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 
flasks in culture medium. Plated cells were incubated at 37 °C in a humidified 
atmosphere of 5% C0 2 and 95% air. With 48 hrs, the culture medium was changed 
in order to remove all non-attached material. Subsequently, cultures were 
incubated for a period 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 
the flask (39). The flasks will be incubated at 37®C for six hours at which 
time the medium will be changed. This procedure will be repeated up to three 
times. After 24 to 48 hours medium containing 300 ug/ml G418 will be added 
directly to the flask and the cells will be grown and subcultured for 7 to 14 
days in G418 containing medium. The G418 concentration may be raised to 1 
mg/ml depending on the health of the culture. 
4. Tests on the transduced tumor population. Following transduction, 
growth and selection of the tumor populations the following tests will be 
performed on the tumor prior to injection into patients. 
1) Cell viability will be greater than 70% as tested by trypan 
dye exclusion. 
2) Sterility will be assured by testing for aerobic and 
anaerobic bacteria, fungus and mycoplasma. 
3) S+/L- assay must be negative. 
4) Southern blot or PCR analysis will be run on the transduced 
tumor to assure that proviral sequences are present. 
5) IL-2 protein assay to assure the production of IL-2 . Cells 
must be producing at least 100 pg IL-2/10 6 cells/24 hours. 
The S+/L- assay, the 3T3 amplification assay and the polymerase chain 
reaction assay are detailed in Appendix A. 
5. Injection of tumor cells . Gene-modified tumor cells will be 
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