5. 4.1. 2 Cell Separation and Enrichment of CD4 and CD8 T Cell 
Subpopulations 
Peripheral blood mononuclear cells (PBMC) from the leukapheresis 
collections will be separated from the red cells and neutrophils by 
Ficoll-Hypaque density gradient centrifugation according to previously 
published techniques (39-43). The PBMC will then be washed and 
counted. PBMC will then be fractionated into populations enriched for 
CD4 or CD8 expressing cells. CD4 enrichment will be accomplished 
by positive selection and/or CD8 depletion. The resulting 
subpopulations will be referred to as “CD4 enriched cells." Similarly, 
CD8 enrichment may be achieved by positive selection and/or CD4 
depletion steps, and the resulting subpopulations will be termed “CD8 
enriched cells." Appendix 1 shows the current fractionation protocol 
used in the ADA deficient SCID protocol using the AIS 
Micro CELLectorTM system. Enrichment will be performed prior to 
transduction of the cellular fractions. The conditions for enrichment of 
cells may be modified by the PI during the course of the protocol in 
order to take advantage of refinements in techniques. 
5.4.2 Growth and transduction of T lymphocytes 
Fractionated cells will be cultured at approximately 5 x 105 cells/well in 
24 well tissue culture plates in AIM-V*, which consists of AIM-V 
(GIBCO) with 2mM glutamine, 50U/ml penicillin, 50 pg/ml streptomycin, 
2.5 pg/ml Fungizone, and 25-1000 units/ml of IL2 (Cetus). Media may 
be supplemented with 5% fetal calf serum or up to 10% human serum 
or plasma obtained either from the donor or from blood bank supplies. 
At the initial plating, 10 ng/ml OKT3 (Ortho) monoclonal antibody will 
be added to each well. The cells will be cultured at 37°C in a 
humidified incubator with 5% CO 2 . The conditions of culture and 
lymphocyte stimulation may be modified by the PI during the course of 
this protocol to take advantage of improvements in technique- or media. 
Once the T lymphocytes have begun to proliferate (usually 24-96 hours 
after initiation of the culture), vector-containing supernatant (containing 
protamine 5-10 pg/ml and up to 1000 U/ml IL2) will be added to the 
wells after aspirating off the top half of the medium. This will be 
repeated 1 to 2 times daily for a period of up to 7 days. After the final 
exposure to retroviral vector, the cells will be fed with fresh AIM-V* and 
cultured another 2 to 7 days to permit the cultures to return to 
exponential growth. Approximately 80% of the culture will then be 
infused into the patient and the remaining cells returned to culture for 
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