HEPATOCELLULAR TRANSPLANTATION AND TARGETING GENETIC MARKERS TO HEPATIC CELLS 
Constituent 
Final Concentration 
COMPONENTS FOR SERUM FREE MEDIA ("SUM + CHOWDHURY") 
Insulin 
Glucagon 
100 ng/ml* 
1 ug/ml 
50 ng/ml 
10 uU/ml 
20 mU/ml 
5 ug/ml 
3 nM 
EGF 
hGF 
Prolactin 
Linoleic acid 
Selenium 
HEPES (pH 7.4) 
10 mM 
lO’M* 
1 uM 
Dexamethasone 
T3 
Trans ferrin 
GHL 
10 ug/ml 
20 ng/ml 
* different from conditions used for rodent cultures . 
We are currently evaluating an alternative culture media described by Lanford et al 
(1989) which has been optimized for primate hepatocyte culture. If this media proves 
superior to the " SUM+CHOUDHURY" in preserving hepatocellular differentiation or 
permitting transduction with vector we will Incorporate the improved media for this 
cultivation. 
For these experiments hepatocytes will be plated at a density of 1.3x10'' cells/ml^ 
(1x10* cells/100 mM plate) and grown at 37° in 5% CO 2 (humidified) incubators. Cell 
morphology will be evaluated by phase contrast microscopy. All cells will be transduced 
with the vector as described below. After three days cells will be washed with a volume 
lOOx greater than the titer of vector used for transduction and harvested by 
tr 3 rpsinization with parvovirus free trypsin. Prior to the infusion of hepatocytes into 
the patient, the cells will be cleansed of media constituents by washing and gentle 
pelletting of cells in isotonic solutions containing 5% human albumin. Sterility is 
maintained throughout the procedure of perfusion and culture. All solutions will be 
endotoxin free. Cells will be deemed suitable for transplantation only if the donor 
organ was free of infectious agents described above and >80% viability by trypan blue 
is demonstrated after trypsinization and washing. 
In this protocol we have chosen to plate all of the cells in tissue culture and to 
expose all of these cells to the recombinant vector. For logistical reasons it will be 
virtually impossible to coordinate the harvest of hepatocytes and subsequent 
transplantation without some means of preserving hepatocytes. Hepatocyte viability 
deteriorates rapidly with >70% loss of viability in non-attached cells at reduced, 
ambient, or physiological temperatures. Cryopreservation of hepatocytes remains a sub- 
optimal procedure with recovery of viable cells often in the range of 50%. It has been 
observed that cultivation of hepatocytes prior to cryopreservation often enhances the 
efficiency of recovery, either by providing cells with the opportunity to recover from 
the relative hypoxia of the preparative process, or by selection of viable cells. We 
have also found high viability of cells (>70%) after trypsinization from culture 
[806] 
Recombinant DNA Research, Volume 14 
