IL-2 gene. 
3) IL-2 production by the producer line will be measured and 
should be significantly above baseline control values. IL-2 will be assayed 
using standard biologic assays on the CTLL sensitive cell line (15) or by 
ELISA assay (R&D Systems, Minneapolis, MN) . 
4) Sterility of the producer line and the supernatant will be 
assured by testing for aerobic and anaerobic bacteria, fungus and for 
mycoplasma. 
■'•5) Viral testing will be performed including: 
a. 
MAP test 
b. 
LCM virus 
c. 
Thymic agent 
d. 
S+/L- assay for ecotropic virus 
e. 
S+/L- for xenotropic 
virus 
■e 
X. • 
S+/L- for amphotropic 
virus 
g* 
3T3 simplification 
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 ass follows. The tumors 
were minced into 1 mm 3 fragments and dissociated with agitation in serum free 
DMEM (Dulbecco Modified Eagle Medium) (Biofluids) containing 2mM glutamine, 
01.1 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 q 
for 5 minutes and the pelle t resuspended in a culture medium consisting of 5ml 
of DMEM high glucose (4.5g/l) with penicillin and glutamine supplemented with.- 
Recombinant DNA Research, Volume 15 
[115] 
