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VOL 66 (4) 2000 IN VITRO INDUCTION OF APOPTOSIS BY CYCLOSPORINE A

investigation aimed to examine CsA action with relation to apoptosis and
necrosis by means of morphological and biochemical tools under
conditions in which specific hepatocellular functions are preserved during
an incubation time between 4 and 20 hours.

MATERIALS AND METHODS

Animals

Permission for animal studies was obtained from the Veterinäramt
Basel-Landschaft, CH-4410 Liestal, and all study protocols were in
compliance with the institutional guidelines. Male Wistar rats were
obtained from Biological Research Laboratories (CH-4414 Füllinsdorf,
Switzerland). They were kept in Macrolon® cages with wood shavings as
bedding under optimal hygienic conditions, at a temperature of 22-23 °C,
a relative humidity of 50-74 %, and fluorescent light for a 12-hour
day/12-hour night cycle. They were given water and rodent pellets ad
libitum.

Hepatocyte isolation and cell-culture conditions

Rat hepatocytes (rats 180-220 g) were isolated according to the
two-step liver perfusion method (Boelsterli et al. 1993). The cells were
seeded in 35 mm six-well culture dishes1 at a density of 0.7 u 106 cells in
2 ml WME2, or in 60 mm culture dishes1 at a density of 2 u 106 cells in 5
ml WME. The culture medium contained 10 % fetal calf serum, penicillin
(100 U/ml), streptomycin (0.1 mg/ml), insulin (10-7 M) and
dexamethasone (10-7 M). After an attachment period of 2 hours at 37 °C
in a 5 % CO2/95 % air atmosphere, the medium was changed. The test
compound was added together with the new medium. CsA3 was dissolved
in DMSO, and this solution was added to the culture medium, resulting in
a final concentration of 1 % DMSO in the culture medium. Control plates
received the DMSO- containing medium without CsA.

The maximum soluble CsA concentration in culture medium in the
presence of BSA, without any observable precipitation, was 50 µM.

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