We previously demonstrated a laboratory model of ex vivo expansion of mobilized peripheral blood mononuclear cells (PBMNC) that acquire the CD8+CD56+ NKT cell phenotype and aggressively destroy targets through the NKG2D receptor (

Cytotherapy.
2006
;
8
:
141
–8
). We developed a large scale culture method for clinical use that allows growth, expansion and cryopreservation of these expanded cells for infusion post-transplant. PBMNC were collected by large volume leukapheresis from healthy donors (n=3). Unselected MNC cells (2.5 × 106/ml) were cultured in Life Cell Bags (Nexell Therapeutics) with serum-free AIM V medium (Invitrogen), IL-2 (Chiron, 1000 IU/ml)and OKT3 (Ortho Biotech, 500 ng/ml) at 37° C and 5% CO2. Six bags were arranged from each collection. Fresh cytokines and medium were added on Days 3 and 5, and the cells were harvested on Day 7. The viability of the ex vivo expanded cell populations was 95% +/− 1.8%. Total MNC cell numbers expanded from 2.5 × 106/ml to 6.4 × 106/ml (2.5 +/− 0.5% fold increase). Phenotypic analyses showed CD3 cells increased from 55% (Day 0) to 94% (Day 7; +/−1.42%); the CD4 cells increased from 30% to 58% (+/− 1.48%); the CD8 cells increased from 11% to 48% (+/−3.98%); the CD56 cells increased from 17.2% to 47.8% (+/− 6.2). Cytotoxicity of Day 7 expanded cells was markedly increased at 74% (+/−7.1%) (Targets: K562 cells; E:T ratio 100:1) compared with 33% (+/− 5.4%) at baseline. We also tested if the ex vivo expanded cells could be cryopreserved for future clinical use. After 7 days in liquid nitrogen, cells were promptly thawed, washed and analyzed. The viability was 91% (+/− 2.1%) and the cytotoxicity against K562 cells was only slightly decreased (65%, +/− 5.4%), when compared to non-cryopreserved expanded cells (p = 0.18). The phenotype was not affected by cryopreservation and thawing. This large scale culture method results in growth and expansion of aggressive effector cells that are cytotoxic against tumor cells. The majority of the expanded cells are CD3+ T cells (equally distributed between CD4+ and CD8+ T cells). NK cells (CD56+) also readily expanded. After cryopreservation of the ex vivo expanded cells and a short-term storage, viability and phenotype of the cells did not change. There is minimal decrease in cytotoxicity that is not statistically significant. This large scale expansion model will be used in clinical trials at our institution to expand cells for adoptive cellular therapy following autologous PBSC transplantation in patients with a hematologic malignancy.

Topics:
cryopreservation, immunotherapy, adoptive, t-lymphocytes, transplantation, cytotoxicity, aldesleukin, carbon dioxide, cd56 antigens, cytokine, hematologic neoplasms

Disclosure: No relevant conflicts of interest to declare.

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2006, The American Society of Hematology
2006
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