b. LCM virus 
c. Thymic agent 
d. S+/L- assay for ecotropic virus 
e. S+/L- for xenotropic virus 
f. S+/L- for amphotropic virus 
g. 3T3 amplification 
6) Electron microscopy will be performed to assure the absence of 
adventitious agents. 
The retroviral supernatant will not be used to transduce tumor 
cells injected into patients until approval is received from the Food and Drug 
Administration . 
3. Preparation of gene-modified tumor cells . Tumor lines will be 
established in tissue culture from tumor fragments or single cell suspensions 
using standard tissue culture techniques (38). Tumor and normal tissue will 
be obtained immediately after surgery and processed as follows. The tumors 
we re minced into 1 mml fragments and dissociated with agitation in serum free 
DMEM (Dulbecco Modified Eagle Medium) (Bio fluids) containing 2mM glutamine, 0.1 
mg/ml hyaluronidase, 0.02 mg/ml Dnase I and 0.1 mg/ml collagenase for 3 hours 
at room temperature. The cell suspension was then centrifuged at 800 g for 5 
min utes and the pellet resuspended in a culture medium consisting of 5rol o f 
DMEM high glucose (4.5g/l) with penicillin and glutamine supplemented with 10% 
fetal calf serum. The cells were either centrifuged prior to being frozen in 
90% FCS , 10% DMSO at - 80° C, or plated in appropriate dishes or culture f lasks 
in culture medium. Plated cells were incubated at 37° C in a humidifie d 
atmosphere of 5% CO z and 95% air. Wi thin 48 hrs, the culture medium wa s 
chan ged in order to remove all non-attached material. Subsequently, cultures 
were incubated for a perio d of 6 to 8 days without medium change . The tumors 
grow as adherent monolayers in tissue culture flasks (Falcon #3028; 175 cm 2 ; 
750 ml) containing about 50 ml of medium. When the cells are actively growing 
and not yet confluent the medium will be poured off and 30-50 ml of medium 
containing the retroviral supernatant with 5 ug/ml protamine will be added to 
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Recombinant DNA Research, Volume 15 
