i . Evaluation for toxicity will take place over the first 48 hours in the 
Clinical Research Center and three times per week for the first week, 
then weekly for three weeks. After the second injection, the patient 
will be again monitored for 48 hours in the Clinical Research Center. 
Followup will then be weekly for three weeks, every two weeks for 
four months, and monthly thereafter. 
- Acquisition of Tissue, Tissue Culture and Transfection 
Techniques 
Acquisition of Human Tissue and Quality Control of Culture 
Procedures All human tissue will be obtained in the operating room using standard surgical 
techniques. Tissue will be transported to a dedicated tissue culture facility in closed, sterile containers. 
All work with tissue and cells will be done in Category II environmental hoods. Laboratory personnel 
will wear dedicated laboratory apparel for work with human tissues. All buffers, tissue and culture 
materials, materials for transfection and for injection into the recipient host will be FDA approved 
pharmaceutical grade. Quality of cell culture procedures will be monitored as follows: 
1. Serum additives will be determined according to specification of the 
manufacturer and validated by two members of the gene therapy team 
prior to use. 
2. The patient's history of hypersensitivities will be coordinated for 
possible cross reactivity with antibiotics or other products routinely 
used in preparation of the transfected cells. The final preparation of 
cells will be labelled appropriately concerning antibiotics used during 
selection of transfected cells. 
3. During the in vitro handling of patient's cells, preparations will be 
routinely cultured for bacteria, fungus, and virus. 
4. Samples from each patient will be verified by both the surgeon and 
tissue culture technician, and logged into appropriate records as 
received. Each sample will be color-coded in the log book, and all tissue 
culture materials used in conjuction with the patient's sample will be 
similarly color-coded. Prior to innoculation into the patient, thelog 
record and color coded data will be reviewed and verifed by two 
independent team members. 
Human Glioma Primary Cell Culture Protocol Primary cultures are 
derived according to the techniques as described by Manfred and Westphal with some 
modifications(46). At surgery, tissue for culture is placed in serum-free Dulbecco's Modified Eagle's 
Medium (DMEM) at room temperature and transported to the culture facility. All cell culture is 
performed in a Category 2 Environmental Laminar Flow Biologic Safety Cabinet. Between 0.5-1 g of 
tissue is placed in a 100 mm dish and minced finely with sterile iridectomy scissors in calcium and 
magnesium-free phosphate buffered saline. This medium is replaced with 10 ml of the same medium to 
which had been added 0.05% Pronase (Calbiochem), 0.03% collagenase (Worthington ClsII), and 0.01% 
Worthington DPFF DNase I. The dish is placed on an orbital shaker at 37° for 20 minutes. The 
suspension is then dispersed with a sterile Pasteur pipette, then in a sterile 10 ml serological pipette. 
At this point the cell number is estimated in a hemocytometer. T ’r.e cell suspension is spun down in a 
table top centrifuge at approximately 250 g for 4 minutes. The supernatant is decanted, the cells are 
resuspended in DMEM containing 10% fetal bovine serum and distributed to 60 mm Nunc tissue culture 
plates (some coated with fibronectin) at a cell density of about 2X 106 cells /dish. The cells are allowed 
to attach for 24 hours before medium change. After attachment and cell division are verified but 
Recombinant DNA Research, Volume 18 M351 
