17 -Gln in the presence of PSC833 and MK571, dog hepatocytes showed only a decelerated efflux of E17 -Gln in cells incubated with MK571. Cryopreserved cells showed no differences on efflux transport in monkey and dog hepatocytes. P-gp mediated transport can be verified with Tal in human and monkey hepatocytes; MRP2 efflux can be verified in dog hepatocytes by using E17 -Gln.