Ficoll plaque premium

The collected whole blood (1.5 mL) samples were diluted in 2.5 mL of Hank’s balanced salt solution (HBSS) and carefully layered into 15 mL SepMate^TM tubes (StemCell Technologies, Vancouver, BC, Canada) containing 3.5 mL Ficoll Plaque Premium (GE Healthcare, Chicago, IL, USA). The tubes were centrifuged for 15 min (min) at 1200× g, 20 °C. The upper layer consisting of PBMCs was decanted into a 15 mL falcon tube containing 7 mL of HBSS. Following mixing, the tubes were centrifuged at 400× g (18 °C) for 15 min. Following centrifugation, the supernatant was discarded, and the pellet was resuspended in 7 mL of HBSS. The resuspended cells were centrifuged again under the same conditions. Then, the supernatant was discarded, and the pellet was resuspended in Roswell Park Memorial Institute medium (RPMI; Gibco, Carlsbad, CA, USA) containing 1% l-glutamine, 1% of antibiotic (100 U/mL penicillin and 100 µg/mL streptomycin) and 10% fetal bovine serum (FBS, Gibco, Carlsbad, CA, USA). The PBMCs in each sample were counted using LUNA^TM Cell Counting Slides (Logos Biosystems, Annandale, VA, USA) with the Luna^TM Automated Cell Counter (Logos Biosystems, Annandale, VA, USA).

Human peripheral blood mononuclear cells (PBMCs) were isolated from leucocyte cones of healthy donors by standard gradient centrifugation using Ficoll Plaque Premium (GE healthcare). PBMCs were then harvested from the interface, washed with PBS at 400g for 10 min and twice at 200g for 20 min. PBMCs were maintained at 37°C in a 5% CO₂ atmosphere in RPMI 1640 medium, supplemented with 10% FCS, 100 U ml⁻¹ penicillin and 100 µg ml⁻¹ streptomycin, 2 mM L-glutamine, 1 mM sodium pyruvate and 1% MEM non-essential amino acids and 25 µM of the thiol-oxidoreductase inhibitor PX-12 when indicated. In a MLR, PBMCs isolated from two donors were mixed at a 1 : 1 ratio to a final concentration of 1–2 × 10⁶ cells ml⁻¹.
2B4 Saito hybridoma T cells [16 (link)] were maintained at 37°C in a 10% CO₂ atmosphere in DMEM medium, supplemented with 10% FCS and 100 U ml penicillin and 100 µg ml⁻¹ streptomycin.

DC vaccination therapy at the Shinshu University Hospital was approved by the Ethics Committee of Shinshu University School of Medicine (approval number 1123 and 1199). The Act on the Safety of Regenerative Medicine in Japan was enforced since November 25, 2014. Class III technologies use somatic cells with accumulated clinical experiences and are regarded as low risk technologies. The DC vaccination therapy (Class III technology) at the Shinshu University was approved on November 25, 2015 (approval number: PC3150643 and PC3150645). Human peripheral blood mononuclear cell (PBMC)-rich fraction was collected from blood samples of patients via leukapheresis with a COM.TEC cell separator (Fresenius Kabi Japan K.K., Tokyo, Japan). PBMCs were subsequently isolated using a Ficoll-Plaque Premium (GE Healthcare, Piscataway, NJ, USA) density gradient. The collection and use of blood complied with relevant guidelines and institutional practices from Ethics Committees of Shinshu University School of Medicine (approval number 2107). Written informed consent was obtained from all patients. All methods in this study were performed in accordance with the Ethical Guidelines for Medical and Health Research involving Human Subjects proposed by Ministry of Health, Labour and Welfare in Japan60 .