fresh medium/20% LAK supernatant. For half the cells, the fresh medium is 
AIMV supplemented with penicillin,, fungizone, and 6000 IU/ml IL-2; for the 
other half, the fresh medium is RPHI supplemented with 10% human serum, 
penicillin, streptomycin, gentamicin, fungizone, and 6000 IU/ml IL-2. The 
cultures are placed into 6-well tissue culture dishes and incubated at 37< 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 s and 6 x 10 5 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. 
If, during the growth of TIL, patients performance status has 
de teriorated to 3 or greater or if they have developed significant card iac , 
renal, pulmonary or hematologic dysfunction than they will be taken of f study 
and will not receive the inf usion of TIL or IL-2. 
When the total lymphocytes for a patient are ready for harvest, 5 x 10 6 
cells are taken for cytological examination. Cytospins are examined for the 
Recombinant DNA Research, Volume 15 
[73] 
