12-16 hour period beginning 48 hours after initial plating. The virus will be removed, 
pooled, and aliquots will be subjected to analyses described in section III.E.3. The hepatocyte 
cultures will be washed extensively with PBS, and released with Trypsin-EDTA as described 
(16). The detached cells will be recovered by centrifugation, pooled and washed extensively in 
isotonic saline. In preparation for transplantation, the cells will be suspended in isotonic 
saline. 
3. Analysis of transduced hepatocytes 
Approximately four hours prior to cell harvest, an extra plate of infected hepatocytes 
will be assayed for recombinant gene expression using the cytochemical assay which measures 
uptake of fluorescent labeled LDL. Fluorescent LDL is added to the cells for 4 hours and removed 
immediately prior to the cell harvest. The monolayer is visualized by fluorescence microscopy 
to identify transduced cells. This provides a minimal estimate of gene transfer. 
An aliquot of the pooled supernatant removed from the hepatocyte cultures will be 
analyzed for gross contamination by fungus or bacteria using the following procedures. A 200 
ml aliquot of this supernatant will be concentrated lOOx by centrifugation. The sediment will 
be gram-stained and visualized under oil immersion microscopy. Absence of detectable gene 
transfer using the in situ LDL receptor assay, recovery of less than 5 x 107 hepatocytes or 
gross contamination in the gram-stained sediment will preclude reinfusion of the hepatocytes. 
A more extensive analysis will be performed on the pooled hepatocyte suspension used 
for reinfusion. The following tests will be performed but the results will not be available until 
after the cells have been infused. 
Test 
Sterility 
Electron Microscopy 
Extended S+L- Focus 
LDLR gene Transduction 
1 Outside Laboratory 
2 Human Applications Laboratory, University of Michigan 
Aliquots of the pooled supernatants removed from the hepatocyte cultures immediately 
prior to harvest will also be analyzed for sterility, electron microscopy, and extended S+L- 
focus by the above Outside Laboratory. 
Performed Bv 
a 
a 
a 
H.A.L. 
F. Delivery of Hepatocytes 
An important issue relates to the number of cells to be infused. Based on the hepatic 
perfusions done to date, it is possible to harvest 1 to 10 x 10 9 cells from a piece of liver that 
one can expect to obtain by removing the left lateral segment of a liver from a 4 yr old 
(approximately 70 gm). Our experience with the WHHL rabbit indicated that 2 x 10 8 cells 
could be safely transplanted into 2 kg animals. In the baboon experiment, 2 x 10 9 cells were 
safely infused into a 17 kg animal. It is reasonable, therefore, to use 1 x 10 8 cells/kg in 
human studies, up to the maximum number harvested. Considerations include the absolute 
volume to be infused, which is usually limited to 25-50 ml. It is also possible that increases in 
the level of LDL receptor expression achieved by transduction would allow a corresponding 
decrease in the number of reinfused cells that are necessary to achieve a therapeutic effect. 
Recombinant DNA Research, Volume 15 
[185] 
