Human serum albumin (hsa)

TOM-1 (DMSZ, ACC 578) and MOLM-13 cells (DMSZ, ACC 554) were cultured in complete RPMI 1640 medium supplemented with 20% or 10% fetal calf serum (FCS), respectively (all purchased from Merck Millipore, Burlington, MA, USA). HEK293T (ATCC, CRL-11268), HT-1080 (ATCC, CCL-121), and OCI-AML3 cells (DMSZ, ACC 582) were cultured in DMEM, high glucose, GlutaMAX (Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 10% FCS. Primary AML blasts and CD34⁺ HSPCs were cultured in stem cell growth medium (SCGM) (CellGenix, Portsmouth, NH, USA) supplemented with 1% human serum albumin (HSA) (Baxter, Deerfield, IL, USA), 10 ng/mL Flt3L, 10 ng/mL SCF, and 10 pg/mL IL-3 (all purchased from PeproTech, Rocky Hill, NJ, USA). ALL blasts were cultured in adapted Iscove’s modified Dulbecco’s medium (IMDM) medium⁵⁹ (link) (Thermo Fisher Scientific, Waltham, MA, USA). Mycoplasma tested cells were maintained at 37°C in a humidified atmosphere of 5% CO₂.

Gemcitabine was got from Eli Lilly and Company (Indiana, USA). HSA (20%, 50 mL) was purchased from the Baxter AG (Vienna, Austria). cRGD (sequence: Arg-Gly-Asp-D-Tyr-Lys) was synthesized by Shanghai Gil Biochemistry Co., Ltd. (Shanghai, China); the purity was greater than 99.9%. Sulfo-SMCC (sulfosuccinimidyl-(4-N-maleimidomethyl)cyclohexane-1-carboxylate) was obtained from Thermo Fisher Scientific (Waltham, MA, USA). Normal saline (NS) was purchased from Shanghai Baxter Healthcare Co., Ltd. (Shanghai, China). Absolute ethanol and chloroform were purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). Deionized water was purchased from Fudan University (Shanghai, China). Roswell Park Memorial Institute (RPMI)-1640 culture medium and fetal bovine serum (FBS) were obtained from Gibco (Carlsbad, CA, USA). Double stain Hoechst/PI kit and myristic acid (C-14) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Cell Counting Kit-8 (CCK-8) was purchased from Dojindo (Kyushu, Japan). PBS and other reagents were prepared in our laboratory. All the solvents and chemicals were of analytical grade.

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.