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 5 ml viable cells in 80% 
fresh medium/20% LAK supernatant. For half the cells, the fresh medium is 
AIMV supplemented with penicillin, streptomycin, fungizone, and 6000 IU/ml IL- 
2; for the other half, the fresh medium is RPHI supplemented with 10% human 
serum, penicillin, gentamicin, fungizone, and 6000 IU/ml IL-2 . The cultures 
are placed into 6-well tissue culture dishes and incubated at 37< C in 
humidified incubators with 5% C0 2 . Cultures initiated from lymph nodes will 
be cocultivated with cryopreserved tumor stimulation cells at an initial ratio 
of at least 1:10 tumor cells to lymphocytes (but not more than 1:1) depending 
on the availability of tumor cells. 
Usually the lymphocyte density is not much increased at the end of • 
seven days in culture, and the cultures are collected, centrifuged, and 
resuspended at 5 x 10 s total viable cells/ml in newly prepared 80%/20% medium 
mixtures of the same type. Occasionally a culture will have increased 
lymphocyte density and need medium replenishment prior to seven days. After 
this first passage, lymphocytes are subcultured by dilution when the density 
is between 1.5 x 10 6 and 2.5 x 10 6 cells per ml; densities of subcultures are 
established between 3 x 10 3 and 6 x 10 3 ml. Cultures are kept in 6-well dishes 
when the volume is less than 1 liter, and transferred to 3 liter polyolefin 
bags (Fenwal) when the volume reaches one liter. The subcultures from bags 
are accomplished with Fluid Fill/Weigh Units (Fenwal), which are programmed 
to pump prescribed weights of TIL culture and fresh medium into a new bag. 
[118] 
Recombinant DNA Research, Volume 15 
