313. Cell Type Specific Media Optimization for Clinical Retroviral Transduction Protocols.

In order to optimize the transduction of CD34+ cells, CD34+ derived Langerhans cells (LCs) and T lymphocytes for upcoming clinical trials, we determined which medium is best suited for each cell type. Our aim is to identify the formulation providing the best cell proliferation, survival and the highest transduction efficiency (TE) while maintaining the biological activity of the cells. To this end, we harvested and screened PG13-derived retroviral vector stocks (VS) collected in various media formulations. High-titer VS were consistently obtained by controlling PG13 cell-plating densities, time of cell growth and vector harvests. We also periodically determined the titer of the frozen VS to determine how long these VS can be stored for clinical applications.CD34+ cells and CD34+-derived LCs. We investigated the vector titers and ex vivo transduction efficiencies obtained with VS collected in six serum-free media formulations (IMDM,QBSF 60, XVivo 10, XVivo 15, DMEM low and high glucose) and their serum containing counterparts. In all experiments, CD34+ cells were pre-stimulated with rhMDGF, rhSCF and rhFLT-3. PG13-derived VS collected in either DMEM or XVivo 10 gave the best results and the transgene expression averaged 45.6±6.03% and 55.3±6.6%, respectively in the total cell population. However, the percentage of cells co- expressing both CD34 and eGFP were significantly lower when the transductions were performed in DMEM (14.3±4.6%) as compared to XVivo 10 (21.8±4%). This data led us to select XVivo10 for pre-stimulating CD34+ cells and manufacturing VS for the transduction of CD34+ cells and CD34+-derived LCs. We have shown that upon freezing these VS are stable for at least 12 months.T lymphocytes. We investigated the vector titers and TE obtained with three media formulations (XVivo10, RPMI and DMEM) in presence or absence of 10% serum. PBMCs were activated with PHA and 20U IL-2 in RPMI+FBS. PG13-derived VS collected in either DMEM+FBS, RPMI+FBS gave better TE (55-56% range) than VS collected in either DMEM, RPMI or XVivo10+FBS (44-49% range) or than VS collected in X-Vivo 10 alone (26% range). Subsequently, we found that at least 5% of serum (FBS or human AB) was required for optimal proliferation and transduction of T lymphocytes and that activation of the PBMCs in XVivo15+5% AB serum gave consistently higher TE (45% ± 8% NIT+) than RPMI (37% ± 4% NIT+) or Yssel’s(33% ± 3% NIT+) using VS collected in DMEM+10% FBS. Finally, we compared the TE in PBMCs using frozen VS collected in either DMEM alone or supplemented with 5%HSA or DMEM+FBS. We observe that some of the VS generated in DMEM+FBS can be stored for up to 41 months with no apparent decrease in TE on T lymphocytes while other VS stored for 24 months show a 3 fold decrease of TE on T lymphocytes. These results will be discussed in the context of various VS coming from PG13 packaging cell clones carrying different transgenes and of various collection media.In conclusion, the collection of VS can be optimized with various media but the optimum medium varies for each cell type.Molecular Therapy (2006) 13, S119–S120; doi: 10.1016/j.ymthe.2006.08.369 [ABSTRACT FROM AUTHOR]