Tregs from the Quantum system demonstrated an 8-fold greater interleukin-10 stimulation index than cells from flask culture following restimulation. 5.58 108) Tregs in flasks (mean viability 71.3%) versus 7.00 109 (range 3.57 109 to 13.00 109) Tregs in the Quantum system (mean viability 91.8%), demonstrating a mean 17.7-fold increase in Treg yield for the Quantum system over that obtained in flasks. The two culture processes gave rise to cells with a memory Treg CD4+CD25+FoxP3+CD45RO+ phenotype of 93.7% for flasks versus 97.7% for the Quantum system. Tregs from the Quantum system demonstrated an 8-fold greater interleukin-10 stimulation index than cells Cor-nuside from flask culture following restimulation. Quantum systemCexpanded Tregs proliferated, maintained their antigenic phenotype, and suppressed effector immune cells after cryopreservation. We conclude that an automated perfusion bioreactor can support the scale-up expansion of functional Tregs more efficiently than diffusion-based flask culture. for 5 min, spent medium was aspirated, and cells were resuspended in 124 ml complete medium. Cells were manually disassociated and removed from flasks by serological pipet and subsequently centrifuged as before prior to counting and cryopreservation as described above. Calculation of Population Cell Rabbit Polyclonal to PKR Doublings and Cell Doubling Time For the purposes of this study, population cell doublings (DS) and cell doubling time (DT) were estimated in terms of the standard exponential growth equation over the period of cell expansion as derived from Sherley23. for 5 min, supernatant was discarded, and pellets were resuspended with fresh Treg medium. To measure the growth curve, Tregs were seeded at a density of 2 106 cells per well in a 6-well plate (Greiner Bio-One N.A., Monroe, NC, USA) coated with goat antimouse IgG antibody (Thermo Fisher Scientific) and mouse anti-CD28 mAb (BD Biosciences) at respective concentrations of 5 and 10 g/ml, as previously described24. The number of viable cells was counted by trypan blue exclusion at each time point using a Neubauer chamber (Cat. 5971R30, Hausser Scientific, Horsham, PA, USA) and visualized with 10 light microscopy (Evos XL core microscope, Invitrogen-ThermoFisher Scientific, Grand Island, NY, USA). Cells Cor-nuside grown for 5 and 13 d were subsequently used in the suppression assay (see below). Animals Sprague Dawley rats constitutively expressing green fluorescent protein (GFP) were acquired from the Rat Resource and Research Center (Columbia, MO, USA; strain SD-Tg(UBC-EGFP) 2BalRrrc). SD-Tg(UBC-EGFP) 2BalRccc are transgenic rats that express enhanced green fluorescent protein (EGFP) from the ubiquitin C promoter in all cells25,26. Animals were acquired, cared for, and used in accordance with the NIH Guide for the Care and Use of Laboratory Animals and followed a protocol approved by the University of Wyoming Institutional Animal Care and Use Committee. Rats were housed at ambient temperature with stable humidity and natural dayCnight cycle, with free access to rodent laboratory food and water. Proliferation Assay Assays were carried out as previously described24. Briefly, Tregs were thawed and expanded as described above and then cocultured with isolated splenocytes from GFP rats. Three days prior to coculture, GFP rat splenocytes were stimulated with 5 g/ml concanavalin A (con A; Sigma-Aldrich Corp.). Tregs were mixed with 20,000 con ACstimulated GFP rat splenocytes at ratios of 1 1:1, 1:2, 1:4, 1:6, 1:8, and 1:16 splenocytes to Tregs (day 0 of co-culture) and proliferation of splenocytes was measured over a 4-d period by GFP fluorescence using a Tecan M200 plate reader (Tecan Group Ltd, M?nnedorf, Switzerland). Co-culture was carried Cor-nuside out in RPMI 1640, 10% FBS, 100 U/ml penicillin, Cor-nuside 100 g/ml streptomycin, and 50 M -mercaptoethanol in 96-well plates. Measurement of proliferation from con ACstimulated GFP splenocytes alone was used as the control. The suppression assay was performed using Tregs from each donor separately after 5 or 13 d of monoculture following thawing. Correlation of rat splenocyte fluorescence in co-culture with Quantum systemCexpanded Tregs was calculated and averaged across all three donor cell products.