Drug Targeting Organ-Specific Strategies

316 12 Use of Human Tissue Slices in Drug Targeting Research
Brendel’s group compared two incubation systems, the roller system (dynamic organ culture
(Figure 12.2)) and the 12-well plate culture (the plates were put on a gyratory shaker),
with respect to their ability to maintain the functionality of rat liver slices over 72 h of culturing.
The slices were evaluated with respect to ATP concentration, potassium retention,
MTT reduction and protein synthesis, in addition to alanine transaminase (ALT) and LDH
leakage. Metabolic function was investigated by oxidative O-deethylation of 7-ethoxycoumarin
(7-EC) [43]. It was concluded that dynamic organ culture was superior to multi-well plate
culture [43]. Recently, another comparison has been made between the DOC-system and 12well
system, showing that the 12-well system was superior to the DOC-system with regard to the
metabolism of xenobiotics following long-term incubations (> 24 h) [68]. However, this study
was performed in 95% air and 5% CO 2, which may have influenced the results obtained. A
high oxygen percentage of at least 40% is essential for optimal incubation of liver slices, as
will be described in more detail below. In addition, both sets of experiments were carried out
using different incubation media, which also may have influenced the results obtained.
12.3.3 Incubation Systems for Human Liver Slices
Various incubation systems have also been tested using human liver slices, these include the
24-well plates with magnetic stirrers [38,66,69], the six-well plates in a shaker [52] and the
DOC roller system [70,71], but no direct comparison has been made as yet. Human liver
slices can be cultured for 72 h in DOC and maintain their ability to respond to specific inducers
of cytochrome P450 such asmethylclofenapate and Aroclor 1254 [71].
12.3.4 Oxygenation and Culture Media for Liver Slice Incubation
In addition to the incubation system itself, the oxygen and nutrient concentration of the
medium are also important for the viability of liver slices [46,47]. It appeared that a nutrientenriched
medium containing bicarbonate maintained the slice viability better than a simpler
medium such as Krebs–HEPES buffer [44]. It was shown that K + -retention, protein synthesis
and LDH leakage was maintained in rat liver slices for 5 days in a Waymouth’s/bicarbonate
medium in a dynamic organ culture system [44]. Oxygenation of the hepatocytes, especially
those in the centre of the slice, has been a major concern. In this respect it is important
to note that oxygen at too high a concentration may be toxic, due to tissue damage by oxygen
radicals, whereas excessively low levels of oxygen may result in ischaemia. Both 95% air/5%
CO 2 and 95% O 2/5% CO 2 are commonly used. In long-term culture up to 5 days [43] the
DOC system or DOC-related incubation systems are recommended, because of the intermittent
exposure of the slice to culture medium and to the gas phase. This feature was
claimed to be important for optimal gas exchange. In our experiments however, slices that
were continuously submerged in medium performed equally well or even better than those
in the DOC system or DOC-related system (six-well culture plate on the rocker platform
[72]). It can be calculated that liver slices consume only 0.3–1% of the dissolved O 2 per
minute. This implies that, provided that the medium is continuously oxygenated, the availability
of O 2 is unlikely to be a limiting factor. In our laboratory we have carried out a study