F. Growth of the GITKSVNa Producer cell line for Clinical Use 
The producer cell lines will be grown in either monolayer in flasks or in an 
artificial capillary system (ACS). 
1. The producer cells are maintained in complete medium which contains Dulbecco’s 
modified MEM with 4.5 gm glucose/liter with L-glutamine (DMEM), 10% FCS, 2 mM 
L-glutamine, 100 U/ml penicillin G sodium and 100 mcg/ml streptomycin sulfate. 
2. Monolayer Cultures 
The monolayer culture will be grown in T-175 flasks at 37°C with 5% CO 2 . When 
the cells are >90% confluent, the cells will be trypsinized and split 10:1 in fresh 
complete medium. A confluent T-1 75 flask generally contains 1 -1 .5X1 0 7 cells. 
3. ACS Cultures 
The producer cells grown in five T-175 flasks are trypsinized, washed in complete 
medium and resuspended in 100 ml of media. The cells are then injected into a 
TM 
autoclaved, CELLMAX 100 bioreactor (Cellco Advanced Bioreactors, Inc.; 
Kensington, MD) and the bioreactor is placed in a 37°C, 5% CO 2 incubator. 
Perfusate is pumped continuously through the 8000 hollow fibers to maintain 
proper nutrition and eliminate waste products of cellular metabolism as previously 
described (19). In brief, complete medium is pumped at a flow rate of 10-300 
ml/minute, increasing as the number of cells increase. Glucose measurements will 
taken periodically from the perfusate to aid in assessing the need for media 
change. The 100 ml cartridge can reach near tissue density, containing 
approximately 1 0 1 0 cells (equivalent to the number of cells contained in 1 000 
confluent flasks). We have grown G1 -based vectors in the ACS on multiple 
occasions without evidence of development of aberrant growth or replication- 
competent retrovirus. 
G. Testing and Harvest of the Producer cell lines for Clinical Use 
1 . 48 hours prior to clinical use 
a. Sterility cultures 
b. Endotoxin level 
2. On the day of cell harvest for clinical use: 
a. An aliquot of supernate will sent for a STAT gram stain and culture. 
b. Supernate will be at saved at -70°C for viral titer and tests for replication 
Recombinant DNA Research, Volume 15 
[811] 
