Further, thawed NK cells did not expand in patients.5 Therefore, we explored the possibility of shipping NK cells formulated for infusion at 1107 cells mL-1 in infusion buffer Buminate 5% (Baxter, Deerfield, IL) at room temperature and at 4C15C (on ?20C frozen insulated ice packs). identified as CD56+CD3- cells in gated populations. TABLE 1 NK cell content in apheresis products with IL-2 for infusion.24,25 Because it is an allogeneic cell line with infinite division potential, these cells must be irradiated before infusion for safety. We have also adapted NK-92 culture to the G-Rex and can achieve up to 200-fold expansion of the cells with minimum manipulation in less than 4 weeks of culture. III. SHIPPING NK CELL PRODUCTS Remote manufacturing of cell therapy products in centralized facilities is likely to be the norm in the future. As a PACT manufacturing facility, we have shipped NK cell products to the clinical site located in another continuing state, and we have had to optimize conditions for overnight cell shipment. We first explored the possibility of shipping expanded NK cells cryopreserved in 10% DMSO, 40% HBSS and 40% human serum albumin solution (containing 25% HSA). Notably, greater than 70% of the cells were viable within several hours of thaw, they were not cytotoxic. While their potency could be regained after overnight culture in IL-2, their viability dropped significantly (Fig. 5). Further, thawed NK cells did not expand in patients.5 Therefore, we explored the possibility of shipping NK cells formulated for infusion at 1107 cells mL-1 in infusion buffer Buminate 5% (Baxter, Deerfield, IL) at room temperature and at 4C15C (on ?20C frozen insulated ice Roburic acid packs). Shipping formulated cells allows infusion upon arrival at the clinical site. In our trial, the clinical site counts the cells, removes cells in excess of Roburic acid the infusion dose, and performs additional sterility testing. We found that expanded NK cells shipped both at RT and on ice in Fenwal transfer packs retained greater than 90% viability and potency within 48 hours after formulation without cell loss (Fig. 5 and Table 3). We finally chose to ship the cells on ice packs because the temperature was more stable (temperature fluctuated from 8 to 11C) during shipping on ice packs compared to room temperature conditions, which fluctuated from 5 to 21C. Open in a separate window FIG. 5 Characterization of fresh and cryo-preserved expanded NK cell products. (A, B) Expanded and cryopreserved NK cells stay viable within hours post thaw; however, killing K562 cells do not. Cryopreserved cells regain their potency after overnight incubation, however, lose their viability. Freshly formulated NK cells (N=3) remain potent after shipment at ambient temperatures or on frozen ice packs (C). TABLE 3 Viability of NK cells (N=3) 48 hours post-formulation at 1107 cell mL-1 in 5% human serum albumin (HSA) expansion would allow lower infusion cell doses, and the choice of supplementary cytokines requires more explanation. Studies are necessary to identify the best way to cryopreserve the NK cells ARFIP2 so that sufficient cells can be grown at one time to allow multiple infusions. This would facilitate third-party applications also. Future clinical studies with NK cells will include genetic modifications to enhance and expand specificity and function likely, while combinatorial treatments with T cells and small molecules should potentiate their function and take advantage of their ability to recruit and activate the adaptive immune response. All scholarly studies should include detailed patient immune monitoring post infusion to measure expansion, cytokine secretion Roburic acid and to identify epitope-spreading that could be a marker of activation of adaptive responses. Acknowledgments We are thankful to all members of the cell processing and vector production facilities and the flow cytometry laboratory of Center for Cell and Gene Therapy for their help with this work. This work was supported in part by NIH-NHLBI- N01 HB37163, NHLBI-1U54 “term_text” :”HL081007″ NIH-NCI and }HL081007,} NIH-NCI PO1 CA55819, and NIHNCI RO1 {“type”:”entrez-nucleotide”,”attrs”:{“text”:”CA134522″,”term_id”:”35021619″,”term_text”:”CA134522″}}CA134522. ABBREVIATIONS AMLacute myeloid leukemiaB-ALLacute B-cell lymphoblastic leukemiaC-ALLcommon acute lymphoblastoid leukemiaH-SCThematopoietic stem cell transplantILinterleukinN/Anot applicableNK cellnatural killerPBMCperipheral blood mononuclear cellsSCGMstem-cell growth medium.