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 transduction is such that subculturing is necessary, 
the cells are diluted to 5xl0^/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 con- 
taining G418 and then cultured as described above. 
When the total TILs for a patient are ready for harvest, 5x10^ 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 characteri- 
zation of cell surface markers and for assessment of cytotoxicity using 
techniques identical to that in our previous protocol (86-C-183c; reference 23, 
attached). Briefly, TILs are stained with fluorescent-labeled antibodies 
(Leu2, Leu3, Leu4, Leu7, Leull, Leul5, Leul9, LeuM3, HLADR, and Tac). Chromium 
release assays are performed with K562, Daudi, autologous tumor, and allogeneic 
tumor targets. 
When the total cell number reaches at least 4 x lO^^ cells the TILs are 
collected in two or more batches by continuous flow centrifugation. The TIL in 
this protocol will then be cryopreserved in 10^*^ cell aliquots. 
Recombinant DNA Research, Volume 14 
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