Drug Targeting Organ-Specific Strategies

312 12 Use of Human Tissue Slices in Drug Targeting Research
production of slices with reproducible thickness. Recently, the so-called ‘Brendel slicer’ was
introduced, which has largely the same characteristics as the Krumdieck slicer, but offers the
advantage of more constant oxygenation. However, with this technique the slices have to be
prepared manually [36]. Liver slices from both tissue slicers have been evaluated. No significant
differences were observed in levels of protein, potassium, total glutathione (i.e. GSH
and GSSG), reduced glutathione (GSH) and cytochrome P450 and activities of 7-ethoxyresorufin
O-deethylase and 7-benzoxyresorufin O-debenzylase in freshly cut rat liver slices
produced by either of the two tissue slicers [37].
To prepare liver slices with the Krumdieck slicer, cylindrical cores of tissue are first isolated
from the liver specimens (Figure 12.1). These tissue cores are prepared preferably by advancing
a sharp rotating metal tube into the liver tissue using a drilling press, thus assuring
the preparation of accurately cylindrical cores. If a biopsy punch is used to prepare the cores,
it is difficult to obtain a uniform cylindrical shape.
The cores are subsequently placed in the slicer, and the slicing procedure is performed by
advancing the core over an oscillating knife in a controlled environment (Figure 12.1). Cold
(4°C) Krebs–Henseleit buffer (pH = 7.4, saturated with 95% O 2 and 5% CO 2) supplemented
with 25 mM glucose is commonly used in preparing the slices [35,38–40], but Williams’
medium E [41], Earle’s balanced salt solutions [37], Sacks preservation medium [42] and V-7
preservation buffer [43,44] are also used.
All these buffers have a glucose concentration of 25 mM, which seems to be essential for
the viability of the slices.
The optimal thickness for liver slices, in order to retain their viability during culture, is approximately
175–250 µm. Price et al. [45] reported that the optimal thickness of liver slices for
drug metabolism studies should be 175 µm. In slices thicker than 250 µm the inner cell layers
suffer from a lack of oxygen and substrates, and in slices thinner than 175 µm the ratio of
damaged cells in the outer cell layers to the living cell mass becomes unfavourable
[36,43,44,46–48]. For cryopreservation slightly thicker slices were reported to give better results
[40], although recent developments show that slices of approximately 200–250 µm can
also be successfully used for cryopreservation (unpublished observation).
12.3 Incubation and Culture of Liver Slices
12.3.1 Incubation Systems
Previously, liver slices were incubated in static organ cultures [1]. Hart et al. [49] cultured rat
liver slices for 24 h spread out on wet filter paper, floating on top of the incubation medium.
Several slice-containing vessels were placed in a box with saturated 95% O 2 and 5% CO 2 at
37°C. However, the slices employed were rather thick (approximately 0.3 mm) and only the
upper cell layers (0.2 mm) in the slice contained viable cells. Together with the introduction
of the Krumdieck slicer [5,46], a new incubation technique for slices, the dynamic organ culture
system (DOC), was introduced [35]. The main characteristic of this system is the intermittent
exposure of the slice to incubation medium and the gas phase. The DOC is in fact a
modified version of the Trowell incubation system [1].