Amphotericin b

A549-Dual™ cells (InvivoGen, Toulouse, France, a549d-nfis), designated hereafter as A549 cells, were cultured at 37 °C under a 5% CO₂ atmosphere in 4.5 g·L⁻¹ glucose Dulbecco’s Modified Eagle’s Medium (DMEM) (PAN Biotech Dutscher, Brumath, France), supplemented with 1% or 10% of heat-inactivated fetal bovine serum (FBS) (Dutscher, Brumath, France), 100 U·mL⁻¹ penicillin, 100 μg·mL⁻¹ streptomycin, 2 mM L-glutamine, and 0.5 μg·mL⁻¹ amphotericin B (PAN Biotech, Dutscher, Brumath, France), and supplemented with 10 μg·mL⁻¹ blasticidin and 100 μg·mL⁻¹ zeocin (InvivoGen, Toulouse, France). Human Embryonic Kidney, HEK 293T (CRL-3216) was cultured at 37 °C under a 5% CO₂ atmosphere in DMEM, supplemented with 5% or 10% heat-inactivated FBS. For exosome purification, cells were grown in 1% FBS media or in Panserin 401 (PAN Biotech Dutscher, Brumath, France). The ZIKV clinical isolate PF-25013-18 (PF13) has been previously used and described [52 (link)]. The DENV2 clinical isolate, strain UVE/DENV2/2018/RE/47099 was provided by the H2020 European Virus Archive goes global (EVAg). The two flaviviruses were amplified on Vero E6 cells and titrated by plaque-forming unit assay before infection of the A549 cells at a multiplicity of infection (moi) of 5 for 48 h for ZIKV and 72 h for DENV2.

Bone marrow MSCs were seeded at density 5.0 × 10⁶ cells in a cell culture medium consisting of Mesenchymal Stem Cell Basal Medium (ATCC, Manassas, VI, USA) supplemented with Mesenchymal Stem Cell Grow Kit—low serum (ATCC, Manassas, VI, USA), 10,000 U/mL Penicillin, 10 mg/mL streptomycin, 25 ug/mL amphotericin B (PAN Biotech GmbH, Aidenbach, Germany) at 37 °C and 5% CO₂.

We used the following pancreatic cancer cell lines: BxPC-3, Capan-1, DanG, HuP-T3, Panc-1, MIA PaCa-2, PSN-1, and AsPC-1. All cell lines were cultured in the appropriate media according to the ATCC recommendations (Gibco^® RPMI 1640 for BxPC-3, Capan-1, DanG, PSN-1, and AsPC-1 or Gibco^® Dulbecco’s Modified Eagle’s Medium (DMEM) for the remaining), supplemented with 10% fetal bovine serum (FBS; Corning, Wiesbaden, Germany), and 1% streptomycin/penicillin (PAN-Biotech, Aidenbach, Germany) at 37 °C in a humidified incubator with 5% CO₂. For all experiments, cell lines were used up to passage number 20. Cell culture medium for pancreatic stellate cells (PSCs) consisted of Gibco^® DMEM/F-12, 1% amphotericin B (PAN-Biotech, Aidenbach, Germany), 10% FBS, and 1% streptomycin/penicillin. Detection of mycoplasma was conducted regularly using conventional PCR technique. All cells tested negative for mycoplasma contamination. All cells were authenticated commercially by IDEX BioResearch (Ludwigsburg, Germany).

Human peripheral blood mononuclear cells (PBMCs) were isolated from whole blood of the RA patients (three donors). Isolation was performed using Histopaque®-1077 (Sigma-Aldrich, Munich, Germany) as density gradient and SepMate™-50 (Stemcell™ Technologies, Vancouver, BC, Canada) tubes according to the manufacturer's protocol. The tubes were centrifuged with 1200 × g for 20 min with full acceleration and break on. After isolation, cells, now referred to as mononuclear osteoclast progenitor cells (OPCs), were cultured in cell-repellent 6-well cell culture plates in Roswell Park Memorial Institute (RPMI; PAN-Biotech, Aidenbach, Germany) 1640 supplemented with 1% amphotericin B, 1% penicillin–streptomycin, 20% FCS and 2% stable glutamine (PAN-Biotech, Aidenbach, Germany). After five days, cell supernatant with non-adherent cells was transferred to 50 ml tubes, each well of the plate was rinsed with PBS, and the tubes were centrifuged at 120 × g for 8 min.
For co-culture experiments, cells were seeded in 12-well cell culture plates with 300.000 cells per well and incubated for 14 days. For differentiation of OPCs in polynuclear osteoclast-like cells (OLCs), the medium was supplemented with 5 µg/mL human sRANK-Ligand and 2,5 µg/mL human macrophage colony-stimulating factor (M-CSF; both: Peprotech Inc., Rocky Hill, NJ, USA). After seven days of incubation, the whole medium was replaced.

Primary HSF were obtained from ScienCell Research Laboratories (ScienCell, 4700; Clinisciences, Carlsbad, CA, USA). Cells were cultured in Modified Eagle’s Medium (MEM eagle, PAN Biotech P0408500, Aidenbach, Germany) supplemented with 10% heat-inactivated fetal bovine serum (FBS, PAN Biotech, 3302 P290907, Aidenbach, Germany), 2 mM L-glutamine (Biochrom AG, K0282, Berlin, Germany), 0.1 mg/mL penicillin-streptomycin (PAN Biotech, P0607100), 1 mM sodium pyruvate (PAN Biotech, P0443100) and 0.5 µg/mL amphotericin B (PAN Biotech, P0601001).
ONNV was provided by the CNR (Centre national de référence) Arbovirus of Marseille (France). Polyinosinic-polycytidylic acid (PIC) was purchased from Amersham Biosciences, Little Chalfont, UK (catalog number: 27-4732-01) and recombinant human IL-1β and TNF-α from Peprotech, Rocky Hill, CT, USA (catalog number: 200-01B and 300-01A, respectively). Irinotecan hydrochloride trihydrate was obtained from “medac Gesellschaft für klinische Spezialpräparate mbH” (Wedel, Germany).

Articular chondrocytes (pACs) of porcine origin were harvested from non-arthritic knee joints from 4- to 6-month-old female and male porcine donors derived from the slaughterhouse (n = 8). Cartilage slices (1–2 mm) were digested in a rotating bioreactor tube overnight with collagenase NB5 synthesized by Clostridium histolyticum (1 mg/mL, Nordmark, Uetersen, Germany) and dissolved in chondrocyte growth medium (96% (v/v) DMEM/Ham’s F-12 (1:1) with stable L-glutamin, 1% (v/v) amphotericin B, 1% (v/v) MEM amino acids, 1% (v/v) penicillin/streptomycin, 1% (v/v) ascorbic acid (all: PAN-Biotech GmbH, Aidenbach, Germany). A cell strainer was used (100 µm pore size, TPP, Trasadingen, Switzerland) to remove undigested ECM remnants. Isolated pACs were washed in PBS and cultured in chondrocyte growth medium containing 10% fetal calf serum (FCS) in T175 flasks (CellPlus, Sarstedt AG, Nümbrecht, Germany). At confluence, pACs were stripped off with 0.05%/0.02% trypsin/ethylenediaminetetraacetic acid (EDTA, Carl Roth GmbH and Ko.KG, Karlsruhe, Germany) and propagated in T175 flasks.