Manufacturing Regulatory T Cells from Umbilical Cord Blood

UCB Tregs were manufactured as described previously [1 (link)]. Cryopreserved UCB units matched at HLA 3/6 to recipient were provided by MDACC UCB bank. Units were thawed and processed in CliniMACS buffer (Miltenyi Biotec, Bergish Gladbach, Germany) containing 0.5% HSA (Baxter Healthcare, Westlake Village, CA. USA) and mono-nuclear cells selected for CD25⁺ using magnetic beads according to manufacturer's instructions (Miltenyi Biotec, Bergish Gladbach, Germany) [23 (link)]. CD25⁺ cells were co-cultured with CD3/28 co-expressing Dynabeads® (Clin Ex Vivo ™ CD3/CD28, Invitrogen, Carlsbad, CA) at 1:1 cell:bead ratio and seeded at 1×10⁶ cells/ml in EX-VIVO 15 medium (Cambrex BioScience, Walkersville, MD) supplemented with 10% human AB serum (Gemini Bio-Products, Sacramento, CA), 2 mM L-glutamine (Sigma, St. Louis, MO), 1% Penicillin-Streptomycin (Gibco/Invitrogen, Grand Island, NY)] and 1000 IU/ml interleukin (IL)-2 (CHIRON Corporation, Emeryville, CA). Cells were cultured in tissue culture flasks at 37°C/5% CO₂ incubator and maintained at 1×10⁶ cells/ml with fresh medium and IL-2 every 48-72 hours. Culture was harvested at 14 days. Release criteria included: Viability ≥70%, Endotoxin <5EU/kg, Gram Stain: No organism seen, Mycoplasma: Negative, Sterility: No organism at time of infusion, CD4⁻CD8⁺ cells: <10 %, CD4⁺CD25⁺ cells >60% and <100 beads per 3×10⁶ cells.