Sodium pyruvate

The mouse BALB/c brain endothelioma cell line b.End5 (ECACC, Salisbury, UK) was used as a simplified BBB in vitro model [41 (link)]. b.End5 cells were grown in Dulbecco’s modified Eagle medium (DMEM high glucose, #41966052, Gibco, Life Technologies, New York, NY, USA) supplemented with 10% (v/v) foetal bovine serum (FBS, Sigma Aldrich, St. Louis, MO, USA), 1% (v/v) non-essential amino acids (Biochrom AG, Berlin, Germany), 2 mM _L-glutamine (Biochrom AG, Berlin, Germany), 1 mM sodium pyruvate (Biochrom AG, Berlin, Germany), and 1% (v/v) antibiotic–antimycotic solution (Sigma Aldrich, St. Louis, MO, USA). Cells were maintained at 37 °C in humid atmosphere enriched with 5% CO₂. For the viability assay, b.End5 cells were seeded using a volume of 200 µL at a density of 2.5 × 10⁴ cells/mL in rat tail collagen-I (100 µg/mL)-coated 96-well plates and incubated for 48 h at 37 °C and 5% CO₂.

The human NK cell lines NK-92 and NKL were a kind gift from Dr. Roland Jacobs (Hannover Medical School). All cells were cultured in RPMI 1640 supplemented with 10% FCS (both from Biochrom AG, Berlin, Germany), 100 U*ml⁻¹ penicillin and 100 mg* ml⁻¹ streptomycin (both from Sigma–Aldrich, St. Louise, USA) 1 mM sodium pyruvate, 2 mM L-glutamine (both from Biochrom AG) in a 5% CO₂ humified incubator (Thermo Fisher Scientific, Waltham, USA) at 37 °C. The medium for the NK cell lines was additionally supplemented with 200 U/ml human Interleukin-2 (IL-2) (Novartis Pharma GmbH, Zwickau, Germany).

The prostate cancer cell line PC3, LNCaP as well as the CHO cell line were purchased from American Type Culture Collection and have not been further authenticated. By FACS analysis an expression of PSCA was not detected in PC3 or LNCaP. PSMA expression was detectable in LNCaP but also not in PC3 cells. Therefore, PC3 cells were transduced with the open reading frame (orf) encoding PSCA or PSMA or both. LNCaP cells were transduced with orf encoding PSCA. For in vivo analysis the PC3 cell line expressing PSCA and PSMA was further modified to express the gene encoding firefly luciferase (Tu-Luc). Transduction was performed using a lentiviral packaging system as described previously [e.g. 15]. All cell lines were cultured in RPMI 1640 medium completed with 10% FCS, 100 U/ml penicillin and 100 μg/ml streptomycin, 2 mM N-acetyl-L-alanyl-L-glutamine, 1% non-essential amino acids and 1 mM sodium pyruvate (Biochrom). Cells were maintained at 37 °C in a humidified atmosphere of 5 % CO₂.

Cells were cultured in RPMI 1640 medium (Life Technologies) supplemented with penicillin (50 U/ml), streptomycin (50 U/ml), HEPES (25 mM), sodium pyruvate (1 mM), β-mercaptoethanol (50 μM) (all purchased from Biochrom) containing 10 % fetal calf serum (Sigma-Aldrich), at 37°C, 5 % CO₂ in 96U bottom plates (Corning).

We obtained AtT‐20 cells from the American Type Culture Collection (ATCC, Wesel, Germany). TtT/GF cells were kindly provided by the former laboratory of Dr. med. habil. Nicolai Savaskan at Erlangen University, Germany. Cells were grown in phenol red‐free Dulbecco's modified Eagle's medium (DMEM; Gibco, Waltham, MA, USA) containing 10% fetal bovine serum (FBS; Gibco), 0.1 mg·mL⁻¹ penicillin/streptomycin (Biochrom, Berlin, Germany), sodium pyruvate (1 mm; Biochrom), and nonessential amino acids (1×; Biochrom). Cells were cultivated under 5% CO₂ at 37 °C.

293T, Rabbit kidney (RK13), Henrietta Lacks (HeLa), African green monkey kidney (Vero), Crandell feline kidney (CrFK) and Madin-Darby canine kidney (MDCK) cells were propagated in Dulbecco’s modified Eagle’s medium (DMEM; Biochrom AG, Berlin, Germany) supplemented with 10% fetal bovine serum (FBS; Biochrom AG), and 1% penicillin-streptomycin. ED, Chinese hamster ovary (CHO)-K1, and CHO cells expressing HevA, HevB, and HevC (CHO-A, CHO-B and CHO-C cells, respectively; a kind gift from Dr. Patricia Spear, Northwestern University, Chicago, IL, USA) were grown in Iscove’s modified Dulbecco’s medium (IMDM; Invitrogen, Paisley, UK) supplemented with 20% FBS, 1% nonessential amino acids (Biochrom AG) and 1% 100 mM sodium pyruvate (Biochrom AG).
For generation of Vero cells, which transiently express EHV-4 gB (Vero/gB4), cells were transfected with the vector pcDNA3 (Invitrogen, Paisley, UK) containing gB4 using Lipofectamine 2000 (Invitrogen, Paisley, UK).
Peripheral blood mononuclear cells (PBMC) were isolated from heparinized blood collected by density gradient centrifugation over Histopaque 1077 (Sigma, St. Louis, MO, USA), following the manufacturer’s instructions. After two washing steps, cells were suspended in RPMI 1640 supplemented with 10% FBS, 0.3 mg/mL glutamine, non-essential amino acids, and 1% penicillin-streptomycin.

To test the biological activity of isolated hsEVs, we used COLO 357 cells (European Collection of Authenticated Cell Cultures, Salisbury, UK) that were once derived from a metastasis of a pancreatic adenocarcinoma and grow as an adhering monolayer with a cell doubling time of 21 h [22 (link)]. These cells were used because of their stable expression of the protease-activated receptors PAR1 and PAR2 and their low spontaneous migratory capacity [23 (link)]. Cells were cultured under serum-free conditions using RPMI1640 (Lonza, Basel, Switzerland) supplemented with 10% panexin (Pan-Biotech, Aidenbach, Germany), 1% penicillin streptomycin with glutamine (Gibco, Thermo Fisher Scientific, Waltham, MA, USA), and 1% sodium pyruvate (Biochrom, Berlin, Germany). We substituted fetal calf serum (FCS) (Biochrom, Berlin, Germany) by panexin to avoid contaminating our experimental preparation with FCS-derived EVs. Therefore, the concentration of FCS was diminished stepwise, and panexin was increased over several weeks. Cells were seeded in cell culture flasks (Sarstedt AG & Co, Nuembrecht, Germany) and incubated at 37 °C, 5% CO₂, and 95% humidity. Cells were free of contamination, and regular tests for detection of mycoplasma infections were performed.