2. Isolation and Transduction of Human Hepatocytes 
A portion of liver tissue from 4 different donors, labeled Z1 through Z4, was used for 
hepatocyte isolation (19). Liver sections from three of the donors were available because the 
organ had been surgically reduced prior to transplantation; the fourth organ had been harvested 
but rejected for transplantation because the donor was hemodynamically unstable and had 
elevated liver function tests. The organs were perfused with UW solution (Z1, Z2, Z3) or Euro- 
Collins solution (Z4) and stored for variable periods of time prior to hepatocyte isolation (30 
min to 36 hrs). The age of the donor ranged from 22 months to 44 years. 
Hepatocytes were isolated by collagenase perfusion of the major vessels using a 
modification of the technique shown to be successful in isolating hepatocytes from rabbits (16). 
The viability of recovered hepatocytes ranged from 85 to 98 %, while the recovery of viable 
cells ranged from 3 to 33 x 10 6 cells/gm wt wet of tissue. The hepatocytes were plated 
overnight in hormonally defined medium (HDM) containing fetal bovine serum onto Primaria 
tissue culture plates, and were subsequently maintained in serum free HDM. Plating 
efficiencies ranged from approximately 50 to greater than 95 %. The cultures were seeded at 
various subconfluent densities and rapidly grew to confluence within 72 hours of the initial 
plating. Hepatocytes were maintained in primary culture for a total of 4 to 5 days. 
A series of experiments were performed to maximize the efficiency of retroviral 
mediated gene transfer. Two recombinant retroviruses were used to infect the primary 
cultures from each patient; both types of virus were produced in the amphotropic packaging cell 
line ¥ CRIP (90). The first recombinant virus was produced from the previously described 
BAG vector that expresses the lacZ gene from a transcript initiated at the 5' LTR and a gene 
encoding Neomycin from SV40 sequences located internal to the viral transcriptional unit (91). 
The second recombinant virus was produced from a vector called AFP-BA-rLDLR which 
expresses rabbit LDL receptor from a transcript initiated at promoter sequences that are 
derived from the chicken p-actin gene along with enhancer sequences from the mouse alpha- 
fetoprotein gene (92). The relative titer of each virus was estimated by infecting subconfluent 
plates of NIH3T3 cells and harvesting total cellular DNA for Southern blot analysis. BAG 
infected cells contain approximately 0.5 copy of proviral DNA per cell while LDL receptor 
infected cells contain approximately 1 copy of proviral DNA per cell. 
Based on our previous experiences with primary cultures of rat (93) and rabbit (14, 
18) hepatocytes, we developed a protocol for efficiently infecting human primary hepatocytes 
(19). The protocol was designed to maximize gene transfer and minimize the time the 
hepatocytes are maintained in culture. Hepatocytes were seeded at different densities (2 or 4 x 
10 6 per 10 cm plate) and exposed to the viruses for a single 12 hour period beginning 48 hrs 
(day 2) or 72 hrs (day 3) after initial plating. Cells were harvested 48 hrs after completion 
of the infection and total cellular DNA was prepared for blot analysis to estimate the abundance 
of integrated proviral DNA in the unselected population. Efficiency of gene transfer with the 
LDL receptor virus ranged from a maximum of 1 proviral copy/diploid genome for Z1 to a 
minimum of 0.1 proviral copies/diploid genome for Z2. Infection efficiency was consistently 
highest when the cells were exposed to virus 48 hrs after plating; density of plating did not 
consistently affect the efficiency of gene transfer. The absolute level of gene transfer was less 
with the lower titer BAG virus although the effects of plating density and time of exposure to 
virus on gene transfer were similar. 
Expression of the integrated LDL receptor provirus in human hepatocytes was studied by 
RNA blot analysis which measures the steady state level of recombinant RNAs. The AFP-BA- 
rLDLR vector contains two transcriptional units: transcription from the 5’ LTR produces a 5.6 
kb RNA responsible for passage of the virus, while a transcript initiated at the p-actin 
Recombinant DNA Research, Volume 15 
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