Transport proteins expressed in cell membranes of various organs are mainly involved in pharmacokinetic processes in the body. Preclinical data about the affinity of a drug candidate to transport proteins should be verified early in drug development since they may cause interactions with other compounds and these informations are necessary for the approval process. In comparison to cell lines, which represent only an isolated transport process, the use of primary cells for these studies is preferred to get as close as possible to the in vivo situation. Since the liver is the central organ for drug metabolism, the primary hepatocytes are as liver functional unit particularly well suited for studying drug transport and hepatic metabolism. This presentation shows the results generated with radiolabeled model substrates to assay the functionality of uptake and efflux transporter in primary hepatocytes from human, Cynomolgus and Beagle in 2D cultures. Estrone-3-sulfate (E1S) is a suitable substrate to verify the functionality of uptake transporters in freshly primary hepatocytes of these species. P-gp mediated transport can be verified with Talinolol (Tal) in human and monkey hepatocytes; MRP2 efflux can be verified in dog hepatocytes by using E17β-Gln (Estradiol-17βGlucuronide). Cryopreservation had almost no effect on transport kinetics in monkey and dog hepatocytes.

1. Characterization of uptake and efflux transporters in freshly
isolated and cryopreserved primary hepatocytes of different
species by using radiolabeled substrates.
Copyright©2014byPRIMACYTCellCultureTechnologyGmbH,Schwerin,Germany
The 18th International Congress on In Vitro Toxicology ESTIV2014
Egmond aan Zee, The Netherlands
Contact Data:
PRIMACYT Cell Culture Technology GmbH, Hagenower Straße 73, 19061 Schwerin, Germany
Phone: +49 (0)385 - 3993 600, Email: info@primacyt.de

2. Background
Primary mammalian hepatocytes are the system of choice for
multiple in-vitro applications such as drug metabolism,
toxicology, and transporter assays.
Membrane transporters can be major determinants for the
uptake and efflux of drugs.
Fig. 1: Human hepatic membrane transport proteins
(Chandra and Brouwer, Pharmaceutical Research, 2004)
• Analysis of concentration dependent uptake
of estrone-3 sulfate E1S in fresh isolated and
cryopreserved plated hepatocytes.
• Characterization of efflux transporter P-gp
with [3H]-talinolol (Tal) in presence or
absence of its specific inhibitor PSC833.
• Examination of efflux transporter MRP2 with
[3H]-estradiol-17β-glucoronide (E17β-Gln),
with and without the inhibitor MK571.
Objectives
Methods
Primary human, monkey (Cynomolgus) and dog (Beagle) hepatocytes have been cultured on
24well plates in HHMM (Human Hepatocyte Maintenance Medium). Assays with radiolabeled
substrates +/- inhibitors were done on day 2 in culture after isolation or after thawing.
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3. Phase contrast images of freshly isolated monkey (A), dog (C), human (E) and
cryopreserved monkey (B), dog (D) hepatocytes on day 1 in culture.
A - freshly isolated monkey
E - freshly isolated Human
D - cryopreserved dog
C - freshly isolated dog
B - cryopreserved monkey
Figure 2
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4. Concentration dependent uptake of E1S in primary human, dog and monkey hepatocytes.
Blue curves: cryopreserved cells;
Red and green curves: fresh hepatocytes in absence (red) or presence (green) of BSP (Bromosulfophthaleine), a
competitive inhibitor for E1S uptake.
Figure 3
Notes: *p<0.05 vs. -BSP
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5. Time dependent efflux of Talinolol and E17β-Gln mediated by P-gp and MRP2, resp.,
in fresh human, dog and monkey hepatocytes.Figure 4
Notes: *p<0.05 vs. –PSC833, †p<0.05 vs. –MK571, $p<0.05 vs. human, §p<0.05 vs. dog
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6. E1S is a suitable model substrate to verify the functionality of uptake
transporters in primary hepatocytes of different species.
Results generated in animal hepatocytes can not be transferred to
humans.
P-gp mediated transport of Tal can be influenced by PSC833 in
hepatocytes of all species.
In dog hepatocytes MRP2 efflux can be verified with E17β-Gln and
inhibited by MK571.
Results and Conclusions
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7. Contributors
PRIMACYT Cell Culture Technology GmbH, Schwerin, Germany
A. Ullrich, J. Jia and D. Runge
Department of Clinical Pharmacology, University Medicine of Greifswald,
Germany
M. Keiser, J.Jia and W. Siegmund
Department of Surgery, University Medicine of Greifswald
M. Patrzyk, A. Busemann and C. D. Heidecke
Major keywords are:
uptake
transporters,
ef.lux
transporters,
radiolabeled
substrates,
membrane
transporters,
inhibitor
PSC833,
inhibitor
MK571,
time
dependent
ef.lux
of
Talinolol,
freshly
isolated
hepatocytes,
cryopreserved
hepatocytes,
human
hepatocytes,
Cynomolgus
monkey,
hepatocytes,
Beagle
hepatocytes,
18th
International
Congress
on
In
Vitro
Toxicology
ESTIV2014.
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