A novel closed system utilizing styrene copolymer bead adherence for the production of human dendritic cells

The goal of this study was to refine dendritic cell (DC) culture methods for use in immunotherapy in cancer patients. Because of the low frequency of DCs in the peripheral blood, ex vivo expansion and maturation of DC precursors are required for clinical application.1 To date, most DC culture systems are initiated from the adherent fraction of PBMNCs, selected by the use of open polystyrene flasks, followed by washing and culture in serum-free medium containing GM–CSF and IL-4 or IL-7 (as well as other maturational cytokines). 2,3 The open system is labor-intensive and poses an increased risk of microbial contamination. We developed a closed system for culturing populations of monocyte-enriched PBMNCs4 using flexible, gaspermeable cell culture bags and sterile connecting devices. Apheresis MNCs were obtained from G–CSF-mobilized donors, with their informed consent. All materials and reagents used for the apheresis and DC culture were sterile and/or endotoxin-free and approved by the FDA for human use, with the exception of the plastic beads and IL-4. The principal requirement of MNCs suitable for DC culture was a maximum number of monocytes and monocyte precursors with a minimum number of RBCs, lymphocytes, and platelets. This was attained by the use of an apheresis system (Spectra, COBE BCT, Lakewood, CO) with the MNC program. This collection scheme utilized a separation fraction of 250, equivalent to a velocity of 635 rpm at an inlet flow of 50 mL per minute. Donors underwent a 10-L apheresis. In our initial experiments, we found that the yield of DCs per unit of culture surface area in closed gas-permeable cell culture bags was less than that in the open-flask systems. In an effort to improve the yields of DCs in the closed system, we introduced styrene copolymer beads (90to 125-μ diameter; density, 1.05 g/cm3; SoloHill Engineering, Ann Arbor, MI) into the bags to increase the available surface area (by 380 cm2) and to supply a surface area similar to that found in the flasks.5 In a biologic safety cabinet, 1 g of gamma-radiation-sterilized beads and 10 × 108 total cells per bag from the MNC product were diluted in 100 mL of medium (AIMV, GIBCO, Grand Island, New York) and loaded into gas-permeable cell culture bags (Lifecell X-fold Cell Culture Container PL2417, 180 cm2, Nexell Therapeutics, Irvine, CA). The tissue culture bags were then incubated in a humidified 37°C, 5-percent CO2 atmosphere. After 4 hours, the contents of the bag were gently resuspended, the beads were allowed to settle for 5 minutes at 1 × g, and the bag was clamped 1 cm above the settled beads. The supernatant was then expressed by the use of a transfer bag and a sterile connecting device (Terumo Corp., Phoenix, AZ). This procedure was repeated three times with 50 mL of AIM-V medium. As control for adherence, a sample of the expressed cells was im-munophenotyped for monocyte markers (CD14 and CD11c). Adherence of MNCs to the bag and bead surface was inferred by a decrease in the percentage of CD14+ and CD11c+ cells in the expressed fraction relative to that in the apheresis product. After removal of nonadherent cells, 100 mL of AIM-V medium containing rHu-GM–CSF (25 ng/mL; Sargramostim, Immunex, Seattle, WA)and rHu-IL-4 (1000 U/mL, Sigma, St. Louis, MO) was introduced into the tissue culture bags. The bags were placed into a dedicated, HEPA-filtered, humidified, 37°C, 5-percent CO2 incubator for 7 days. On Day 3 or 4, the bags were visually inspected for media color change or bacterial or fungal contamination. On Day 7, the tissue culture bags were taken from the incubator and samples removed for viability staining, microbial analyses, cell enumeration with a hematology analyzer (Beckman-Coulter, Hialeah, FL), microscopic examination of DC morphology, and immunophenotyping to determine the purity of the DC preparation. Immunophenotyping was performed with a flow cytometer (FACSCalibur, Becton Dickinson, San Jose, CA) and software (CellQuest, Becton Dickinson). The MoAb panel used included antibodies to CD45/CD14, CD3/CD19, CD1a, CD11c, HLA-DR, CD83, CD86, and CD123. On microscopic examination, plastic beads were not visible in the supernatant. The yields of DCs were improved over those in the other systems studied (Table 1). The immunophenotype of the recovered cells (Table 2) matches established DC phenotypes that are effective in adjuvant vaccine therapy. Culture supernatants were routinely negative for microbial contamination. We have developed a novel technique for the reproducible generation of DCs. The quantities of cells produced are