streptomycin (50 /ig/ml) (Flow) and gentamicin (50 ng /ml) (Gibco) Enzymatic 
digestion is conducted at room temperature with stirring for 18 hrs. The tumor 
single cell suspension is washed 5x in Hanks Balanced Salt Solution. 
3.1.2. Establishment of Tumor Cell Line 
An attempt to establish a tumor cell lines in tissue culture for each melanoma 
biopsied . The tumor cell suspension will be placed in RPMI 1640, 10% heated- 
inactivated pooled human AB serum and antibiotics at a concentration of 5X10 5 
viable tumor cells/ml and cultured in a 5% C0 2 incubator. Successfully 
established cell lines will be characterized by histology, immunohistochemistry 
(when indicated), and tested for bacterial, fungal or mycoplasma prior to use. 
In all instances, freshly isolated tumor cells will be cryopreserved as described 
below in 6.1.3. These frozen tumor cells will be needed for skin testing and, if 
sufficient numbers are available, as vaccine immunogen, if a cell line cannot be 
established. 
3.1.3. Cryopreservation of Fresh Autologous Tumor 
The single cell suspension will be cryopreserved at-70°c in 90% heat-inactivated 
autologous serum, 10% DMSO at a final concentration of 10 7 tumor cells/ml. 
Autologous serum will be prepared from the pretreatment blood draw. 
3.2 Human IL-2 - Producing M-24 
M-24 cells transduced with an IL-2 retroviral vector (GINa CV IL-2), selected in G418, 
and cloned for high production will be used as the IL-2 producing cell line to be included 
in the tumor cell vaccine. This replication-defective retroviral vector will be provided 
by Genetic Therapy, Inc., Gaithersburg, MD. This cell line M-24 (IL-2) will be 
maintained in the Cellular and Genetic Therapy Core Laboratory on the 14th floor of the 
Factor Building in the Jonsson Cancer Center. A large master cell bank of sufficient 
numbers of cells to complete this protocol will be prepared and cryopreserved in 90% 
fetal bovine serum and 10% DMSO. This cell bank will pass the following tests: 
a. hLL-2 production by ELISA assay (not less than 10 4 pg/10 6 cells/24 hr) 
b. Absence of aerobic, anaerobic, fungal and mycoplasma contamination 
c. Free of replication competent retrovirus using S+L-assay with 3T3 amplification 
d. Viability >95% 
3.2.1. Preparation of M-24 (IL-2) 
On the day of vaccine preparation, an aliquot of M-24 (IL-2) will be thawed, 
washed 5x in Hank’s Balance Salt Solution and adjusted to the appropriate 
numbers to produce either 10 3 , 10 4 , or 10 5 pg/24 hr IL-2 depending on the 
vaccine treatment group. 
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Recombinant DNA Research, Volume 18 
