Cellgro

Leukapheresis was performed using a Cobe Spectra separator (Cobe BCT, Lakewood, CO, USA). All of the following operations were performed under Good Manufacturing Practice (GMP) conditions in the GMP facility of University Hospital Motol using the protocol for DC generation that was approved by the State Institute for Drug Control, as previously described [25 (link), 26 (link)]. The leukapheretic product was diluted in PBS + 1 mM EDTA (Lonza, Verviers, Belgium), and mononuclear cells were separated by Ficoll-Paque Premium (GE Healthcare, Waukesha, WI, USA) gradient centrifugation. Collected mononuclear cells (PBMC) were washed in PBS + 1 mM EDTA (Lonza), resuspended in CellGro medium (CellGenix, Freiburg, Germany) and plated in triple flasks (Thermo Scientific, Waltham, MA, USA) at 1 × 10⁶ cells per cm² of surface area. After 2 h, non-adherent cells were washed with PBS (Lonza). Adherent monocytes were cultured for 6 days in CellGro (CellGenix) medium with 20 ng/ml of IL-4 (Gentaur, Kampenhout, Belgium) and 500 U/ml of GM-CSF (Gentaur); fresh cytokines were added on day 3. Immature DCs were harvested on day 6, washed in PBS (Lonza) and resuspended in CellGro (CellGenix).

Blood obtained from each study volunteer at the Austrian Red Cross Blood Transfusion Service of Upper Austria was used to produce autologous APOSEC according to current GMP guidelines. PBMCs were separated from whole blood samples of the participants by density centrifugation using LSM 1077 (Lymphocyte Separation Medium, Lonza, Switzerland). Removal of LSM was achieved by two washing steps using Dulbecco’s phosphate-buffered saline (Lonza, Switzerland). PBMCs were resuspended in phenol red–free CellGro GMP DC medium (CellGenix, Freiburg, Germany) containing no xenogeneic proteins. A sample was drawn for complete blood count to adjust white blood cells to a concentration of 25 × 10⁶ cells/ml. Irradiation with 60 Gy induced apoptosis of PBMCs. By cultivation of apoptotic PBMCs in CellGro GMP DC medium, release of the secretome was achieved. After incubation for 24 h ± 2 h, cells were removed by centrifugation. The supernatant containing the secretome was sterile filtered at a pore size of 0.22 μm. The adequate production of APOSEC was defined by appropriate secretion of the following important cytokines: interleukin (IL)-8 (0–5214 pg/ml), epidermal growth factor (EGF; 25–226 pg/ml), and transforming growth factor-β (TGF-β; 2575–21732 pg/ml).
Lyophilized culture medium not containing any cells (CellGro, CellGenix, Freiburg, Germany) served as placebo.

Human peripheral blood mononuclear cells (PBMC) were isolated from four healthy male volunteers by venous blood draw and density gradient centrifugation with Ficoll-Paque (GE Healthcare Bio-Sciences AB, Sweden). PBMCs (25 × 10⁶ cells/ml) were resuspended in serum-free medium (CellGro, CellGenix, Freiburg, Germany). An automated cell counter (Sysmex Inc., USA) was used to determine cell count. PBMCs were gamma-irradiated with 60 Gy to induce apoptosis. Induction of apoptosis was confirmed by annexin V-fluorescein/propidium iodide (FITC/PI) co-staining (Becton Dickinson, Franklin Lakes, NJ, USA) using a flow cytometer. At 20h after irradiation 58% of PBMCs were annexin V/PI positive (supplementary Fig. S1). CM was collected from cultures at 2, 4, and 20 h time points, and then, centrifuged (500 × g for 9 min) to remove cell debris. CM was stored at −80 °C for subsequent protein and lipid analyses. Fresh CM was used for microparticle and exosome separations.