TIL cultures are Initiated at 5x10^ ml viable cells (tumor plus lympho- 
cytes) in 80% fresh medium/ 20% LAK supernatant. For half the cells, the fresh 
medium is AIMV supplemented with penicillin, fungizone, and 6000 lU/ml IL-2;‘for 
the other half, the fresh medium is RPMI supplemented with 10% human serum, 
penicillin, gentamicin, fungizone, and 6000 lU/ml IL-2. The cultures are placed 
into 6-well tissue culture dishes and incubated at 37° in humidified incubators 
with 5% CO 2 . 
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 5x10^ 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, 
TILs are subcultured by dilution when the density is between 1.5x10^ and 
2.5x10^ cells per ml; densities of subcultures are established between 3x10^ and 
6x10^ 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. When subculture volumes exceed 
3 liters, the fresh medium used is AIMV. Cultures growing in serum-containing 
medium are thus diluted into AIMV, and no further LAK supernatant is added to 
cultures growing in serum-containing or serum-free medium. 
Tumor-infiltrating lymphocytes will be transduced when the total number 
of lymphocytes is about 1-5x10^ or higher. Up to one half of the TIL culture 
is centrifuged, the medium is saved, and the cells are resuspended in the viral 
supernatant with 5 ug/ml protamine (27). Multiplicities of infection are about 
2 to 8. The cells in viral supernatant are placed into 800 ml tissue culture 
flasks at 200 ml/flask and incubated at 37° for 2 hours. During incubation. 
Recombinant DNA Research, Volume 14 
