lateral segment of the liver will be divided. Ligamentous attachments to the left lateral segment 
of the liver will be taken down and mattress-type sutures will be placed in the parenchyma to 
occlude the vascular supply. The liver parenchyma will be transected along the line of 
demarcation of blood supply with a scalpel. Individual blood vessels and bile ducts will be suture 
ligated. The specimen will be immediately transferred to the Human Applications Laboratory for 
hepatocyte isolation. To alleviate the need for a second laparotomy, the left gastric (coronary) 
vein will be identified and isolated. A Broviac catheter will be inserted and secured into the 
coronary vein. The catheter will be tunnelled through the abdominal wall and secured to the skin 
with monofilament absorbable suture at a site distant to the laparotomy incision. After 
hemostasis is achieved, the abdominal wall will be closed in two layers with absorbable sutures. 
E. Isolation of Hepatocytes 
1. Hepatocyte Isolation and Primary Cultures. 
The resected liver will be immediately transported on ice to the Human Applications 
Laboratory. Major vessels will be cannulated with 16 gauge angiocatheters and perfused with 
IX Leffert's solution (102), pH 7.4, containing 0.5 mM EGTA for 10 minutes. This will be 
followed by perfusion with IX Leffert's solution, pH 7.4, without EGTA for 5 minutes, and then 
IX Leffert's, pH 7.4, 5 mM CaCl2, 5 mg/ml BSA (fraction V), 0.5 mg/ml collagenase D 
(Boehringer Mannheim) for 18 to 24 minutes. The collagenase perfusion will be terminated 
when the liver softens and begins to dissociate. All solutions will be filter sterilized prior to 
use and oxygenated at 37° during the perfusions. Flow rates will vary from 100 to 125 
ml/min. The perfused liver will be teased apart with a rubber policeman and forceps, and 
filtered through a presterilized 85 mm nylon mesh at 4° into a sterile flask containing RPMI 
1640 medium containing 10% fetal bovine serum and 1% penicillin/streptomycin (Medium 
A). The filtered hepatocytes will be pelleted by centrifugation three times at 50 x g for 1 min, 
and resuspended in medium A, and viable cells will be quantified by exclusion of trypan blue. 
Hepatocytes will be plated at 7 x 10^ cells per cm 2 overnight in hormonally defined 
medium (HDM, Ref. 103) containing 10% fetal calf serum and 1% penicillin/streptomycin. 
The following day the media will be replaced with fresh HDM without serum. The preparation of 
hepatocyte cultures will be performed in a laminar flow hood. The specific components of HDM 
are listed under Appendix J. 
There are three possible supports onto which the cells can be plated. The approach we 
have the most experience with involves plating 4 x 10 9 cells onto 10 cm Primaria plates (16, 
17, 19, 93). The expected isolation of hepatocytes from the resected liver is 2 x 10 9 cells. 
This will require 500 10 cm plates, a number we have easily managed in our WHHL and baboon 
experiments. An alternative approach we are actively exploring is to grow the cells in roller 
bottles or bioreactors. 
All procedures involving the isolation, cultivation and transduction of the hepatocytes 
will be performed in a dedicated laboratory located in the Clinical Research Center at the 
University of Michigan. This facility, called the Human Applications Laboratory, is currently 
being renovated to become a GLP/BL2+ facility. 
2. Retroviral transduction procedure of primary FH hepatocytes 
FDA certified, frozen supernatant will be thawed in preparation for hepatocyte 
transduction. The thawed virus will be supplemented with polybrene ( 8 ug/ml ) and placed on 
the primary cultures of hepatocytes. Virus will be exposed to the cultured hepatocytes for a 
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