Blood cell separator

A total of 5-10 μg/kg granulocyte colony-stimulating factor (G-CSF) was administered subcutaneously to the donors on a daily basis for 5 days.. Peripheral blood stem cells were collected from donors on the fourth and fifth days using a blood cell separator (Fresenius kabi LLC). The peripheral blood leukocyte count was 20-40×10⁹/L.
For patients who underwent bone marrow transplantation (BMT), bone marrow was harvested from the posterior ilium of donors under local anesthesia on the fourth day, at 5 ml/kg of patient, according to standard procedures. The collection criteria included: mononuclear cells ≥5×10⁸/kg and CD34⁺ cells ≥2×10⁶/kg. The ratio of bone marrow cells to peripheral blood cells was approximately 1:2 in the case of BMT+PBSCT. Among the transplantation cases, seven received BMT, 89 received PBSCT and 28 underwent PBSCT+BMT. Red blood cells or blood plasma in the bone marrow were eliminated in patients with mismatched blood types. Bone marrow suspension was transfused immediately into patients without in vitro T-lymphocyte manipulation.

Approximately 40-60ml of white cells was obtained from each patient using a blood-cell separator (FreseniusKabi, Germany). The yield of peripheral blood mononuclear cells (PBMCs) ranged from 1×10⁹-3×10⁹ cells. Cells were washed twice with AIM-V cell-culture medium (Gibco, America), transferred into culture flasks, and cultured for 2 hours. Adherent cells were used for induction of DCs by adding 100ng/ml GM-CSF (PeproTech, America) and 100ng/ml IL-4 (PeproTech, America) to the AIM-V cell-culture solution. Remaining non-adherent cells from the remainder of the PBMCs were stored at -80℃ and thawed for co-cultivation. On day 6, recombinant adenoviruses encoding different TAAs were used to infect DCs (MOI=10). On day 7, IL-1β (25 ng/ml; Novoprotein, China) and TNF-α (100 ng/ml; Novoprotein, China) were added into the culture for maturation of DCs.