harvested from the culture flasks by exposure to 0.25% versene (EDTA) for 10 
minutes. The cells will be washed three times by suspension in 50 mis normal 
saline and centrifugation. The final cell pellet will be suspended in normal 
saline and counted. 2 x 10® viable cells in 1 ml normal saline will be 
injected subcutaneously just beneath the skin in the anterior mid thigh and 
the overlying skin marked with a tatoo dot. If 2 x 10® cells are not 
available, fewer may be given but not less than 2 x 10 7 cells will be 
injected. About 3 cm lateral or vertical to this injection the patient will 
receive two intradermal injections (separated by 1 cm) of 2 x 10 7 gene 
modified tumor cells in 0.1 ml normal saline and these sites also marked by a 
tatoo dot. These sites will be monitored weekly by a physician. At three 
weeks the patient will undergo excisional biopsy of superficial inguinal lymph 
nodes (without formal dissection) in the area draining the inoculation site 
for growth of lymphocytes. If tumor grows at any of these sites they will be 
excised when they reach 1 to 2 cm for growth of TIL. If no tumor growth is 
evident then the sites of tumor injection will be excisionally biopsied at 8 
weeks after injection for pathologic analysis. 
6. Growth of lymphocytes: The procedures used here are the same as 
those used in our previous protocols (40) involving the infusion of TIL (86-C- 
183) and is taken virtually verbatim from that protocol. 
At least two days prior to surgery, peripheral blood lymphocytes are 
collected by leukapheresis for four hours. These are Ficoll-Hypaque separated 
and the mononuclear cells collected from the interface, washed in saline, and 
placed in culture in roller bottles at 10 6 cells/ml. Half are placed into 
AIMV (a serum free medium, Gibco Laboratories) with 6000 IU/ml IL-2 (Cetus), 
and half are placed into RPMI supplemented with 2% type-compatible human 
serum, penicillin (unless the patient is allergic), gentamicin, and 6000 IU/ml 
IL-2. After 3 to 4 days cells are centrifuged and the supernatants are 
collected and filtered. These are referred to as LAK supernatants. 
Immediately upon resection of tumor or lymph nodes, the specimen(s) is 
transported to the laboratory in a sterile container and placed on a sterile 
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