The most common techniques for isolating human hepatocytes from donor
livers involve a two-step collagenase perfusion and digestion (Fabre et al.,
1988; LeCluyse et al., 2000; Strom et al., 1982). Encapsulated liver tissue
(25–100 g) is perfused with calcium-free buffer containing 5.5 mM glucose and
0.5 mM EGTA for 20 min at a flow rate of 40 mL/min/cannula followed by
perfusion with buffer containing 1.5 mM calcium and collagenase (0.4 mg/mL)
for 25 min at a flow rate of 35 mL/min/cannula. Hepatocytes are dispersed
from the digested liver in Dulbecco’s modified Eagle medium (DMEM)
containing 5% fetal calf serum, insulin (4mg/mL), and dexamethasone
(1.0mM) (Supplemented DMEM) and washed by centrifugation at 70 g
for 4 min. The cell pellets are resuspended in 30 mL Supplemented DMEM and
12 mL 90% isotonic Percoll. The cell suspensions are centrifuged at 100 gfor
5 min. The resulting pellets are resuspended in fresh medium and washed once
by low speed centrifugation. Hepatocytes are resuspended in Supplemented
DMEM and cell viability is determined by trypan blue exclusion (LeCluyse
et al., 2000). Cell yields are normally between 5 and 12 million cells per gram of
wet liver tissue, and cell viability varies from 75% to 91% (Kostrubsky et al.,
1999; LeCluyse et al., 2000).
Hepatocytes are plated in 6-well plates (1 106 viable cells/well) and
maintained at 37oC, 95% humidity, and 5% CO 2 in Hepatocyte Culture
Medium (JRH Bioasciences, Lenexa, KS) supplemented with L-glutamine
(0.292 mg/mL), nonessential amino acids (10mM), insulin (6.25mg/mL),
transferrin (6.25mg/mL), selenium (6.25 ng/mL), bovine serum albumin
(125 mg/mL), linoleic acid (5.35mg/mL), penicillin (100 U/mL), streptomycin
(100mg/mL), and dexamethasone (0.1mM) (Prueksaritanont et al., 2005).
After a 2-day recuperation period, the cultured hepatocytes are treated for
three consecutive days with test compounds dissolved in DMSO. The final
DMSO concentration in the medium is 0.1% or less. Cells are normally
exposed to drugs at concentrations of 2, 10, and 20mM. However, some drugs
(e.g., phenobarbital) may require 100mM or higher concentrations to produce
induction. Rifampicin (10mM) is a positive control for CYP3A4 induction,
and 0.1% DMSO is used as a negative or vehicle control (Luo et al., 2002).
The extent of CYP3A4 expression and induction is quantitatively assessed
by measuring microsomal metabolism of a CYP3A4-specific probe substrate.
Testosterone and midazolam are the most well accepted and commonly
employed substrates (Bjornsson et al., 2003; Hariparsad et al., 2004; Tucker
et al., 2001). The level of CYP3A4 protein expression can be qualitatively and
quantitatively determined by Western immunoblotting with CYP3A4- specific
antibodies, or by measuring CYP3A4 mRNA by means of RT–PCR, Northern
blot, or branched DNA signal amplification technology (bDNA) (Czerwinski
et al., 2002; Desai et al., 2002; Luo et al., 2002; Prueksaritanont et al., 2005).
For the measurement of CYP3A4-specific metabolic activity at the end of
the drug treatment, the drug-containing medium is removed from the wells,
and replaced with drug-free medium for 4 h. Cells are then exposed to
testosterone (250mM) in Williams’s E medium (1 mL/well) for 30 min. This
554 TESTING DRUG CANDIDATES FOR CYP3A4 INDUCTION