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 
dissection board in a laminar flow hood. A small representative portion is 
taken for pathologic analysis, and the rest is minced into pieces roughly 4 mm 
in diameter. These are placed into an enzyme solution of collagenase, DNAse 
type I, and hyaluronidase type V as previously described for overnight 
digestion at room temperature. The resulting suspension is filtered through a 
wire mesh to remove any large debris, washed in saline, and placed on Ficoll- 
Hypaque gradients. The interface containing viable lymphocytes is collected 
and washed in saline, and a portion is frozen for subsequent use as targets in 
cytotoxicity assays. 
Lymphocyte cultures are initiated at 5 x 10 3 ml viable cells in 80% 
[72] 
Recombinant DNA Research, Volume 15 
