Drug Targeting Organ-Specific Strategies

12.3 Incubation and Culture of Liver Slices 315
Based on these various findings under a variety of conditions a thorough comparison of
five incubation systems (Figure 12.2) was made by us in a collaborative study of four laboratories
[52]. The five systems that were evaluated included: the shaken flask (a 25-ml Erlenmeyer
flask in a shaking water bath [40]), the stirred well (24-well culture plate equipped
with stainless steel grids and magnetic stirrers [38,64]), the rocker platform (a DOC system
using six-well culture plates with Netwell inserts, rocked on a platform [41]), the roller system
(dynamic organ culture rolled on an insert in a glass vial [35]) and the six-well shaker (sixwell
culture plates in a shaking water bath). In the rocker platform 40% O 2/5% CO 2/55% N 2
was used whereas in the other four systems 95% O 2/5% CO 2 was used to oxygenate the tissue.The
liver slices were incubated in these incubation systems for 0.5, 1.5 and 24.5 h and subsequently
subjected to viability and metabolic function tests. The viability of the incubated
liver slices was evaluated by potassium content, MTT assay, energy charge, histomorphology
and lactate dehydrogenase (LDH) leakage.Their metabolic functions were studied by determination
of the metabolism of lidocaine (Figure 12.3), testosterone and antipyrine. Up to
1.5 h of incubation, all five incubation systems gave similar results with respect to viability
and metabolic function of the slices. However, after 24 h, the shaken flask, the rocker platform
and the six-well shaker incubation systems, appeared to be superior to the stirred well
and the roller incubator. It is notable that the cytochrome P450-dependent metabolism of
testosterone and lidocaine was retained at the same levels as found after 0.5 and 1.5 h of incubation
in the shaken flask, rocker platform and six-well incubation systems. This suggests
that the de-differentiation seen after 24 h in pure hepatocyte culture does not occur in slices
for at least 24 h.
nmol MEGX/mg wet weight liver slice
20
15
10
5
0
*
0.5 1.5 24.5
Incubation time (hours)
**
**
Shaken flask
Stirred well
Rocker platform
Roller system
6 Well shaker
Figure 12.3. Metabolism of lidocaine to MEGX (in nmol MEGX mg –1 wet weight liver slice) in liver
slices after different incubation times (h). *p < 0.05 versus shaken flask, rocker platform, roller system
and six-well shaker. **p < 0.05 versus shaken flask, rocker platform and six-well shaker. Data are the
mean of three separate experiments ± SEM.