Gentamycin

The use of human tissue was approved by the ethics committee of the Medical University Vienna, Austria (EK Nr. 957/2011, 30 January 2013), and all donors gave written consent. Human MSCs were isolated within 8 h after surgery as previously described (Egger et al., 2017 (link)). MSCs from 6 donors (aged 20–70) were cultivated in standard medium composed of MEM alpha (Thermo Fisher Scientific, Waltham, MA, USA), 0.5% gentamycin (Lonza, Basel, Switzerland), 2.5% human platelet lysate (PL BioScience, Aachen, Germany; filtered through 0.2 μm filters according to the data sheet provided by the manufacturer; Supplementary Figure 1) and 1 IU/ml heparin (Ratiopharm, Ulm, Germany) in humidified atmosphere at 37°C, 5% CO₂ and 21% or 5% O₂, and cryo-preserved in liquid nitrogen as previously described (Neumann et al., 2014 (link)). Upon use, MSCs were thawed and subcultivated once, resulting in passage 2. Cells intended for cultivation at 5% O₂ were isolated and subcultivated at 5% O₂ until seeding. To characterize MSC-derived EVs, MSCs (passage 2) were seeded at a density of 3,000 cells/cm² into 12-well plates (TPP, Trasadingen, Switzerland) (n = 4 each) and cultivated in 2 ml standard medium at 21 or 5% O₂ for 6 days. The medium was completely exchanged every second day, and medium without cells served as control. The supernatants were stored at −20°C until further use.

Human naïve CD4⁺ T cells were purified from peripheral blood mononuclear cells by non-T cell depletion using human naïve CD4⁺ T-cell isolation kit and AutoMACS (all from Miltenyi Biotec). T cells were resuspended in serum-free X-VIVO 15 medium supplemented with gentamycin (Lonza), seeded onto flat bottom polystyrene 48-well plates (Costar, Corning Inc.) at 10⁶ cells/ml in a volume of 1 ml per well and stimulated as described above. After 48 h supernatants were harvested, supplemented with 0,5% bovine serum albumin (Sigma-Aldrich) and frozen. Later, cytokine concentrations in the supernatants were measured using Bio-Plex Pro assay on the Bio-Plex 200 system (all from Bio-Rad) according to the manufacturer’s protocol.

‘Plating medium’ was composed as follows: 10% newborn calf serum, 2 mM L-glutamine, penicillin/streptomycin (100 units/mL and 0.1 mg/mL respectively), 0.1 mg/mL gentamycin (Biological industries, Kibbutz Beit-Haemek, Israel) in Earl’s Medium 199 (Sigma). ‘Assay medium’ contained: 2 mM L-glutamine, penicillin/streptomycin (100 units/mL and 0.1 mg/mL respectively), 0.1 mg/mL gentamycin, 550 nM hydrocortisone (Sigma) in Dulbecco-modified Earl’s medium (DMEM) diluted 1:1 in Ham’s F12 medium (Biological industries, Israel). For growing hCMEC/D3 (brain endothelial cell line) EGM-1 medium was used: EGM-2 medium (Lonza) supplemented with FBS, bFGF, gentamycin, ascorbic acid and hydrocortisone according to the manufacture instructions (EGM-2 SingleQuots, LONZA).

Numbers of freshly isolated neutrophils were adjusted to 5 × 10⁶ cells/mL in Roswell Park Memorial Institute (RPMI) medium supplemented with 10% sterile fetal calf serum (HyClone, South Logan, UT), 200mM glutamax (Thermofisher, Waltham, MA), 100μM pyruvate (ThermoFisher, Waltham, MA), and 50μg/ml gentamycin (Lonza, Basel, Switzerland) in a cell-repellent surface 48-well plate (Greiner Bio-one, Kremsmünster, Austria). Cells were then stimulated for 2 h at 37°C with 5% CO2 and 95% humidity with lipopolysaccharide (LPS; Escherichia coli 0111:B4 Ultrapure, 100 ng/mL, Invivogen, Toulouse, France) or heat-killed (30 min at 70°C) Klebsiella pneumoniae (ATCC43816; equivalent of 12.5 x 10⁶ CFU/mL). After 2 h neutrophils were centrifuged for 8 min at 1400 revolutions per minute, after which cell supernatants were stored at −80°C until further analysis. MPO release was measured in the stored supernatants using Luminex (R&D, Minneapolis, MN).

The use of human tissue was approved by the Ethics Committee of the Medical University Vienna, Austria (EK Nr. 957/2011, 30 January 2013), and all donors (female, 50–65 years old) gave written consent. Human adipose-derived MSCs were isolated within 8 h after surgery as described before [43 (link)]. MSCs were cultivated in standard medium composed of MEM alpha (Thermo Fisher Scientific, Waltham, MA, USA), 0.5% gentamycin (Lonza, Basel, Switzerland), 2.5% human platelet lysate (PL BioScience, Aachen, Germany), and 1 U/mL heparin (Ratiopharm, Ulm, Germany) in a humidified incubator at 37 °C, 5% CO₂ and cryo-preserved in liquid nitrogen as described before (Neumann et al., 2014). Upon use, MSCs were thawed at passage 2 or 3 and subcultivated once. Then, MSCs were seeded at 2000 cells/cm² in 24 well plates (TPP, Trasadingen, Switzerland) with 2 mL standard medium (n = 4). For the cytotoxicity testing, each resin was printed in the shape of discs (diameter of 5 mm, height of 2 mm). One disc per well was added and cells together with the discs were cultivated for 4 days. MSCs without additional discs served as the control.

The functional plasticity of the patients’ MO population was assessed by generating immature DC. Fresh monocytes were incubated in X-VIVO 15™ Media with Gentamycin and Phenol Red (Lonza, Cohasset, MN) supplemented with human interleukin 4 (hIL-4; Peprotech, Rocky Hill, NJ) at 500 IU/ml and human granulocyte monocyte colony stimulation factor (hGM-CSF; Peprotech, Rocky Hill, NJ) 1000 IU/ml 1 at 37 °C 5% CO₂ in the dark. On day 3, 50% of the X-VIVO15/10™ was replenished with fresh media along with 50% of the initial cytokine concentration. The cultures were terminated on day 5 [22 ].