HEPATOCELLULAR TRANSPLANTATION AND TARGETING GENETIC MARKERS TO HEPATIC CELLS 
Hepatocytes are obtained through perfusion of the liver with collagenase solutions 
essentially as described (Berry and Friend, 1969). The liver is flushed with isotonic 
saline containing divalent cation chelators (EGTA) , followed by perfusion with 
collagenase solution. If perfusion is not possible, as in samples obtained from reduced 
liver transplants which do not retain major vessels, perfusion is performed through all 
visible vessels on the cut surface of the liver using a 20 gauge catheter. 
Table 4. EGTA solution 
Earle's Balanced Salt Solution (GIBCO) . 
Without calcium and magnesivun. 
With 0.5 mM EGTA. 
Table 5. Collagenase solution 
Earle's balanced salt solution (GIBCO). 
With calcium and magnesium. 
180 mg/400 mis collagenase (Boehringer Mannheim, collagenase H # 1074 024).’ 
18 mg/400 mis soybean trypsin inhibitor (SIGMA) . 
* Tested batchwise for ability to harvest viable mouse hepatocytes. 
The perfusion requires approximately 15-20 minutes after which time the hepatocytes 
can be dispersed into small cell clumps and single cells. Hepatocytes are placed on 
tissue culture plastic (Primaria, Falcon Plastics Company) in the presence of fetal calf 
serum to aid in attachment. In our most recent preparation of human hepatocytes from 
a right liver lobe made available to us preserved in Belzer's solution 24 hours after 
a reduced liver transplant at the Southwestern Medical School, Dallas, we obtained 7.2 
X 10 * cells from approximately 70 grams of liver. These cells exhibited 70-90% trypan 
blue exclusion. After six hours, the medium is removed and replaced with a nutrient 
solution deficient in tyrosine which contains no serum additives. The absence of 
tyrosine selects for hepatocytes which uniquely express the enzyme phenylalanine 
hydroxylase and can synthesize their own tyrosine from phenylalanine. Hormonally 
defined media selects for growth of hepatocytes over non-hepatocytes (which grow better 
in the presence of serum) and contributes to maintaining the differentiated state of 
hepatocytes in culture (Jefferson et al, 1984). The culture media is composed of: 
Table 6, Composition of tissue culture media. 
Constituent Final Concentration 
BASAL MEDIA 
Minimal Essential Media (MEM, Hazelton, w/o tyrosine 75% 
Weymouths Media (Hazelton, w/o tyrosine) 25% 
Recombinant DNA Research, Volume 14 
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