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^ 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 1.5 x 10^ and 2.5 x 10° 
cells per ml; densities of subcultures are established between 3 x 10° and 6 x 
10° ml. Cultures are kept in 6 -well dishes when the volume is less than 1 liter, 
and transferred to 3 liter polyolefin bags (Fenwal) or into hollow fiber 
cartridges (30a) as previously described when the volume reaches one liter. The 
subcultures from bags may be 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- inf iltrating lymphocytes will be transduced no later than when the 
total number of lymphocytes is about 1-5 x 10®. Up to one -half of the TIL 
culture is centrifuged, the medium is saved, and the cells are resuspended in the 
viral supernatant with 5ug/ml protamine (31) . Multiplicities of infection are 
at least 2. and up to 20. 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, the flasks are agitated every 15 minutes to resuspend the cells. The 
original medium is centrifuged to remove any remaining cells and decanted into 
new containers. At the end of 2 hours, the cells are centrifuged and resuspended 
in the original cleared medium. If the density is such that subculturing is 
necessary, the cells are diluted slightly to a density of about 10° ml and placed 
into fresh 6 -well tissue dishes for continued incubation. The following day, the 
above transduction procedure is repeated. If the cell density at the conclusion 
of this second tr^sduction is such that subculturing is necessary, the cells are 
diluted to 5 X 10°/ml for continued incubation. 
When TIL are to be selected in G418, the TIL are cultured for 3 to 5 days 
after the second transduction and then G418 is added directly to the culture bags 
to a final concentration of 300 ug/ml G418. After 10 to 12 days the cells are 
washed and resuspended at 3 to 6 x 10° cells/ml in fresh medium not containing 
G418 and then cultured as described above. 
After each split of the TILs (approximately once a week) cultures are sent 
to microbiology to detect bacterial organisms. Mycoplasma testing will be 
performed one week before infusion by a commercial testing service. When the 
total TILs for a patient are ready for harvest, 5 x 10° cells are taken for 
cytological examination. Cytospins are examined for the presence of remaining 
tumor. At least 200 cells are studied and therapy proceeds only when no tumor 
cells are found. Other TIL samples are taken for characterization of cell 
surface markers and for assessment of cytotoxicity using techniques identical to 
that in our previous protocol. Briefly, TILs are stained with fluorescent- 
labeled antibodies (Leu2, Leu3 , Leu4, Leu7, Leull, Leul5, Leul9 , LeuM3 , HLA-DR, 
and Tac) . Chromium release assays are performed with K562, Daudi , autologous 
tumor, and allogeneic tumor targets. 
At the time of cell collection, one liter of saline for injection is 
pumped through the collection chamber and the centrifuge is stopped. TILs are 
resuspended in the collection bag, the centrifuge is started again, and another 
liter of saline is pumped through to fully wash the TILs free of tissue culture 
medium components. The cells are then filtered through a platelet administration 
set into 600 ml transfer packs (Fenwal), and 50 ml of 25% albumin and 450,000 lU 
of IL-2 are added to the 200 to 300 ml volume of cells in saline. The TIL are 
infused over 30 to 60 minutes through a central venous catheter. 
Recombinant DNA Research, Volume 14 
[841] 
