Heparin

The right kidney was removed either at nephrectomy or at the time of termination. The mice were injected with 5000 U/kg BW heparin i.p. (Ratiopharm GmbH, Ulm, Germany), and 3 min later they were sacrificed by cervical dislocation. The chest was opened and after cross-section of the vena cava, blood was collected from the thoracic cavity. Blood was washed out from the blood vessels by intracardial injection of 10 mL 4 °C saline. The kidneys were removed and decapsulated. One third of the upper pole of the kidneys was placed in 500 µL TRI Reagent (TR 118, Molecular Research Center, Inc., Cincinnati, OH, USA) and was snap frozen in liquid nitrogen and kept at −80 °C for RNA isolation. A 1 mm cross-section of the kidney at the hilus level including all layers of cortex and medulla was fixed in 4% buffered formaldehyde. On the next day, the section was dehydrated and embedded in paraffin (FFPE) for histology. The remaining parts of the kidney were cut into pieces and snap frozen in liquid nitrogen and kept at −80 °C for any further molecular biology analysis.

For whole mount staining and qualitative RT-PCR, the trachea was removed and opened by cutting the trachealis muscle longitudinally to expose the lining epithelium. The trachea was then pinned on a piece of wax to align the epithelial layer approximately in the same plane. Then, the samples were either used for RT-PCR (n = 7) as described later or for whole mount immunostaining (n = 19). Samples subjected to whole mount staining were fixed overnight by immersion in Zamboni’s fixative, composed of 2% formaldehyde (Carl Roth) and 15% picric acid (Merck, Darmstadt, Germany), and then washed in 0.1 M phosphate buffer.
Trachea, lung, and spleen processed for sectioning were dissected either freshly or after transcardiac perfusion with Zamboni solution, after initial perfusion with a rinsing solution containing heparin (2 ml/l; 10,000 U; Ratiopharm, Ulm, Germany) and procaine hydrochloride (5 g/l; Merck), pH 7.4. Subsequently, all tissues were immersed in Zamboni’s fixative overnight at 4 °C, washed in 0.1 M phosphate buffer, and, if processed for cryosectioning, stored overnight in 18% sucrose in the same buffer (Merck). Afterward, specimens were embedded in an OCT cryostat sectioning medium (Sakura Finetek, Staufen, Germany) and stored at −20 °C until further processing. Samples of three animals were routinely embedded in paraffin after immersion fixation and buffer wash.

Polyplexes containing 2 μg pDNA were formed at N/P ratios of 6 and 12 in a total volume of 200 μL HBG. lPEI polyplexes formed at N/Ps 6 and 12 served as a positive control. HBG buffer (200 μL) served as blank, and 2 μg pDNA in 200 μL HBG buffer was considered as maximum ethidium bromide (EtBr) fluorescence intensity (100% value). These samples were prepared in parallel to the polyplexes. After 30 min of incubation at room temperature, 700 μL of EtBr solution (c = 0.5 μg/mL) were added. The fluorescence intensity of EtBr was measured after an additional 3 min incubation using a Cary Eclipse spectrophotometer (Varian, Germany) at the excitation wavelength λ_ex = 510 nm and emission wavelength λ_em = 590 nm. The fluorescence intensity of EtBr was determined in relation to the 100% value. As a further experiment, 250 IU of heparin (Ratiopharm, Ulm, Germany) was added to the samples after EtBr addition to investigate polyplex stability against polyanionic stress.

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.

One hundred and twenty-nine/Sv mice (2–3 months old, 20–30 g body weight) were anaesthetized by IP injection containing ursotamin (100 mg/kg body weight, Serumwerk, Saale, Germany), xylazin (20 mg/kg body weight, Ceva Tiergesundheit, Düsseldorf, Germany), and heparin (50 I.E./g body weight, Ratiopharm, Ulm, Germany). Pulmonary arterial smooth muscle cells (PASMCs) were isolated from mouse precapillary pulmonary arterial vessels using iron particles (Sigma-Aldrich, Taufkirchen, Germany) according to a previously reported protocol [23 (link)]. Primary calls were cultured for 6–8 days in smooth muscle cell growth medium (Promocell GmbH, Heidelberg, Germany) supplemented with Normocin (Invivogen, Toulouse, France) and 10% FBS (Sigma-Aldrich). After the first passaging, cells were seeded in 6-well plates (100,000 cells/well) and left for 24h to attach followed by 24 h starvation in smooth muscle cell basal medium (Promocell GmbH) without supplements. The cells were further put in a hypoxic (1% O₂, 5% CO₂) or normoxic (21% O₂, 5% CO₂) condition in growth medium for 72 h. Afterwards the cells were lysed on ice in cell lysis buffer (Cell Signaling Technology, Leiden, Netherlands) containing PMSF. Protein samples were collected and stored at −20 °C until further analysis.

At 6 weeks post human CD4⁺ T cell transfer, NSG-DR1 mice were sacrificed by cervical dislocation, then whole-body perfusion with PBS + 50 U/ml heparin (Ratiopharm, Ulm, Germany) was conducted and the organs were extracted for ex vivo analysis. FACS analysis of freshly isolated cells (heart and spleen) was performed. The samples intended for RNA analysis were stored in RNAlater (Qiagen, Hilden, Germany) for 24 h and then stored at -80°C. Samples intended for histological analysis were embedded in Tissue-TeK optimum cutting temperature medium (Sakura Finetek, Alphen ann den Rijn, The Netherlands) and then stored at -80°C. Heart DNA extraction was performed from heart slices by trimming the Tissue-TeK content and performing tissue digestion and DNA purification using a GeneJET genomic DNA purification kit (Thermo Scientific, Vilnius, Lithuania) according to the manufacturer’s instructions.

The GSC lines ZH-161, ZH-305, S24, T-325, and T-269 were established from freshly resected tumors and cultured as sphere cultures under stem cell conditions. All other cell lines were cultured under standard adherent conditions. Stem cell conditions for culturing GSCs were neurobasal medium supplemented with B-27 (20 μL/mL; Invitrogen), GlutaMAX (10 μL/mL; Invitrogen), fibroblast growth factor-2, epidermal growth factor (20 ng/mL each; PeproTech), and heparin (32 IU/mL; Ratiopharm). Growth factors were replenished twice weekly. The non-GSC LN-18, LN-229, LN-308, LN-319, and LN-428 glioma cell lines were kindly provided by N. de Tribolet, and T98G, U87MG, and A172 glioma cell lines were purchased from the American Type Culture Collection. Non-GSC cell lines were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% FCS. Dimethyl sulfoxide was utilized as a solvent for TMZ (Merck) and Grm2/3 negative allosteric modulators RO1 and RO2 at final concentrations of 0.2%. Grm2/3 negative allosteric modulators RO1 and RO2 were provided by F. Hoffmann-La Roche.